Iterative and regenerative DNA sequencing method

An iterative and regenerative method for sequencing DNA is described. This method sequences DNA in discrete intervals starting at one end of a double stranded DNA segment. This method overcomes problems inherent in other sequencing methods, including the need for gel resolution of DNA fragments and the generation of artifacts caused by single-stranded DNA secondary structures. A particular advantage of this invention is that it can create offset collections of DNA segments and sequence the segments in parallel to provide continuous sequence information over long intervals. This method is also suitable for automation and multiplex automation to sequence large sets of segments.

BACKGROUND OF THE INVENTION 
Analysis of DNA with currently available techniques provides a spectrum of 
information ranging from the confirmation that a test DNA is the same or 
different than a standard sequence or an isolated fragment, to the express 
identification and ordering of each nucleotide of the test DNA. Not only 
are such techniques crucial for understanding the function and control of 
genes and for applying many of the basic techniques of molecular biology, 
but they have also become increasingly important as tools in genomic 
analysis and a great many non-research applications, such as genetic 
identification, forensic analysis, genetic counseling, medical diagnostics 
and many others. In these latter applications, both techniques providing 
partial sequence information, such as fingerprinting and sequence 
comparisons, and techniques providing full sequence determination have 
been employed (Gibbs et al., Proc. Natl. Acad. Sci USA 1989; 86:1919-1923; 
Gyllensten et al., Proc. Natl. Acad. Sci USA 1988; 85:7652-7656; Carrano 
et al., Genomics 1989; 4:129-136; Caetano-Annoles et al., Mol. Gen. Genet. 
1992; 235:157-165; Brenner and Livak, Proc. Natl. Acad. Sci USA 1989; 
86:8902-8906; Green et al., PCR Methods and Applications 1991; 1:77-90; 
and Versalovic et al., Nucleic Acid Res. 1991; 19:6823-6831). 
DNA sequencing methods currently available require the generation of a set 
of DNA fragments that are ordered by length according to nucleotide 
composition. The generation of this set of ordered fragments occurs in one 
of two ways: chemical degradation at specific nucleotides using the Maxam 
Gilbert method (Maxam A M and W Gilbert, Proc Natl Acad Sci USA 1977; 
74:560-564) or dideoxy nucleotide incorporation using the Sanger method 
(Sanger F, S Nicklen, and A R Coulson, Proc Natl Acad Sci USA 1977; 
74:5463-5467) so that the type and number of required steps inherently 
limits both the number of DNA segments that can be sequenced in parallel, 
and the number of operations which may be carried out in sequence. 
Furthermore, both methods are prone to error due to the anomalous 
migration of DNA fragments in denaturing gels. Time and space limitations 
inherent in these gel-based methods have fueled the search for alternative 
methods. 
Several methods are under development that are designed to sequence DNA in 
a solid state format without a gel resolution step. The method that has 
generated the most interest is sequencing by hybridization. In sequencing 
by hybridization, the DNA sequence is read by determining the overlaps 
between the sequences of hybridized oligonucleotides. This strategy is 
possible because a long sequence can be deduced by matching up distinctive 
overlaps between its constituent oligomers (Strezoska Z, T Paunesku, D 
Radosavljevic, I Labat, R Drmanac, R Crkvenjakov, Proc Natl Acad Sci USA 
1991; 88:10089-10093; Drmanac R, S Drmanac, Z Strezoska, T Paunesku, I 
Labat, M Zeremski, J Snoddy, W K Funkhouser, B Koop, L Hood, R 
Crkvenjakov, Science 1993; 260:1649-1652). This method uses hybridization 
conditions for oligonucleotide probes that distinguish between complete 
complementarity with the target sequence and a single nucleotide mismatch, 
and does not require resolution of fragments on polyacrylamide gels 
(Jacobs K A, R Rudersdorf, S D Neill, J P Dougherty, E L Brown, and E F 
Fritsch, Nucleic Acids Res. 1988; 16:4637-4650). Recent versions of 
sequencing by hybridization add a DNA ligation step in order to increase 
the ability of this method to discriminate between mismatches, and to 
decrease the length of the oligonucleotides necessary to sequence a given 
length of DNA (Broude N E, T Sano, C L Smith, C R Cantor, Proc. Natl. 
Acad. Sci. USA 1994;91:3072-3076, Drmanac R T, International Business 
Communications, Southborough, Mass.). Significant obstacles with this 
method are its inability to accurately position repetitive sequences in 
DNA fragments, inhibition of probe annealing by the formation of internal 
duplexes in the DNA fragments, and the influence of nearest neighbor 
nucleotides within and adjacent to an annealing domain on the melting 
temperature for hybridization (Riccelli P V, A S Benight, Nucleic Acids 
Res 1993;21:3785-3788, Williams J C, S C Case-Green, K U Mir, E M 
Southern. Nucleic Acids Res 1994;22:1365-1367). Furthermore, sequencing by 
hybridization cannot determine the length of tandem short repeats, which 
are associated with several human genetic diseases (Warren S T, Science 
1996; 271:1374-1375). These limitations have prevented its use as a 
primary sequencing method. 
The base addition DNA sequencing scheme uses fluorescently labeled 
reversible terminators of polymerase extension, with a distinct and 
removable fluorescent label for each of the four nucleotide analogs 
(Metzker M L, Raghavachari R, Richards S, Jacutin S E, Civitello A, 
Burgess K and R A Gibbs, Nucleic Acids Res. 1994; 22:4259-4267; Canard B 
and R S Sarfati, Gene 1994; 148:1-6). Incorporation of one of these base 
analogs into the growing primer strand allows identification of the 
incorporated nucleotide by its fluorescent label. This is followed by 
removal of the protecting fluorescent group, creating a new substrate for 
template-directed polymerase extension. Iteration of these steps is 
designed to permit sequencing of a multitude of templates in a solid state 
format. Technical obstacles include a relatively low efficiency of 
extension and deprotection, and interference with primer extension caused 
by single-strand DNA secondary structure. A fundamental limitation to this 
approach is inherent in iterative methods that sequence consecutive 
nucleotides. That is, in order to sequence more than a handful 
nucleotides, each cycle of analog incorporation and deprotection must 
approach 100% efficiency. Even if the base addition sequencing scheme is 
refined so that each cycle occurs at 95% efficiency, one will have &lt;75% of 
the product of interest after only 6 cycles (0.95.sup.6 =0.735). This will 
severely limit the ability of this method to sequence anything but very 
short DNA sequences. Only one cycle of template-directed analog 
incorporation and deprotection appears to have been demonstrated so far 
(Metzker M L, Raghavachari R, Richards S, Jacutin S E, Civitello A, 
Burgess K and R A Gibbs, Nucleic Acids Res. 1994; 22:4259-4267; Canard B 
and R S Sarfati, Gene 1994; 148:1-6). A related earlier method, which is 
designed to sequence only one nucleotide per template, uses radiolabeled 
nucleotides or conventional non-reversible terminators attached to a 
variety of labels (Sokolov B P, Nucleic Acids Research 1989;18:3671; 
Kuppuswamy M N, J W Hoffman, C K Kasper, S G Spitzer, S L Groce, and S P 
Bajaj, Proc. Natl. Acad Sci. USA 1991; 88:1143-1147). Recently, this 
method has been called solid-phase minisequencing (Syvanen A C, E Ikonen, 
T Manninen, M Bengstrom, H Soderlund, P Aula, and L Peltonen, Genomics 
1992; 12:590-595; Kobayashi M, Rappaport E, Blasband A, Semeraro A, 
Sartore M, Surrey S, Fortina P., Molecular and Cellular Probes 1995; 
9:175-182) or genetic bit analysis (Nikiforov T T, R B Rendle, P Goelet, Y 
H Rogers, M L Kotewicz, S Anderson, G L Trainor, and M R Knapp, Nucleic 
Acids Research 1994; 22:4167-4175), and it has been used to verify the 
parentage of thoroughbred horses (Nikiforov T T, R B Rendle, P Goelet, Y H 
Rogers, M L Kotewicz, S Anderson, G L Trainor, and M R Knapp, Nucleic 
Acids Research 1994; 22:4167-4175). 
An alternative method for DNA sequencing that remains in the development 
phase entails the use of flow cytometry to detect single molecules. In 
this method, one strand of a DNA molecule is synthesized using 
fluorescently labeled nucleotides, and the labeled DNA molecule is then 
digested by a processive exonuclease, with identification of the released 
nucleotides over real time using flow cytometry. Technical obstacles to 
the implementation of this method include the fidelity of incorporation of 
the fluorescently labeled nucleotides and turbulence created around the 
microbead to which the single molecule of DNA is attached (Davis L M, F R 
Fairfield, C A Harger, J H Jett, R A Keller, J H Hahn, L A Krakowski; B L 
Marrone, J C Martin, H L Nutter, R L Ratliff, E B Shera, D J Simpson, S A 
Soper, Genetic Analysis, Techniques, and Applications 1991; 8:1-7). 
Furthermore, this method is not amenable to sequencing numerous DNA 
segments in parallel. 
Another DNA sequencing method has recently been developed that uses 
class-IIS restriction endonuclease digestion and adaptor ligation to 
sequence at least some nucleotides offset from a terminal nucleotide. 
Using this method, four adjacent nucleotides have reportedly been 
sequenced and read following the gel resolution of DNA fragments. However, 
a limitation of this sequencing method is that it has built-in product 
losses, and requires many iterative cycles (International Application 
PCT/US95/03678). 
Another problem exists with currently available technologies in the area of 
diagnostic sequencing. An ever widening array of disorders, 
susceptibilities to disorders, prognoses of disease conditions, and the 
like, have been correlated with the presence of particular DNA sequences, 
or the degree of variation (or mutation) in DNA sequences, at one or more 
genetic loci. Examples of such phenomena include human leukocyte antigen 
(HLA) typing, cystic fibrosis, tumor progression and heterogeneity, p53 
proto-oncogene mutations, and ras proto-oncogene mutations (Gullensten et 
al., PCR Methods and Applications, 1:91-98 (1991); International 
application PCT/US92/01675; and International application PCT/CA90/00267). 
A difficulty in determining DNA sequences associated with such conditions 
to obtain diagnostic or prognostic information is the frequent presence of 
multiple subpopulations of DNA, e.g., allelic variants, multiple mutant 
forms, and the like. Distinguishing the presence and identity of multiple 
sequences with current sequencing technology is impractical due to the 
amount of DNA sequencing required. 
SUMMARY OF THE INVENTION 
The present invention provides an alternative approach for sequencing DNA 
that does not require high resolution separations and that generates 
signals more amenable to analysis. The methods of the present invention 
can also be easily automated. This provides a means for readily analyzing 
DNA from many genetic loci. Furthermore, the DNA sequencing method of the 
present invention does not require the gel resolution of DNA fragments 
which allows for the simultaneous sequencing of cDNA or genomic DNA 
library inserts. Therefore, the full length transcribed sequences or 
genomes can be obtained very rapidly with the methods of the present 
invention. The method of the present invention further provides a means 
for the rapid sequencing of previously uncharacterized viral, bacterial or 
protozoan human pathogens, as well as the sequencing of plants and animals 
of interest to agriculture, conservation, and/or science. 
The present invention pertains to methods which can sequence multiple DNA 
segments in parallel, without running a gel. Each DNA sequence is 
determined without ambiguity, as this novel method sequences DNA in 
discrete intervals that start at one end of each DNA segment. The method 
of the present invention is carried out on DNA that is almost entirely 
double-stranded, thus preventing the formation of secondary structures 
that complicate the known sequencing methods that rely on hybridization to 
single-stranded templates (e.g., sequencing by hybridization), and 
overcoming obstacles posed by microsatellite repeats, other direct 
repeats, and inverted repeats, in a given DNA segment. The iterative and 
regenerative DNA sequencing method described herein also overcomes the 
obstacles to sequencing several thousand distinct DNA segments attached to 
addressable sites on a matrix or a chip, because it is carried out in 
iterative steps and in various embodiments effectively preserves the 
sample through a multitude of sequencing steps, or creates a nested set of 
DNA segments to which a few steps are applied in common. It is, therefore, 
highly suitable for automation. Furthermore, the present invention 
particularly addresses the problem of increasing throughput in DNA 
sequencing, both in number of steps and parallelism of analyses, and it 
will facilitate the identification of disease-associated gene 
polymorphisms, with particular value for sequencing entire genomes and for 
characterizing the multiple gene mutations underlying polygenic traits. 
Thus, the invention pertains to novel methods for generating staggered 
templates and for iterative and regenerative DNA sequencing as well as to 
methods for automated DNA sequencing. 
Accordingly, the invention features a method for identifying a first 
nucleotide n and a second nucleotide n+x in a double stranded nucleic acid 
segment. The method includes (a) digesting the double stranded nucleic 
acid segment with a restriction enzyme to produce a double stranded 
molecule having a single stranded overhang sequence corresponding to an 
enzyme cut site; (b) providing an adaptor having a cycle identification 
tag, a restriction enzyme recognition domain, a sequence identification 
region, and a detectable label; (c) hybridizing the adaptor to the double 
stranded nucleic acid having the single-stranded overhang sequence to form 
a ligated molecule; (d) identifying the nucleotide n by identifying the 
ligated molecule; (e) amplifying the ligated molecule from step (d) with a 
primer specific for the cycle identification tag of the adaptor; and (f) 
repeating steps (a) through (d) on the amplified molecule from step (e) to 
yield the identity of the nucleotide n+x, wherein x is less than or equal 
to the number of nucleotides between a recognition domain for a 
restriction enzyme and an enzyme cut site. 
In another aspect, the invention features a method for sequencing an 
interval within a double stranded nucleic acid segment by identifying a 
first nucleotide n and a second nucleotide n+x in a plurality of staggered 
double stranded molecules produced from the double stranded nucleic acid 
segment. The method includes (a) attaching an enzyme recognition domain to 
different positions along the double stranded nucleic acid segment within 
an interval no greater than the distance between a recognition domain for 
a restriction enzyme and an enzyme cut site, such attachment occurring at 
one end of the double stranded nucleic acid segment; (b) digesting the 
double stranded nucleic acid segment with a restriction enzyme to produce 
a plurality of staggered double stranded molecules each having a single 
stranded overhang sequence corresponding to the cut site; (c) providing an 
adaptor having a restriction enzyme recognition domain, a sequence 
identification region, and a detectable label; (d) hybridizing the adaptor 
to the double standard nucleic acid having the single-stranded overhang 
sequence to form a ligated molecule; (e) identifying a nucleotide n within 
a staggered double stranded molecule by identifying the ligated molecule; 
(f) repeating steps (b) through (e) to yield the identity of the 
nucleotide n+x in each of the staggered double stranded molecules having 
the single strand overhang sequence thereby sequencing an interval within 
the double stranded nucleic acid segment, wherein x is greater than one 
and no greater than the number of nucleotides between a recognition domain 
for a restriction enzyme and an enzyme cut site. 
In another aspect, the invention features a method for identifying a first 
nucleotide n and a second nucleotide n+x in a double stranded nucleic acid 
segment. The method includes (a) digesting the double stranded nucleic 
acid segment with a restriction enzyme to produce a double stranded 
molecule having a 5' single stranded overhang sequence corresponding to an 
enzyme cut site; (b) identifying the nucleotide n by template-directed 
polymerization with a labeled nucleotide or nucleotide terminator; (c) 
providing an adaptor having a cycle identification tag and a restriction 
enzyme recognition domain; (d) ligating the adaptor to the double stranded 
nucleic acid to form a ligated molecule; (e) amplifying the ligated 
molecule from step (d) with a primer specific for the cycle identification 
tag of the adaptor; and (f) repeating steps (a) through (b) on the 
amplified molecule from step (e) to yield the identity of the nucleotide 
n+x, wherein x is less than or equal to the number of nucleotides between 
a recognition domain for a restriction enzyme and an enzyme cut site. 
Yet another aspect of the invention pertains to a method for sequencing an 
interval within a double stranded nucleic acid segment by identifying a 
first nucleotide n and a second nucleotide n+x in a plurality of staggered 
double stranded molecules produced from the double stranded nucleic acid 
segment. The method includes (a) attaching an enzyme recognition domain to 
different positions along the double stranded nucleic acid segment within 
an interval no greater than the distance between a recognition domain for 
a restriction enzyme and an enzyme cut site, such attachment occurring at 
one end of the double stranded nucleic acid segment; (b) digesting the 
double stranded nucleic acid segment with a restriction enzyme to produce 
a plurality of staggered double stranded molecules each having a 5' single 
stranded overhang sequence corresponding to the cut site; (c) identifying 
a nucleotide n within a staggered double stranded molecule by 
template-directed polymerization with a labeled nucleotide or nucleotide 
terminator; (d) providing an adaptor having a restriction enzyme 
recognition domain; e) ligating the adaptor to the double stranded nucleic 
acid to form a ligated molecule; (f) repeating steps (b) through (c) to 
yield the identity of the nucleotide n+x in each of the staggered double 
stranded molecules having the single strand overhang sequence thereby 
sequencing an interval within the double stranded nucleic acid segment, 
wherein x is greater than one and no greater than the number of 
nucleotides between a recognition domain for a restriction enzyme and an 
enzyme cut site. 
The invention also pertains to a method for removing all or a part of a 
primer sequence from a primer extended product. The method includes (a) 
providing a primer sequence encoding a methylated portion of a restriction 
endonuclease recognition domain, wherein recognition of the domain by a 
restriction endonuclease requires at least one methylated nucleotide; (b) 
polymerizing by a template-directed primer extension using the primer and 
a nucleic acid segment to generate a primer extended product; and (c) 
digesting the primer extended product with a restriction endonuclease that 
recognizes the resulting double-stranded restriction endonuclease 
recognition domain encoded by the primer sequence in the primer extended 
product. 
A still further aspect of the invention pertains to a method for blocking a 
restriction endonuclease recognition domain in a primer extended product. 
The method includes (a) providing a primer with at least one modified 
nucleotide, wherein the modified nucleotide blocks an enzyme recognition 
domain, and at least a portion of the enzyme recognition domain sequence 
is encoded in the primer; (b) polymerizing by a template-directed primer 
extension using the primer and a nucleic acid segment to generate a primer 
extended product; and (c) digesting the primer extended product with an 
enzyme that recognizes a double-stranded enzyme recognition domain in the 
primer extended product. 
In another aspect of the invention there is provided a method and device 
for automated sequencing of double-stranded DNA segments with nested 
single strand overhang templates, wherein a plurality of double-stranded 
DNA segments are immobilized at sites of a microtiter support or chip 
array having a plurality of sample holders arrayed in a matrix of 
positions on the support. Each DNA segment has an end comprising a 
single-strand overhang template sequence no longer than about twenty 
nucleotides in length. The device then implements a protocol 
simultaneously treating all sample holders with one or more reagents which 
selectively react with at least one nucleotide of the single-strand 
overhang template to effectively label the material at each holder, then 
reading the array by automated detection to determine at least one 
nucleotide of the single-strand overhang template at each position. 
Thereafter, the method proceeds by reducing length of each strand of the 
DNA segment at each holder by a fixed number n&gt;1 at the overhang end, thus 
yielding a homologously ordered array of shorter and nested DNA segments, 
each with a single-strand overhang template sequence, which preferably 
remain immobilized at the same positions on the support where the 
treatment protocol is repeated to determine at least one nucleotide at 
each single-strand overhang sequence. The steps of treating, reading and 
reducing the length of the strands of the DNA segment at each holder by a 
number of n&gt;1 nucleotides are iteratively performed as automated process 
steps to produce nested and progressively shorter DNA segments and to 
sequence the plurality of DNA segments immobilized at the array of sample 
holders in situ. 
In another aspect the invention includes a method for automated sequencing 
of double stranded DNA segments by attaching a recognition domain to each 
segment to form a set of DNA segments having the recognition domain nested 
at an interval no greater than the distance between the recognition domain 
and its cut site for a given enzyme that recognizes the recognition 
domain; treating the DNA segments with an enzyme that recognizes the 
attached recognition domain and cuts each strand of each DNA segment to 
create an overhang template at a distance of &gt;1 nucleotide along the DNA 
segment from the recognition domain so as to generate a set of nested 
overhang templates; and determining at least one nucleotide of each of the 
nested overhang templates. Thereafter, the method proceeds by reducing 
length of each strand at the end of the DNA segment with the overhang 
template by &gt;1 nucleotide to produce a corresponding set of shorter DNA 
segments each with an overhang template. The step of reducing is performed 
by removing a block of nucleotides, so that each shorter DNA segment with 
an overhang template is a known subinterval of a previous DNA segment with 
overhang. 
In another aspect of the invention there is provided a method and device 
for automated sequencing of double-stranded DNA segments, wherein a 
plurality of double-stranded DNA segments are immobilized at sites of a 
microtiter support or chip array having a plurality of sample holders 
arrayed in a matrix of positions on the support. Each DNA segment has an 
end comprising a single-strand overhang template sequence no longer than 
about twenty nucleotides in length. The device then simultaneously treats 
all sample holders with one or more reagents which selectively react with 
at least one nucleotide of the single-strand overhang template to 
effectively label the material at each holder, and reading the array by 
automated detection to determine at least one nucleotide of the 
single-strand overhang template at each position. Thereafter, the method 
proceeds by regenerating material at the respective sample holders by DNA 
amplification in vitro and reducing length of each strand of the 
regenerated DNA segment at each holder by a fixed number n.gtoreq.1 at the 
overhang end, thus yielding a homologously ordered array of shorter and 
nested DNA segments, each with a single-strand overhang template sequence, 
which preferably remain immobilized at the same positions on the support, 
and the treatment protocol is repeated to determine at least one 
nucleotide at each single-strand overhang sequence. The steps of treating, 
reading, regenerating and reducing the length of the strands of the DNA 
segment at each holder by a number of n.gtoreq.1 nucleotides are 
iteratively performed as automated process steps to produce nested and 
progressively shorter DNA segment ends and to sequence the plurality of 
DNA segments immobilized at the array of sample holders in situ. 
In another aspect the invention includes a method for automated sequencing 
of double stranded DNA segments by attaching a recognition domain to each 
segment to form DNA segments having the recognition domain, regenerating 
the template precursor by DNA amplification in vitro, treating the DNA 
segments with an enzyme that recognizes the attached recognition domain 
and cuts each strand of each DNA segment to create an overhang template at 
a distance of .gtoreq.1 nucleotide along the DNA segment from the 
recognition domain, and determining at least one nucleotide of the 
overhang template. The method includes the step of reducing length of each 
strand at the end of the DNA segment with the overhang template by 
.gtoreq.1 nucleotide to produce a corresponding set of shortened DNA 
segments each with an overhang template, the step of reducing being 
performed by removing a block of nucleotides, so that each shortened DNA 
segment with an overhang template is a known subinterval of a previous DNA 
segment with overhang. 
The invention further contemplates an automated instrument for effectively 
performing the sequencing, wherein a stage carries the support on a device 
equipped for providing the respective buffers, solutions and reagents, for 
stepping or positioning the array for reading, and in some embodiments 
robotic manipulation for sample transfer, and heating for amplification, 
e.g., treating at least a portion of material at each sample holder with a 
primer and heat cycling to regenerate material at the respective sample 
holders. The stage may be rotatable, spinning to cause fluid provided at a 
central position to centrifugally flow across the array to alter material 
immobilized in the sample holders. Preferably the stage holds plural 
support arrays, and may operate robotically to transfer material from the 
sites of one support array to the sites of another support array, so that 
all the samples on one support may undergo one set of process steps in 
common (e.g., washing, digestion, labeling) while those on the other 
support undergo another (e.g., heating/amplification or scintillation 
reading). 
Generally, the methods of the invention are applicable to all tasks where 
DNA sequencing is employed, including medical diagnostics, genetic 
mapping, genetic identification, forensic analysis, molecular biology 
research, and the like.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention pertains to an iterative and regenerative method for 
sequencing DNA that exploits the separation of the restriction enzyme 
recognition and cleavage domains in class-IIS restriction endonucleases, 
as well as adaptor ligation, to generate a series of sequencing templates 
that are separated from each other by a discrete interval. These 
sequencing templates constitute a set of single-strand overhangs that can 
then be sequenced by template-directed ligation, template-directed 
polymerization, or by stringent hybridization of oligonucleotides or 
oligonucleotide analogs. 
The present invention features a method for identifying a first nucleotide 
n and a second nucleotide n+x in a double stranded nucleic acid segment. 
The method includes (a) digesting the double stranded nucleic acid segment 
with a restriction enzyme to produce a double stranded molecule having a 
single stranded overhang sequence corresponding to an enzyme cut site and 
(b) providing an adaptor having a cycle identification tag, a restriction 
enzyme recognition domain, a sequence identification region, and a 
detectable label. The method further includes (c) hybridizing the adaptor 
to the double stranded nucleic acid having the single-stranded overhang 
sequence to form a ligated molecule, (d) identifying the nucleotide n by 
identifying the ligated molecule, and (e) amplifying the ligated molecule 
from step (d) with a primer specific for the cycle identification tag of 
the adaptor. The method also includes (f) repeating steps (a) through (d) 
on the amplified molecule from step (e) to yield the identity of the 
nucleotide n+x, wherein x is less than or equal to the number of 
nucleotides between a recognition domain for a restriction enzyme and an 
enzyme cut site. As is described more fully below the order of steps (a) 
through (f) may vary with different embodiments of the invention. 
As used herein, the term "nucleotide n" refers to a nucleotide along a 
given nucleic acid segment. "Nucleotide" is an art-recognized term and 
includes molecules which are the basic structural units of nucleic acids, 
e.g., RNA or DNA, and which are composed of a purine or pyrimidine base, a 
ribose or a deoxyribose sugar, and a phosphate group. A "modified 
nucleotide," as used herein, refers to a nucleotide that has been 
chemically modified, e.g., a methylated nucleotide. "Analogs " in 
reference to nucleotides includes synthetic nucleotides having modified 
base moieties and/or modified sugar moieties, e.g., as described generally 
by Scheit, Nucleotide Analogs (John Wiley, New York, 1980). Such analogs 
include synthetic nucleotides designed to enhance binding properties, 
reduce degeneracy, increase specificity, and the like. In the methods 
described herein, n designates a fixed position within a single stranded 
overhang sequence extending from each double stranded nucleic acid 
segment. Preferably, nucleotide n is selected by digesting a given double 
stranded nucleic acid segment with a restriction enzyme, e.g., a class IIS 
restriction endonuclease, to generate a 5' or a 3' single stranded 
overhang sequence corresponding to the cut site, and n is the first or the 
last unpaired nucleotide in the overhang sequence. 
As used herein, the term "nucleotide n+x" refers to a second nucleotide in 
a given nucleic acid segment which is separated from nucleotide n by x 
nucleotides along a nucleic acid segment. For methods described herein, "x 
" is a number which is less than or equal to the number of nucleotides 
between a restriction enzyme recognition domain and the corresponding 
enzyme cut site for a given enzyme. By convention, "x " is defined by two 
integers which give the number of nucleotides between the recognition site 
and the hydrolyzed phosphodiester bonds of each strand of a nucleic acid 
segment. Preferably, x is no longer than about 9 nucleotides, more 
preferably x is no longer than about 18, 20 or 30 nucleotides, and 
advantageously it is in the range between about 40 and 60 nucleotides in 
length. For example, the recognition and cleavage properties of FokI are 
typically represented as "GGATG(9/13)" because it recognizes and cuts a 
double stranded nucleic acid as follows: 
##STR1## 
where the bolded nucleotides are FokI's recognition site and the N's are 
arbitrary nucleotides and their complements. 
As used herein, the language "restriction enzyme recognition domain " 
refers to a nucleotide sequence that allows a restriction enzyme to 
recognize this site and cut one or both strands of a nucleic acid segment 
at a fixed location with respect to the recognition domain. For class IIS 
restriction endonucleases, the cut site lies x nucleotides outside the 
recognition domain. Generally, the nucleotide sequence of the recognition 
domain is about 4 to about 10, more preferably about 4 to about 6, 
nucleotides in length. For example, for a class IIS restriction 
endonuclease, e.g., BseRI , the recognition domain is 6 nucleotides in 
length. 
The language "enzyme cut site, " refers to the location of a strand 
cleavage by an enzyme where this cleavage occurs in a fixed location with 
respect to the restriction enzyme recognition domain. For class IIS 
restriction endonuclease, the enzyme cut site is located x nucleotides 
away from the recognition domain. In one embodiment, the enzyme cut site 
is the site located the farthest from the restriction enzyme recognition 
domain. Preferably, the enzyme cut site is the site located closest to the 
restriction enzyme recognition domain. 
"Enzyme " as the term is used in accordance with the invention means an 
enzyme, combination of enzymes, or other chemical reagents, or 
combinations chemical reagents and enzymes that when applied to a ligated 
molecule, discussed more fully below, cleaves the ligated molecule to 
generate a double stranded molecule having a single stranded overhang 
sequence corresponding to a cut site. An enzyme of the invention need not 
be a single protein, or consist solely of a combination of proteins. A key 
feature of the enzyme, or of the combination of reagents employed as an 
enzyme, is that its (their) cleavage site be separate from its (their) 
recognition site. It is important that the enzyme cleave the nucleic acid 
segment after it forms a ligated molecule with its recognition site; and 
preferably, the enzyme leaves a 5' or 3' protruding strand on the nucleic 
acid segment after cleavage. 
Preferably, enzymes employed in the invention are natural protein 
endonucleases whose recognition site is separate from its cleavage site 
and whose cleavage results in a protruding strand on the nucleic acid 
segment. Most preferably, class IIS restriction endonucleases are employed 
as enzymes in the invention, e.g., as described in Szybalski et al., Gene, 
100:13-26 (1991); Roberts et al., Nucleic Acids Research, 21:3125-3137 
(1993); and Lovak and Brenner, U.S. Pat. No. 5,093,245. Class-IIS 
restriction endonucleases are a subclass of class-II restriction 
endonucleases that cut at precise distances away from their recognition 
domains, so that the recognition domains and cleavage domains are 
separated on the substrate DNA molecule (Szybalski W, S C Kim, N Hasan, A 
J Podhajska Gene 1991; 100:13-26). Following digestion with class-IIS 
restriction endonucleases, the sequence of the single-stranded end is 
independent of the recognition domain sequence. Class-IIS restriction 
endonucleases usually have asymmetric recognition domains, and class-IIS 
restriction endonucleases typically cut on one side of the recognition 
domain, resulting in double-stranded cut per recognition site. Over 70 
class-IIS restriction endonucleases have been isolated. Because the 
cleavage domain is separate from the recognition domain, methylation of 
nucleotides that lie within the cleavage domain will not effect cleavage, 
so long as the corresponding recognition domain is not methylated 
(Podhajska A J, W Szybalski Gene 1985; 40: 175-182, Podhajska A J, S C 
Kim, and W Szybalski Methods in Enzymology 1992; 216:303-309, Posfai G, W 
Szybalski Gene 1988; 69:147-151). Exemplary class IIS restriction 
endonucleases for use with the invention include AccBSI, AceIII, AciI, 
AclWI, AlwI, Alw26I, AlwXI, Asp26HI, Asp27HI, Asp35HI, Asp36HI, Asp40HI, 
Asp50HI, AsuHPI, BaeI, BbsI, BbvI, BbvII, Bbv16II, Bce83I, BcefI, BcgI, 
Bco5I, Bco116I BcoKl, BinI, Bli736I, BpiI, BpmI, Bpu10I, BpuAI, Bsal, 
BsaMI, Bsc9II, BscAI, BscCI, BseII, Bse3DI, BseNI, BseRI, BseZI, BsgI, 
BsiI, BsmI, BsmAI, BsmBI, BsmFI, Bsp24I, Bsp423I, BspBS3II, BspIS4I, 
BspKT5I, BspLU11III, BspMI, BspPI, BspST5I, BspTS514I, BsrI, BsrBI, BsrDI, 
BsrSI, BssSI, Bst11I, Bst71I, Bst2BI, BstBS32I, BstD102I, BstF5I, BstTS5I, 
Bsu6I, CjeI, CjePI, Eam1104I, EarI, Eco31I, Eco57I, EcoA4I, EcoO44I, 
Esp3I, FauI, FokI, GdiII, GsuI, HgaI, HphI, Ksp632I, MboII, MlyI, MmeI, 
Mn1I, Mva1269I, PhaI, PieI, RleAI, SapI, SfaNI, SimI, StsI, TaqII, TspII, 
TspRI, Tth111II, and VpaK32I, and isoschizomers thereof. Preferred 
endonucleases include FokI and BseRI. 
Class-IIS restriction endonucleases have several applications, as outlined 
below. Class-IIS restriction endonucleases have been used in conjunction 
with an adaptor to act as a universal restriction endonuclease that can 
cut a single-stranded substrate at almost any predetermined site 
(Podhajska A J, W Szybalski Gene 1985; 40:175-182, Podhajska A J, S C Kim, 
and W Szybalski Methods in Enzymology 1992; 216:303-309, Szybalski W. Gene 
1985; 40:169-173). The adaptor consists of a double-stranded hairpin 
portion containing the recognition domain for the class IIS restriction 
endonuclease, and a single stranded end that is complementary to the 
single-stranded template to be cleaved. Following annealing of the adaptor 
to the single-stranded template (e.g. M13), the class-IIS restriction 
endonuclease can cleave this site. A hairpin adaptor has also been used to 
attach a radiolabel to one end of a single-stranded phagemid DNA, to 
facilitate Maxam-Gilbert sequencing (Goszcynski B, McGhee J D Gene 1991; 
104:71-74). 
Class-IIS restriction endonucleases have been used to trim vector inserts 
in order to generate deletions in a vector insert (Morrneneo S, R Knott, D 
Perlman Gene 1987; 61:21-30, Hasan N, J Kur, W Szybalski Gene 1989; 
82:305-311, Hasan N. S C Kim, A J Podhajska, W Szybalski Gene 1986; 
50:55-62). In this application, restriction endonuclease digestion removes 
a portion of the insert, and the resulting single-stranded ends are 
converted to blunt ends prior to intra-molecular ligation and the 
transformation of E. coli, generating a deletion mutant in the construct. 
If the class-IIS restriction endonuclease recognition domain is 
reconstituted, this process can be carried out again, generating a series 
of deletion mutants in the plasmid insert. This is not a sequencing 
method, and the single-strand overhangs that could act as sequencing 
templates are eliminated during the generation of each new plasmid 
construct. 
Class-IIS restriction endonuclease digestion has been used as a mapping 
tool in a fluorescent fingerprinting procedure (Brenner S, Livak K J Proc 
Natl AcadSci USA 1989; 86:8902-8906). In this method, 5' overhangs are 
generated by cleavage with a class IIS restriction endonuclease, using the 
recognition domains that already exist in the original DNA. Digestion is 
followed by labeling these ends using convention dNTPs and ddNTPs tagged 
with distinct fluorescent labels. This labeling constitutes conventional 
Sanger sequencing with fluorescently labeled terminators. The restriction 
fragments are then analyzed by denaturing polyacrylamide gel 
electrophoresis, with detection of emissions using a DNA sequencer. The 
labeled fragments are characterized by both size and terminal sequence, 
increasing the information content in DNA fingerprinting, allowing this 
method to distinguish restriction fragments that cannot be resolved by 
size alone. 
The ability of class-IIS restriction endonucleases to generate ambiguous 
ends has also been used to amplify single restriction fragments from large 
DNA molecules ranging from about 50-250 kb in size (Smith D R Methods and 
Applications 1992; 2:21-27). In this method, digestion of the DNA molecule 
with a class-IIS restriction endonuclease that generates a 5' overhang is 
followed by ligation to a single adaptor, under conditions such that only 
a small subset of digested fragments have single-stranded ends that will 
successfully mediate template-directed ligation to this single adaptor. 
The ligated adaptor provides one target for subsequent PCR amplification 
of an unknown fragment. The second target is provided by a vectorette unit 
(bubble-tag) ligated to blunt ends produced by another restriction 
endonuclease. This strategy permits the amplification of a single unknown 
fragment from the relatively complex mixture. It is designed so that 
specific fragments can be isolated without prior knowledge of the 
nucleotide sequence of the target. These amplified fragments arise from 
random locations within the target. A similar strategy has been developed 
in which adaptors ligated to the class-IIS restriction endonuclease cut 
sites are called DNA indexers (Kato K. Nucleic Acids Research 1996; 
24:394-395, Unrau P, Deugau K V Gene 1994; 145:163-169). 
Restriction endonuclease digestion is frequently used to generate cohesive 
ends for cloning DNA segments into a vector. This can be accomplished by 
attaching restriction endonuclease recognition domains to the ends of a 
DNA fragment by ligation of a linker or adaptor. Alternatively, a 
recognition domain can be incorporated into the end of a nucleic acid 
sequence using a primer whose 5' end contains the restriction endonuclease 
recognition site of interest, followed by primer directed synthesis of the 
opposite strand. One limitation inherent in such primer directed 
incorporation of a restriction endonuclease recognition domain is that the 
fragment of interest cannot contain the recognition domain for this enzyme 
if the intact fragment is to be cloned by digestion with this restriction 
endonuclease, as cutting of internal sites would generate shorter 
segments. This particular obstacle was solved by Han and Rutter (Han J, 
Rutter W J Nucleic Acids Res 1988; 16:11837). They incorporated a 
recognition domain for the restriction endonuclease SfiI into an end of 
DNA segments by primer directed DNA synthesis. A primer encoding this 
recognition domain was used during first strand cDNA synthesis, but during 
this polymerization step methylated-dCTP was substituted for dCTP. This 
was followed by primer mediated synthesis of the opposite strand using all 
four normal dNTPs. Since the SfiI recognition domain contains the cytosine 
nucleoside, the primer extension with 6-methyl dCTP methylates one strand 
of each recognition domain for SfiI lying outside of this primer sequence, 
blocking cleavage mediated by any recognition domain lying outside of this 
primer sequence. Hemi-methylation of the recognition domains lying outside 
of the primer sequence allowed this restriction endonuclease to be used to 
clone intact segments containing recognition domains for this restriction 
endonuclease. 
Padgett and Sorge have adapted primer directed hemi-methylation of 
recognition domains lying outside a primer encoded recognition domain, to 
a polymerase chain reaction (PCR) (Mullis K, Faloona F, Scharf S, Saiki R, 
Horn G, Erlich H. Cold Spring Harbor Symposia on Quantitative Biology, 
Cold Spring Harbor Laboratory, LI:263-273) format (Padgett K A, J A Sorge 
Gene 1996; 168:31-35). This strategy requires a recognition domain in 
which each strand has at least one nucleotide that is not contained in the 
other strand of this domain. A recognition domain with this characteristic 
allows one to use primer extension during the polymerase chain reaction 
(PCR) to hemi-methylate each of the recognition domains except for that 
recognition domain encoded by the amplifying primer. This is accomplished 
by using a methylated nucleotide that is not present in the recognition 
domain sequence that is antisense to the primer encoding this domain. By 
using a methylated dNTP that does not lie in the strand antisense to the 
recognition domain encoded in the amplifying primer, all the recognition 
domains in the PCR product are methylated except the recognition domain 
that is encoded by the amplifying primer. This strategy hemi-methylates 
each recognition domain in the PCR product except the primer-encoded 
recognition domain. This approach has been applied using a recognition 
domain for a class II-S restriction endonuclease, to generate recombinant 
constructs (Padgett K A, J A Sorge Gene 1996; 168:31-35). 
The above described strategies permit a class-IIS recognition domain to be 
appended to the end of a DNA segment through primer extension, while 
hemi-methylating each recognition domain that lies within the original 
target, and they can be used to block cutting mediated by internal 
recognition domains without blocking cutting mediated by the 
primer-encoded recognition domain. The two strategies outlined above 
constitute portions of the preferred embodiments of the invention. 
Preferably, prior to enzyme digestion, usually at the start of the 
sequencing operation, the nucleic acid segment is treated by blocking the 
enzyme recognition domains of the enzyme being employed. The blocking 
prevents undesired cleavage of the nucleic acid segment because of the 
fortuitous occurrence of enzyme recognition domains at interior locations 
in the nucleic acid segment. Blocking can be achieved in a variety of 
ways, including in vitro primer extension or in vitro primer extension 
with hemi-methylation, e.g., in vitro DNA amplification, or methylation of 
the enzyme recognition domain. For example, the DNA amplification can 
occur during or following the amplification of the ligated molecule. 
Hemi-methylation can be achieved in a variety of ways, including in vitro 
primer extension with a methylated nucleotide using a primer having the 
portion of an enzyme recognition domain that blocks enzyme recognition if 
it is hemi-methylated. Preferably, the restriction endonuclease employed 
recognizes a hemi-methylated enzyme recognition domain and a primer 
contains at least one methylated nucleotide in the methylated portion of 
the recognition domain. 
The language "nucleic acid segment " or "a double stranded nucleic acid 
segment " is used interchangeably herein and refers to a double stranded 
polynucleotide of any length. In one embodiment of the invention, the 
nucleic acid segment can contain a single stranded overhang, a nick or a 
gap. For example, the nucleic acid segment of the invention can be a 
genomic DNA, a cDNA, a product of an in vitro DNA amplification, e.g., a 
PCR product, a product of a strand displacement amplification, or a vector 
insert. The length of the nucleic acid segment can vary widely; however, 
for convenience of preparation, lengths employed in conventional 
sequencing are preferred. Preferably, the nucleic acid segment of the 
invention is about 60 basepairs in length, more preferably it is about 
100, 120, 150, 200, 300 or 600 basepairs in length, and most preferably it 
is about 1 to 2, or more kilobase pairs in length. Examples of other 
ranges of lengths include: from about 60 basepairs to about 1 or 2 
kilobase pairs; from about 60 basepairs to about 600 basepairs; from about 
60 basepairs to about 200 or 300 basepairs; and from about 60 basepairs to 
about 120 or 150 basepairs. 
The nucleic acid segments can be prepared by various conventional methods. 
For example, the nucleic acid segments can be prepared as inserts of any 
of the conventional cloning vectors, including those used in conventional 
DNA sequencing. Extensive guidance for selecting and using appropriate 
cloning vectors is found in Sambrook et al., Molecular Cloning: A 
Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory, New 
York, 1989), and the like references Sambrook et al and Innis et al., 
editors, PCR Protocols (Academic Press, New York, 1990) also provide 
guidance for using polymerase chain reactions to prepare nucleic acid 
segments. Preferably, cloned or PCR-amplified nucleic acid segments are 
prepared which permit attachment to magnetic beads, or other solid 
supports, for ease of separating the nucleic acid segment from other 
reagents used in the method. Protocols for such preparative techniques are 
described fully in Wahlberg et al., Electrophoresis, 13:547-551 (1992); 
Tong et al., Anal. Chem. 64:2672-2677 (1992); Hultman et al., Nucleic 
Acids Research, 17:4937-4946 (1989); Hultman et al., Biotechniques, 
10:84-93 (1991); Syvanen et al., Nucleic Acids Research, 16:11327-11338 
(1988); Dattagupta et al., U.S. Pat. No. 4,734,363; Uhlen, PCT application 
PCT/GB89/00304. Kits are also commercially available for practicing such 
methods, e.g. Dynabeads.TM. template preparation kit from Dynal AS (Oslo, 
Norway). 
In one preferred embodiment of the invention, the nucleic acid segment is 
attached to a solid matrix. As used herein, the term "solid matrix " 
refers to a material in a solid form to which a DNA molecule can attach. 
Examples of a solid matrix include a magnetic particle, e.g., a magnetic 
streptavidin or a magnetic glass particle, a polymeric microsphere, a 
filter material, or the like. Preferably, the solid matrix used in the 
methods of the invention permits the sequential application of reagents to 
a DNA molecule without complicated and time-consuming purification steps. 
The nucleic acid segments of the invention can also be used to generate a 
plurality of staggered double stranded nucleic acid molecules having a 
single stranded overhang sequence. This is desirable when the sequencing 
interval is designed to be more than one nucleotide, and one nucleotide is 
sequenced from a single template during each cycle. The language "double 
stranded nucleic acid molecules having a single stranded overhang sequence 
" is intended to include a nucleic acid molecule created by the following 
method: attachment of an enzyme recognition domain at different positions 
within an interval of a selected double stranded nucleic acid segment, and 
digestion of the selected double stranded nucleic acid segment with a 
corresponding restriction enzyme. Preferably, the interval is no greater 
than the distance between a restriction enzyme recognition domain and an 
enzyme cut site. The resulting double stranded nucleic acid molecules 
having a single stranded overhang sequence constitute a plurality of 
staggered double stranded nucleic acid molecules. The single strand 
overhang sequence in the staggered nucleic acid molecule may be either 5' 
or 3'. Preferably, the number of nucleotides in the overhang portion of 
the strand is in the range from about 2 to about 6 nucleotides depending 
on the enzyme used to digest the nucleic acid segment. 
The language "sequencing an interval within a double stranded nucleic acid 
segment " is intended to include the sequencing which occurs by 
identifying nucleotides n and n+x in a plurality of staggered double 
stranded molecules produced from the selected double stranded nucleic acid 
segment. This allows one to sequence all of the nucleotides in a selected 
nucleic acid segment between the nucleotide n and nucleotide n+x. For 
example, for a class IIS restriction enzyme, e.g., FokI, that has a 
restriction enzyme recognition domain nine nucleotides away from its 
enzyme cut site, e.g., x=9, starting with nine staggered double stranded 
nucleic acid molecules will generate sequence information for all 
nucleotides found in the interval between nucleotide n and nucleotide n+x. 
The staggered double stranded nucleic acid molecules having a single 
stranded overhang sequence can be prepared by various methods. For 
example, they can be generated by ligation of the initial nucleic acid 
segment to each of several adaptors with offset class-IIS recognition 
domains (Wu R, T Wu, R Anuradh, Enzymology 1987; 152:343-349). This 
initial DNA segment to be sequenced can be a PCR product or a vector 
insert. If the PCR product is amplified using a DNA polymerase with 
terminal extendase activity, the resulting single nucleotide 3' overhang 
can be removed using a DNA polymerase with 3' exonuclease, such as T.sub.4 
DNA polymerase or Pfu DNA polymerase, prior to blunt end ligation to 
adaptors (Costa G L, M P Weiner, Nucleic Acids Research 1994; 22:2423). 
Offset recognition domains can also be encoded into the amplification 
primers (Mullis K, Faloona F, Scharf S, Saiki R, Horn G, Erlich H., Cold 
Spring Harbor Symposia on Quantitative Biology, Cold Spring Harbor 
Laboratory, LI:263-273), resulting in distinct amplification products with 
offset recognition domains. 
There are a variety of ways in which offset recognition domains can be 
appended to each of numerous inserts in a DNA library. For example, if a 
complete digest were carried out on genomic DNA with the frequent cutter 
Sau3AI, followed by a partial fill-in with dGTP and dATP, each insert 
would contain non-self-complementary DNA ends (Hung M-C, P C Wensink. 
Nucleic Acids Research. 1984; 12:1863-1874). The vector could be digested 
with SalI and undergo a partial fill-in reaction with dCTP and dTTP, 
resulting in linearized vectors with non-self-complementary DNA ends. In 
this case each insert DNA end is complementary to each vector DNA end, so 
that during DNA ligation with cut and partially filled-in inserts and 
vectors, the vast majority of the resulting clones will contain one insert 
(Zabarovsky E R, R L Allikmets. Gene. 1986; 42:119-123). Following the 
isolation of individual clones, each insert can undergo PCR amplification 
using primers that anneal to the vector sequence, with one of the primers 
disabling the Sau3AI site in one side of each amplified insert by having a 
base mismatch to the Sau3AI site near its 3' end, or, preferably, a 
methylated nucleotide in the 3' end region of the primer (this primer's 3' 
end encoding at least part of the Sau3AI recognition domain (GATC), so 
that it will prime efficiently and its methylated nucleotide will block 
Sau3AI cutting of this end of the PCR product, allowing cutting of the 
opposite end of the PCR product). If the adenine is methylated, cutting 
can be done using MboI or DpnII, which share the recognition domain of 
Sau3AI but are blocked by dam methylation. Following digestion, one end of 
each insert will have a four nucleotide long end that can undergo ligation 
to an initial adaptor, so that ligations to distinct initial adaptors can 
append staggered recognition domains (for the class-IIS restriction 
endonuclease that will be used for sequencing) to each of the numerous 
inserts in the library. 
An alternative approach is to generate a library of clones using randomly 
sheared DNA. These DNA fragments can be dephosphorylated and efficiently 
cloned with one insert per vector using a vector that requires 
inactivation of a selectable marker by DNA insertion to be viable in a 
given E. coli host (Bernard P. BioTechniques. 1996; 21:320-323). 
Alternatively, a pool of inserts can be size selected over an agarose gel 
prior to cloning into a vector (Fleischmann R D, et al. Science. 1995; 
269:496-512). Using either approach, or other cloning strategies, each 
vector insert could be amplified using one primer that contains a 
methylated strand of the recognition domain for a restriction endonuclease 
that recognizes a hemi-methylated domain but does not recognize a 
non-methylated domain. This can be accomplished by using a primer that has 
one strand of the recognition domain sequence, with at least one 
methylated nucleotide, so that digestion with the corresponding 
restriction endonuclease will cut that one end of each amplified product, 
and no other sites. This can be carried out by amplification with a primer 
that contains one strand of the recognition domain for DpnI (with a 
methylated adenine). This strategy allows PCR amplification with normal 
nucleotides, as PCR with normal nucleotides effectively blocks internal 
DpnI recognition domains. Alternatively, each end could be amplified and 
digested using the strategy of Padgett and Sorge (Padgett K A, J A Sorge 
Gene 1996; 168:31-35), with either a regular class-II restriction 
endonuclease or with a class-IIS restriction endonuclease. 
In this method, the opposite end of each nucleic acid segment is shared 
between each of the initial template precursors for a given nucleic acid 
segment to be sequenced. Each initial template precursor is attached to a 
solid matrix. A wide range of methods have been used to bind DNA to a 
solid matrix. If the template precursor is a PCR product, one primer can 
contain a moiety that is used to attach the PCR product to a solid matrix. 
For example, this primer can contain a biotin moiety or another reactive 
moiety such as an amine group or thiol group, permitting the attachment of 
the PCR product to a solid matrix (Syvanen A C, M Bengstrom, J Tenhunen 
and H Soderlund, Nucleic Acids Research 1988; 16:11327-11338; Stamm S, J 
Brosius, Nucleic Acids Research 1991; 19:1350; Lund V, R Schmid, D 
Rickwood and E Hornes, Nucleic Acids Research 1988; 16:10861-10880; Fahy 
E, G R Davis, L J DiMichele, Ss Ghosh, Nucleic Acids Research 1993; 
21:1819-1826; and Kohsaka H, A Taniguchi, D D Richmnan, D A Carson, 
Nucleic Acids Research 1993; 21:3469-3472). The solid matrix can be either 
immobile or dispersible. For example, for a DNA segment with a 
biotinylated end, an immobile solid matrix can be an avidin or 
streptavidin coated microtiter plate (Jeltsch A, A Fritz, J Alves, H 
Wolfes, A Pingoud, Analytical Biochemistry 1993; 213:234-240; Holmstrom K, 
L Rossen, O F Rasmussen, Analytical Biochemistry 1993; 209:278-283) or 
manifold support (Lagerkvist A, J Stewart, M Lagerstrom-Fermer, U 
Landegren, Proc Natl Acad Sci USA 1994; 91:2245-2249). The most readily 
available dispersible solid matrix is beads that can be suspended through 
shaking. Beads can be designed to be magnetically pelleted (Lund V, 
RSchmid, D Rickwood and E Hornes Nucleic Acids Research 1988; 
16:10861-10880, Hultman T, S Stahl, E Hornes, M Uhlen Nucleic Acids 
Research 1989; 17:4937-4946, Dawson B A, T Herman, J Lough Journal of 
Biological Chemistry 1989; 264:12830-12837)or they can be pelleted through 
centrifugation (Syvanen A C, M Bengstrom, J Tenhunen and H Sodelund, 
Nucleic Acids Research 1988; 16:11327-11338; Stamm St, J Brosius, Nucleic 
Acids Research 1991; 19:1350). Use of a dispersible solid matrix 
diminishes steric obstacles in enzymatic reactions, and facilitates 
removal of a small aliquot to be amplified. An alternative approach that 
allows a small aliquot of a reaction to be removed and used as a template 
for amplification is to use a method of reversible capture. Reversible 
capture can be accomplished by using a cleavable linkage arm (such as a 
chemically cleavable linkage arm or a photocleavable linkage arm (Dawson B 
A, T Herman, J Lough Journal of Biological Chemistry 1989; 
264:12830-12837, Olejnik J, E Krzymanska-Olejnik, K J Rothschild, Nucleic 
Acids Research 1996; 24:361-366), by using a primer-encoded DNA binding 
domain that can be unbound by denaturation (Lew A M, D J Kemp, Nucleic 
Acids Research 1989; 17:5859; Kemp D J, D B Smith, S J Foote, N Samaras, M 
G Peterson, Proc Natl Acad Sci USA 1989; 86:2423-2427; Kemp D J, Methods 
in Enzymology 1992; 216:116-126), or by the generation of a single 
stranded end during PCR, as such an end can reversibly anneal to its 
complement that is bound to a solid phase (Newton C R, D Holland, L E 
Heptinstall, I Hodgson, M D Edge, A F Markham, M J McLean, Nucleic Acids 
Research 1993; 21:1155-1162; Khudyakov Y E, L Gaur, J Singh, P Patel, H A 
Fields, Nucleic Acids Research 1994; 22:1320-1321). 
Another important aspect of the invention is the adaptor employed within 
the present invention. An adaptor of the invention is a double stranded or 
a single stranded polynucleotide having one or more of a cycle 
identification tag, a restriction enzyme recognition domain and a sequence 
identification region. Preferably, the adaptor may also include a 
detectable label, which in the particular embodiment of FIG. 1 is 
illustrated at the end opposite of the sequence identification region. 
As used herein, the language "a cycle identification tag " refers to a 
unique nucleotide sequence that generates a primer annealing site, and a 
primer can anneal either to the unique sequence or its complement. The 
cycle identification tag is of a length which allows it to perform its 
intended function. Examples of lengths include: from about 8 to about 60 
nucleotides in length; from about 8 to about 30 or 40 nucleotides in 
length; and from about 8 to about 15 or 20 nucleotides in length. Ligation 
of this unique sequence to each double stranded nucleic acid segment 
having the single stranded overhang sequence permits regeneration of each 
nucleic acid segment using primer-directed DNA amplification in vitro 
(e.g., PCR), ameliorating the major limitations inherent in iterative 
methods for product generation, e.g., product losses and the accumulation 
of incompletely processed products. 
The language "restriction enzyme recognition domain " has been defined 
above. In one embodiment of the invention, the adaptor contains only a 
single strand of a restriction enzyme recognition domain, because a single 
strand of the domain can function as a template for the generation of a 
double stranded restriction enzyme recognition domain through 
hybridization to its complement or through template-directed polymerase 
generation of its complement. 
As used herein, the language "sequence identification region " refers to a 
region used to identify nucleotide n and/or nucleotide n+x in a selected 
nucleic acid segment. Preferably, the region used to identify nucleotide n 
and/or nucleotide n+x is a protruding nucleotide strand, e.g., a 5' or a 
3' nucleotide strand. In one embodiment of the invention, the sequence 
identification region is capable of forming a duplex with the single 
stranded overhang sequence of the double stranded nucleic acid segment. 
Preferably, the sequence identification region comprises a number of 
degenerate nucleotides, usually between 1 and 4 degenerate nucleotides. In 
addition, the sequence identification region can also include a fixed 
nucleotide, e.g., a nucleotide whose sequence is known, at its most 
terminal nucleotide. Preferably, at each cycle, only those adaptors whose 
sequence identification regions form duplexes with the single stranded 
overhang sequence of the double stranded nucleic acid segment, are 
hybridized to the one end of the nucleic acid segment to form a ligated 
molecule. 
As used herein, the term "a ligated molecule " refers to a double stranded 
structure formed after the sequence identification region of an adaptor 
and the single strand overhang sequence of the nucleic acid segment anneal 
and at least one pair of the identically oriented strands of the adaptor 
and the nucleic acid segment are ligated, i.e., are caused to be 
covalently ligated to one another. In one embodiment of the invention, the 
ligated molecule is labeled with a detectable label on at least one strand 
of the molecule and detection occurs following the removal of an unligated 
labeled adaptor. 
As used herein, the term "hybridization " refers to annealing of a nucleic 
acid sequence to its complement. Hybridization can occur in the presence 
of a non-annealing region or a nucleotide analog. In one embodiment of the 
invention, hybridization can also entail ligation. In another embodiment 
of the invention, hybridization precedes ligation. The term "ligation, " 
as used herein, refers to a ligation of two molecules using conventional 
procedures known in the art. Ligation can be accomplished either 
enzymatically or chemically. Chemical ligation methods are well known in 
the art, e.g., Ferris et al., Nucleotides & Nucleotides, 8:407-414 (1989); 
Shabarova et al., Nucleic Acid Res. 19:4247-4251 (1991). Preferably, 
however, ligation is carried out enzymatically using a ligase in a 
standard protocol. Many ligases are known and are suitable for the use in 
the present invention, e.g., Lehman, Science 186:790-797 (1974); Boyer, 
ed., The Enzymes Vol. 15B (Academic Press, New York, 1982). Preferred 
ligases include nucleic acid ligases, e.g., T4 DNA ligase, T7 DNA ligase, 
E. coli DNA ligase, Taq ligase, Pfu ligase and Tth ligase. Protocols for 
their use are well known, e.g., Sambrook et al. Molecular Cloning: A 
Laboratory Manual, 2nd Edition (cold Spring Harbor Laboratory, New York, 
1989); Barany, PCR Methods and Applications 1:5-16 (1991). Generally, 
ligases are require that a 5' phosphate group be present for ligation to 
the 3' hydroxyl of an abutting strand. This is conveniently provided for 
at least one strand of the nucleic acid segment by selecting a restriction 
endonuclease which leaves a 5' phosphate, e.g., a FokI restriction 
endonuclease. For example, T.sub.4 DNA ligase is highly specific in its 
ability to ligate the 3' end of one oligonucleotide to the phosphorylated 
5' end of another oligonucleotide using a DNA template, because a mismatch 
between the oligonucleotide substrates at the ligation junction greatly 
reduces the ligation efficiency (Alves A M, F J Carr, Nucleic Acids Res 
1988; 16:8723, Wu D Y, R B Wallace Gene 1989; 76:245-254, Somers VAMC, 
PTM, Moekerk, J J Murtagh, Jr., and FBJM Thunnissen, Nucleic Acids 
Research 1994; 22:4840-4841, and Samiotaki M, M Kwiatkowski, J Parik and U 
Landegren, Genomics 1994; 20:238-242). This permits highly selective 
ligation of an oligonucleotide whose end nucleotide is complementary to 
the template at the ligation junction, allowing template-directed DNA 
ligation to discriminate between single nucleotides in a designated 
position of the DNA template. This forms the basis for point mutation 
discrimination by the ligase chain reaction using either T.sub.4 DNA 
ligase (Wu D Y, R B Wallace, Genomics 1989; 4:560-569) or a heat-stable 
DNA ligase (Barany F. Proc Natl Acad Sci USA 1991; 88:189-193). E. coli 
DNA ligase can also discriminate between mismatches at a ligation junction 
(Kato K, Nucleic Acids Research 1996; 24:394-395), and other DNA ligases 
can be anticipated to share this characteristic. The ligase chain 
reaction, and related earlier methods for nucleotide discrimination using 
a DNA ligase, detect point mutations at a single position. Each position 
assessed requires a unique set of annealing oligonucleotides, so that a 
method based solely on DNA ligation steps can only provide very limited 
sequence information. 
In another embodiment of the invention, template-directed polymerization is 
used instead of template-directed ligation described above. For example, 
double stranded molecule having a single stranded overhang sequence 
generated following FokI digestion can be sequenced by template-directed 
polymerization in the presence of four deoxynucleotide terminators (e.g. 
ddNTPs), each tagged with a distinct fluorescent label. Following 
polymerization and washing, which removes unincorporated terminators, 
identification of the incorporated terminator can be accomplished by 
fluorometry, revealing the sequence of nucleotide n in the nucleic acid 
segment. 
After adaptor ligation, an enzyme recognizing the adaptor via the enzyme 
recognition domain digests the ligated molecule at the site one or more 
nucleotides from a ligation site along the nucleic acid segment leaving a 
double stranded molecule having a single strand overhang sequence 
corresponding to the cut cite capable of participating in the next cycle 
of legation and digestion. 
As used herein, the term "amplify " refers to an in vitro method which can 
be used to generate multiple copies of a nucleic acid, e.g., a DNA duplex 
or single-stranded DNA molecule, its complement, or both. Amplification 
techniques, therefore, include both cloning techniques, as well as PCR 
based amplification techniques. Preferably, the nucleic acid amplification 
is linear or exponential, e.g., PCR amplification or strand displacement 
amplification. These techniques are well known to those of skill in the 
art. Amplification products are compositions which include a greater 
number of properly ligated molecules than the number of original nucleic 
acid segments. 
The term "primer" refers to a linear oligonucleotide which specifically 
anneals to a unique polynucleotide sequence and allows for amplification 
of that unique polynucleotide sequence. In one embodiment of the 
invention, the primer specifically anneals to the unique sequence in a 
cycle identification tag and allows for amplification of a ligated 
molecule. The primer is of a length which allows it to perform its 
intended function. Examples of lengths include: from about 8 to about 60 
nucleotides in length; from about 8 to about 30 or 40 nucleotides in 
length; and from about 8 to about 15 or 20 nucleotides in length. In one 
embodiment of the invention, a primer is said to encode a restriction 
endonuclease recognition domain if it contains a portion of that 
recognition domain, when the primer undergoes primer extension to generate 
a complete strand of that recognition domain. 
A strategy can be implemented to remove one of the amplifying primers, and 
its complement, from each product of amplification, e.g., PCR 
amplification, thus, preventing the sequencing of DNA encoded by this 
primer. 
Selective removal of primer encoded sequence from a PCR product can be 
accomplished by restriction endonuclease digestion, without cutting 
internal recognition domains, using the method of Padgett and Sorge 
(Padgett K A, J A Sorge, Gene 1996; 168:31-35), as described herein. 
Alternatively, a primer can encode the recognition domain for a 
restriction endonuclease that requires a methylated nucleotide for 
cleavage, and recognizes a hemi-methylated recognition domain (see Example 
4). Using this strategy, only the primer directed end is cut by the 
restriction endonuclease because only the primer encoded recognition 
domain is methylated. Therefore, this strategy does not require 
substitution of a free methylated nucleotide for its normal counterpart in 
the PCR mixture, or the recognition domain to contain less that all four 
nucleotides in a given strand, distinguishing it from the method of 
Padgett and Sorge. 
Technology for removing primer encoded sequence from PCR products can also 
be used to facilitate the generation of initial nucleic acid segments from 
clone libraries. For example, the restriction endonuclease recognition 
domain can be incorporated into the vector adjacent to or within several 
basepairs of each vector insert, as already described so that following 
PCR amplification, restriction endonuclease digestion is used to remove 
primer encoded sequence, prior to ligation of initial adaptors (containing 
offset recognition domains for the class-IIS restriction endonuclease 
recognition domain used for sequencing). This will facilitate sequencing 
of clone libraries because sequencing cycles will not be wasted sequencing 
the removed primer encoded end of PCR amplified vector inserts. Once a 
class-IIS recognition domain is discovered that requires a methylated 
nucleotide and recognizes a hemi-methylated recognition domain, the 
strategy of using a methylated primer to hemi-methylate the recognition 
domain in only that primer encoded end of a PCR product will be he 
predominant method for removing an entire primer sequence from PCR 
products in those applications for which current class-IIS restriction 
endonucleases are used, including for the generation of site-directed 
mutants and recombinant constructs. (Beck R, H Burtscher, Nucleic Acids 
Research 1994; 22:886-887; Stemmer W P C, S K Morris, B S Wilson, 
BioTechniques 1993; 14:256-265; Stemmer W P C, S K Morris, C R Kautzer, B 
S Wilson, Gene 1993; 123:1-7; Tomic M, I Sunjevaric, E S Savtchenko, M 
Blumenberg, Nucleic Acids Research 1990; 18:1656.) 
Removal of the amplifying primer can also be accomplished by incorporating 
a dUTP at the 3' end of this amplifying primer. dUTP is a nucleotide 
analog that is readily available and can be incorporated into a primer 
sequence at or near its 3' end during oligonucleotide synthesis. dUPT can 
prime from the extreme 3' end of a primer even when mismatched (Kwok S, 
S-Y Chang, J J Sninsky, A Wang, PCR Methods and Applications 1994; 
3:S39-S47). Uracil DNA Glycosylase is used to cleave the N-glycosylic bond 
between the deoxyribose moiety and uracil, resulting in an abasic site 
(Varshney U, T Hutcheon, J H van de Sande, J Biol Chem 1988; 
263:7776-7784). Subsequent heating hydrolyzes the DNA strand at this site, 
generating a phosphorylated 5' end at the nucleotide located immediately 
3' to the dUMP in the original primer, and this phosphorylated 5' end can 
undergo DNA ligation (Day P J R, M R Walker, Nucleic Acids Res 1991; 
19:6959, Liu H S, H C Tzeng, Y J Liang, and Cc Chen, Nucleic Acids Res 
1994; 22:4016-4017). Heating to hydrolyze the primer at the abasic site 
also removes nucleotides located 5' to the dUMP in the original primer, 
resulting in a 5' phosphorylated end with a 3' overhang sequence. 
An alternative method for removing the primer uses a primer with a 3' 
terminal ribose residue. A 3' terminal ribose residue is incorporated into 
the primer using the RNA residue as the solid support during standard 
phosphoramidite synthesis, and the 3' terminal ribose does not interfere 
with PCR amplification (Walder R Y, J R Hayes, J A Walder, Nucleic Acids 
Res 1993; 21:4339-4343, Silveira M H, and L E Orgel, Nucleic Acids Res 
1995; 23:1083-1084). Following PCR amplification, a ribose linkage is 
created in the PCR product that can be readily cleaved by alkaline 
treatment or by digestion with RNase A for 3'-terminal ribose residues 
that are C or U. Cleavage of the ribose linkage results in a 3' overhang 
sequence. 
Using either method for primer removal, generation of a blunt end suitable 
for ligation to an adaptor can then be accomplished by incubating with a 
single-strand specific exonuclease (e.g. Mung bean exonuclease), or with a 
DNA polymerase with a 3' exonuclease activity (e.g. T.sub.4 DNA 
Polymerase) in the presence of the four dNTPs (Stoker A W, Nucleic Acids 
Res 1990; 18:4290), permitting the removal of a primer sequence and its 
complement from PCR products prior to sequencing. Following adaptor 
ligation, a subsequent PCR step can use the ligated adaptor to generate a 
primer annealing site, so that only successfully ligated products are 
regenerated. Using any of the above strategies, with or without removal of 
one of the initial primers and its complement, initial template precursors 
can be generated. 
As is described more fully below, in the course of such cycles of ligation 
and digestion, preferably the first or farthest unpaired nucleotide the 
first unpaired nucleotide in the overhang sequence of the double stranded 
nucleic acid segment is identified. For example, this nucleotide can be 
identified using an adaptor with a detectable label. As used herein, the 
term "detectable label " refers to a material that can attach to a DNA 
molecule and generate a signal. The adaptors may be labeled by a variety 
of means and at variety of locations. The adaptors of the invention can be 
labeled by methods known in the art, including the direct or indirect 
attachment of radioactive labels, fluorescent labels, colorimetric labels, 
chemiluminescent labels and the like, as described in Matthews et al., 
Anal. Biochem., Vol. 169, pgs. 1-25 (1988); Haugland, Handbook of 
Fluorescent Probes and Research Chemicals (Molecular Probes, Inc., Eugene, 
1992); Keller and Manak, DNA Probes, 2nd Edition (Stockton Press, New 
York, 1993); and Eckstein, editor, Oligonucleotides and Analogues: A 
Practical Approach (IRL Press, Oxford, 1991); Wetmur, Critical Reviews in 
Biochemistry and Molecular Biology, 26:227-259 (1991); and the like. Many 
more particular methodologies applicable to the invention are disclosed in 
the following sample of references: Connolly, Nucleic Acids Research, Vol. 
15, pgs. 3131-3139 (1987); Gibson et al., Nucleic Acids Research, Vol. 15, 
pgs. 6455-6467 (1987); Spoat et al., Nucleic Acids Research, Vol. 15, pgs. 
4837-4848 (1987); Fung et al., U.S. Pat. No. 4,757,141; Hobbs, Jr. et al., 
U.S. Pat. No. 5,151,507; Cruickshank, U.S. Pat. No. 5,091,519; (synthesis 
of functionalized oligonucleotides for attachment of reporter groups); 
Jablonski et al., Nucleic Acid Research, 14:6115-6128 (1986) 
(enzyme-oligonucleotide conjugates); and Urdea et al., U.S. Pat. No. 
5,124,246 (branched DNA). Preferably, the adaptors are labeled with one or 
more fluorescent dyes, e.g., as described in U.S. Pat. No. 5,188,934 and 
PCT application PCT/US90/05565. In a preferred embodiment of the 
invention, the adaptor is attached to a solid matrix, such as a magnetic 
particle, e.g., magnetic streptavidin or magnetic glass particle, 
polymeric microsphere, filter material, or the like. 
FIGS. 1, 2, 3 and 4 illustrate four embodiments of the present invention. 
FIG. 1 illustrates the use of a class-IIS restriction endonuclease that 
generates a 5' overhang, and sequences a nucleotide at each interval by 
template-directed ligation. In FIG. 1, this embodiment is illustrated 
using the class-IIS restriction endonuclease FokI, and the template 
precursor has a biotinylated end that allows it to be bound to 
streptavidin. In Step 1, the template precursor is cleaved with FokI. Fok 
I has the following recognition domain and cut site: 
5' GGATG (N).sub.9 
3' CCTAC (N).sub.13 
FokI generates a four nucleotide long 5' overhang positioned nine 
nucleotides away from one side of the recognition domain, so that 
sequencing can be carried out in intervals of nine nucleotides. Fok I 
digestion cleaves both strands of the double-stranded DNA, generating a 
DNA template with a 5' overhang sequence. The bound template is washed to 
remove the cleaved ends. In Step 2 the 5' overhang sequence mediates 
ligation to one of four adaptors. These adaptors contain the sequence for 
the recognition domain for Fok I and have an adjacent four nucleotide long 
and phosphorylated 5' overhang consisting of three nucleotides with 4-fold 
degeneracy and a 5' terminus with one of the four normal nucleotides. 
Since the four adaptors each have three degenerate nucleotides and four 
distinct 5' terminal nucleotides, there are 256 distinct sequences. The 
adaptors shown are double-stranded, because this increases the ligation 
efficiency, probably due to stacking interactions (Lin S-B, K R Blake, P S 
Miller, Biochemistry 1989; 28:1054-1061). In this embodiment of the method 
there is one ligation reaction during each sequencing cycle. In each 
ligation, all four adaptors are present, and each adaptor is preferably 
tagged with a distinct fluorescent label (e.g. Fama-NHS ester, Rox-NHS 
ester, Tamra-NHS ester, or Joe-NHS ester; Applied Biosystems Division of 
Perkin-Elmer, Foster City, Calif.); each label identifying the nucleotide 
at the single-stranded 5' end of the adaptor. Ligation occurs for the 
adaptor for which the above mentioned 5' nucleotide is complementary to 
the nucleotide on the 5' end of the DNA template at the ligation junction. 
Following ligation, and washing to remove the unligated adaptors, 
identification of the ligated adaptor can be accomplished by fluorometry, 
revealing the sequence of the DNA template at the ligation junction (Step 
3). In step 4, the ligated template from Step 2 undergoes PCR 
amplification using a biotinylated primer and using a primer that is 
complementary to a unique portion of the adaptor's ligated lower strand. 
An alternative approach would sequence via ligation of the adaptor's upper 
strand. In this approach, the fixed nucleotide in the single strand 
extension in each adaptor is the fourth nucleotide 3' to the 5' end. The 
label is preferably in the upper strand, and this label identifies the 
lower strand's fixed nucleotide in the single strand overhang, with the 
remaining nucleotides in this single strand being promiscuous nucleotides 
(degenerate or universal nucleotides). In this embodiment of the 
invention, one of the primers would be homologous to a unique portion of 
the adaptor's ligated upper strand. 
This unique region, and its corresponding amplification primer, may differ 
during every sequencing cycle, or during every several sequencing cycles. 
By using ligated adaptors and corresponding amplifying primers that differ 
in each cycle, uncut products from Step 1 are not amplified, preventing 
uncut products from generating background signal in subsequent cycles. The 
PCR product is bound to streptavidin, and the entire process is repeated, 
sequencing a nucleotide nine nucleotides within the original nucleic acid 
segment during each cycle of cutting, template-directed ligation, and 
amplification of the desired template precursor. During Step 1 of the 
subsequent cycle digestion with FokI cleaves both strands of the DNA and 
generates a new 5' overhang sequence with each strand shortened by nine 
nucleotides when compared to the template at the end of the prior Step 1. 
(This shortening of the template precursor following each cycle is not 
shown in FIGS. 1-4). 
Additional steps can be taken to increase the efficiency of each step, and 
may prove necessary in implementing a protocol that does not use 
amplification to regenerate the template precursor during each cycle. 
These additional steps include: 
1) Treating the template with alkaline phosphatase following restriction 
endonuclease cutting (Step 1 of FIG. 1). This de-phosphorylates the 5' end 
of each template, preventing ligation of one template to another. 
2) Using adaptors with upper strand 3' ends that are blocked by a 3' 
phosphate or blocked by a 3' dideoxy nucleotide. This prevents ligation of 
one adaptor to another during Step 2 of the method of FIG. 1. 
3) Incubating with a DNA polymerase and the four ddNPTs following the 
adaptor ligation step (Step 2 in FIG. 1). This fills in the recessed 3' 
end of those templates that escaped adaptor ligation, and caps these ends 
so that they cannot undergo ligation (Atkinson M R, M P Deutscher, A 
Kornberg, A F Russell, J G Moffatt, Enzymatic Synthesis of DNA 1969; 
8:4897-4904). This additional step prevents templates that failed to 
undergo adaptor ligation during a given cycle from undergoing adaptor 
ligation in subsequent cycles, thus eliminating background signal 
resulting from incomplete ligation of templates. 
4) Retained fluorescent label resulting from incomplete cutting by Fok I 
can be quenched by photo-bleaching immediately prior to Step 1, or through 
cleavage of the label by using a labile linkage (Dawson B A, T Herman, J 
Lough Journal of Biological Chemistry 1989; 264:12830-12837, Olejnik J. E 
Krzymanska-Olejnik, K J Rothschild Nucleic Acids Research 1996; 
24:361-366) thus decreasing background fluorescent signal from previous 
cycles. 
If the lower strand of the adaptor is ligated, and the upper strand's 3' 
end is not blocked, non blocked and added later, or is de-blocked (via 
dephosphorylating a 3' phosphate, Cameron V, O C Uhlenbeck Biochemistry 
1977; 16:5120-5126 or, for example, by the method described in Metzker M 
L, Raghavachari R, Richards S, Jacutin S E, Civitello A, Burgess K and R A 
Gibbs, Nucleic Acids Res. 1994; 22:4259-4267 and Canard B and R S Sarfati, 
Gene 1994; 148:1-6), an intact double-stranded segment can be generated, 
without nicks, using a DNA polymerase with a 5' exonuclease activity, in a 
nick translation reaction (Rigby P W J, M Dieckmann, C Rhodes, P Berg Mol. 
Biol. 1977; 113:237-251). Such nick translation could occur with 
concurrent hemi-methylation of internal recognition domain for the 
class-IIS restriction endonuclease using the primer extension strategy of 
Han and Rutter (Han J, Rutter W J, Nucleic Acids Res 1988; 16:11837). 
If the upper strand of the adaptor is ligated, an intact double-stranded 
segment could be generated, without nicks, by using a DNA polymerase to 
generate the complement to the adaptor's ligated upper strand. This 
polymerization could occur with concurrent hemi-methylation of the adaptor 
encoded recognition domain for the class-IIS restriction endonuclease 
using the polymerase extension in the presence of a methylated nucleotide 
(when sequencing with a class-IIS restriction endonuclease that recognizes 
a hemi-methylated recognition domain; also, if the ligated upper-strand's 
recognition domain sequence were methylated, both strands of the 
recognition domain would be methylated using this method). If the adaptor 
were double-stranded, the unligated lower strand of the adaptor could be 
digested by nick translation using a DNA polymerase with 5' exonuclease 
activity, or by using a DNA polymerase with strand displacement activity. 
FIG. 2 illustrates a second embodiment of the sequencing method of this 
invention wherein a class-IIS restriction endonuclease generates a 3' 
overhang, and sequences a nucleotide at each interval by template-directed 
ligation. In FIG. 2, this embodiment is illustrated using the class-IIS 
restriction endonuclease BseRI, and the template precursor has a 
biotinylated end that allows it to be bound to streptavidin. In Step 1, 
the template precursor is cleaved with BseRI. BseRI has the following 
recognition domain and cut site: 
5' GAGGAG (N).sub.10 
3' CTCCTC (N).sub.8 
BseRl generates a two nucleotide long 3' overhang positioned eight 
nucleotides away from one side of the recognition domain, so that 
sequencing can be carried out in intervals of eight nucleotides. BseRI 
digestion cleaves both strands of the double-stranded DNA, generating a 
DNA template with a 3' overhang sequence. The bound template is washed to 
remove the cleaved ends. In Step 2 the DNA template (3' overhang sequence) 
undergoes ligation in the presence of four adaptors. These adaptors 
contain the sequence for the recognition domain for BseRI and have an 
adjacent two nucleotide long 3' overhang consisting of one nucleotide 
4-fold degeneracy and a 3' terminus with one of the four normal 
nucleotides. Since the four adaptors each have one degenerate nucleotide 
and four distinct 3' terminal nucleotides, there are 16 distinct 
sequences. The adaptors are double-stranded, because this increases the 
ligation efficiency. There is one ligation reaction during each sequencing 
cycle. In each ligation, all four adaptors are present, and each adaptor 
is preferably tagged with a distinct fluorescent label; each label 
identifies the single-stranded nucleotide at the single-stranded 3' end of 
the adaptor. Ligation of the upper strand of the adaptor occurs if the 
above mentioned 3' nucleotide is complementary to the nucleotide on the 3' 
end of the DNA template at the ligation junction. Following ligation and 
washing to remove the unligated adaptors, identification of the ligated 
adaptor can be accomplished by fluorometry, revealing the sequence of the 
DNA template at the ligation junction (Step 3). In step 4, the ligated 
template from Step 2 undergoes PCR amplification using a biotinylated 
primer and using a primer that is homologous to a unique portion of the 
adaptor's ligated upper strand. If the lower strand underwent the ligation 
reaction that sequenced the DNA, by using an upper strand that had its 
fixed nucleotide in its 3' single stranded portion of the adaptor 
immediately adjacent to the double-stranded portion of the adaptor, the 
non-biotinylated primer would be complementary to a unique portion in the 
ligated adaptor's lower strand. This unique region, and its corresponding 
amplification primer, may differ during every sequencing cycle, or during 
every several sequencing cycles, preventing uncut products from a prior 
cycle from generating background signal in subsequent cycles. The PCR 
product is bound to streptavidin, and the entire process is repeated, 
sequencing a nucleotide eight nucleotides further within the original 
nucleic acid segment during each cycle of cutting, template-directed 
ligation, and in vitro amplification of the desired template precursor. 
During Step 1 of each subsequent cycle, digestion with BseRI cleaves both 
strands of the DNA and generates a new 3' overhang sequence with each 
strand shortened by eight nucleotides when compared to the template at the 
end of the prior Step 1. 
Another step can be taken to prevent templates that do not undergo ligation 
during a given cycle from undergoing ligation in a subsequent cycle. 
Following adaptor ligation (Step 2 of FIG. 2) incubation with alkaline 
phosphatase will dephosphorylate the 5' end of those templates that did 
not undergo ligation to an adaptor, preventing these templates from 
undergoing adaptor ligation in subsequent cycles. If amplification (Step 4 
of FIG. 2) is not used, following ligation of the adaptor's upper strand 
(Step 2 of FIG. 2), the lower strand of the DNA being sequenced can prime 
template-directed polymerase extension using a DNA polymerase with a 3' 
exonuclease activity, in the presence of the four dNTPs recognizing that 
the DNA polymerase preferably has a 5' exonuclease activity or a strand 
displacement activity if the adaptor has a lower strand. This will 
re-synthesize the lower strand of the attached adaptor, eliminating the 
nick and any mismatches while generating a template precursor. Also, those 
templates which did not undergo adaptor ligation will be rendered blunt 
ended by the 3' exonuclease activity of the DNA polymerase preventing 
adaptor ligation in subsequent cycles. When using a restriction 
endonuclease that generates a 3' overhang, a terminal transferase can be 
used to add a single dideoxy nucleotide to the end of the template. This 
terminal nucleotide can act as a barb in a hook to help hold the adaptor 
in place, as each adaptor can share a nucleotide complementary to the 
dideoxy nucleotide in each adaptor's annealing strand, so that this will 
increase the efficiency of adaptor ligation. In this case, sequencing 
occurs in an interval that is one nucleotide shorter than the distance 
between the recognition domain and the cleavage domain. 
When a DNA polymerase is used to generate the complement to the adaptor's 
ligated upper strand, this polymerization may be performed with concurrent 
hemi-methylation of the adaptor encoded recognition domain for the 
class-IIS endonuclease using the polymerase extension in the presence of a 
methylated nucleotide (when sequencing with a class-IIS restriction 
endonuclease that recognizes a hemi-methylated recognition domain; also, 
if the ligated upper-strand's recognition domain sequence were methylated, 
both strands of the recognition domain would be methylated using this 
method). If the adaptor were double-stranded, the unligated lower strand 
of the adaptor could be digested by nick translation using a DNA 
polymerase with 5' exonuclease activity, or by using a DNA polymerase with 
strand displacement activity. 
If the lower strand of the adaptor is ligated, an intact double-stranded 
segment could be generated, without nicks, by using a DNA polymerase with 
a 5' exonuclease activity, in a nick translation reaction (Rigby, P W J, M 
Dieckmann, C Rhodes, P Berg Mol. Biol 1977; 113:237-251) using the upper 
strand of the adaptor as a primer. Such nick translation could occur with 
concurrent hemi-methylation of internal recognition domain for the 
class-IIS restriction endonuclease using the primer extension strategy of 
Han and Rutter (Han J, Rutter W J Nucleic Acids Res 1988; 16:11837). 
FIG. 3 shares with FIG. 1 the use of a class-IIS restriction endonuclease 
that generates a 5' overhang, but sequences a nucleotide at each interval 
by template-directed polymerization instead of template-directed ligation. 
In Step 2 of FIG. 3, the DNA template generated following FokI digestion 
is sequenced by template-directed polymerization in the presence of four 
deoxynucleotide terminators (e.g. ddNTPs), each tagged with a distinct 
fluorescent label. Following polymerization and washing, which removes 
unincorporated terminators, identification of the incorporated terminator 
can be accomplished by fluorometry, revealing the sequence of one 
nucleotide in the DNA template, as shown in Step 3. Step 4 illustrates the 
ligation of an adaptor containing the sequence for the recognition domain 
for Fok I and an adjacent three nucleotide long 5' overhang consisting of 
three nucleotides with 4-fold degeneracy. The ligation illustrated in FIG. 
3 is template-directed but is not used to discriminate between nucleotides 
at the ligation junction. Since the single adaptor has three degenerate 
nucleotides, there are 64 distinct sequences. The adaptors shown are 
double-stranded, as this increases the ligation efficiency. The 
amplification shown in Step 5 of FIG. 3 corresponds to Step 4 of FIG. 1, 
except that the amplifying primer is homologous to the ligated strand of 
the adaptor, which is the upper strand in FIG. 3. 
Since the upper strand of the adaptor undergoes ligation, an intact 
double-stranded segment could be generated, without nicks, by using a DNA 
polymerase to generate the complement to the adaptor's ligated upper 
strand. The lower strand of the DNA segment being sequenced can de-blocked 
(via dephosphorylating a 3' phosphate, or by the method described in 
Metzker M L, Raghavachari R, Richards S, Jacutin S E, Civitello A, Burgess 
K and R A Gibbs, Nucleic Acids Res. 1994; 22:4259-4267 and Canard B and R 
S Sarfati, Gene 1994; 148:1-6), allowing it to act as a primer. This 
polymerization could occur with concurrent hemi-methylation of the adaptor 
encoded recognition domain for the class-IIS endonuclease using the 
polymerase extension in the presence of a methylated nucleotide (when 
sequencing with a class-IIS restriction endonuclease that recognizes a 
hemi-methylated recognition domain; also, if the ligated upper-strand's 
recognition domain sequence were methylated, both strands of the 
recognition domain would be methylated using this method). 
In the strategy illustrated in FIG. 3, if the class II-S restriction 
endonuclease generates a single nucleotide 5' end extension, 
template-directed polymerization will generate a blunt end, so that 
adaptor ligation is blunt ended, as opposed to the template-directed 
ligation illustrated in FIG. 3. Furthermore, if a class-IIS restriction 
endonuclease is discovered that generates a blunt end, or a blunt end is 
generated using a single strand exonuclease, a nucleotide at this end 
could be sequenced by template-directed polymerization through a 
nucleotide exchange reaction, in which the 3' exonuclease activity of DNA 
polymerase is used to generate a recessed 3' end that can undergo 
template-directed polymerization, incorporating a labeled nucleotide and 
once again generating a blunt end that would undergo ligation to the 
adaptor (Atkinson M R, M P Deutscher, A Kornberg, A F Russell, J G Moffatt 
Enzymatic Synthesis of DNA 1969; 8:4897-4904, Englund P T Journal of 
Biological Chemistry 1971; 246:3269-3276). In this case, the template is 
formed fleetingly, through the 3' exonuclease activity of a DNA polymerase 
during the exchange reaction that constitutes the DNA sequencing step. If 
the incorporated labeled terminator inhibits adaptor ligation, only a 
fraction of a given terminator needs to carry a label, and only a fraction 
of a given template needs to undergo labeling, because only a fraction of 
a template must undergo adaptor ligation to allow regeneration of the 
desired template precursors by DNA amplification in vitro. This 
illustrates how product regeneration allows separation of the template 
generation and template sequencing elements of this method without 
physical separation of these elements into separate aliquots. 
FIG. 4 illustrates a variation of the method of FIG. 3 in which the 
overhang appended to the adaptor-encoded sequence is attached to a solid 
phase. In this variation, the PCR primer that varies between cycles 
carries the biotin moiety. Following FokI cutting, the end encoded by the 
adaptor is attached to the solid matrix, and a nucleotide in this end is 
sequenced by template-directed polymerization. In addition, this end could 
be sequenced by template-directed ligation, in which case the class-IIS 
restriction endonuclease could generate a 5' overhang or a 3' overhang. 
Another variation that could be carried out would be to combine sequencing 
by template-directed polymerization with sequencing by template-directed 
ligation. For example, if the adaptor undergoing template-directed 
ligation in Step 4 of FIG. 4 were a sequencing adaptor, as shown in FIG. 
1, sequencing could be accomplished by template-directed ligation and 
template-directed polymerization during each cycle using the same template 
precursor. Also, it is clear that the process of sequencing each template 
can be separated from the process of generating each template, so that a 
FokI generated four nucleotide overhang could be sequenced, for example, 
by template-directed ligation and in a separation reaction by fill-in with 
labeled ddNTPs. 
Variants of protocols shown in FIGS. 1-4 not requiring the exponential 
amplification step (Step 4 of FIGS. 1 and 2 and Step 5 of FIGS. 3 and 4) 
can be developed using steps that optimize completion of each step and 
that "cap " incomplete reactions, as described previously in conjunction 
with striding. For example MmeI has a recognition domain that is separated 
from its cleavage domain by 18 bp. Therefore, one could sequence over a 
span of 90 nucleotides over five iterative cycles, as opposed to only 5 
nucleotides when using a method that sequences consecutive nucleotides. 
Other measures that may increase the number of sequencing cycles that can 
be carried out without using exponential in vitro amplification, include: 
1) Modification of a restriction endonuclease recognition domain by use of 
a base analog to improve binding to the restriction enzyme, so that a 
modified double-stranded oligonucleotide binds to its restriction 
endonuclease more effectively than the naturally occurring recognition 
domain (Lesser D R, M R Kurpiewski, T Waters, B A Connolly, and L 
Jen-Jacobson, Natl. Acad. Sci. USA 1993; 90:7548-7552). Using a ligated 
adaptor with a modified class-IIS recognition domain may improve 
restriction endonuclease binding and cutting efficiency. For example, a 
hybrid restriction endonuclease could be generated in which a protein that 
recognizes a certain DNA sequence or moiety is attached to the cleaving 
domain of a class-IIS restriction endonuclease, generating a new 
specificity with a defined distance between a cleavage domain and a 
recognition domain (Kim Y G, J Cha, S Chandrasegaran, Proc. Natl. Acad. 
Sci. USA. 1996; 93:1156-1160). 
2) Ligating adaptors that are covalently attached to a class-IIS 
restriction endonuclease. A variety of enzymes have been covalently 
attached to oligonucleotides (Jablonski E, E W Moomaw, R H Tullis, J L 
Rith, Nucleic Acids Res 1986; 14:6115-6128, Li P, P P Medon, D C Skingler, 
J A Lanser, R H Symons, Nucleic Acids Res 1987; 15:5275-5287, Ghosh S S, P 
M Kao, D Y Kwoh, Anal Biochem 1989;78;178:43-51). Use of a double-stranded 
recognition domain with the class-IIS restriction endonuclease attached to 
it could be used to target cutting to the cleavage domain adjacent to the 
ligated adaptor's recognition domain, so long as buffer conditions during 
the prior ligation do not permit cutting. Since the restriction 
endonuclease would only be positioned immediately adjacent to the desired 
recognition site, digestion would not be mediated by internal recognition 
domains, so that methylation of internal recognition domains would not be 
necessary. 
3) Using a class-IIS restriction endonuclease that requires a methylated 
recognition domain, and will recognize a hemi-methylated recognition 
domain. In this case, the recognition domain can be hemi-methylated during 
adaptor ligation using an adaptor strand that contains a methylated strand 
of this domain, so that only this recognition domain would be recognized. 
A class-IIS restriction endonuclease that requires a methylated 
recognition domain could be used in this method and would be advantageous, 
as it would obviate the need to block internal recognition domains for 
this class-IIS restriction endonuclease. 
Restriction endonucleases and DNA ligases have been used in this invention, 
but different enzymes or reactive chemicals could be used to generate the 
templates described in this invention. Mutated enzymes that carry out the 
same role can substitute for their naturally occurring counterparts (Kim J 
J, K T Min, M H Kim, S J Augh, B-D Dim, D-S Lee Gene 1996; 171:129-130). 
Furthermore, various entities can substitute for DNA ligase and 
restriction endonucleases. Template-directed ligation has carried out 
through chemical condensation (Gryaznov S M, R Schultz, S K Chaturvedi, R 
L Letsinger, Nucleic Acids Research 1994 22:2366-2369, Dolinnaya N G, M 
Blumenfeld, I N Merenkova, T S Oretskaya, N F Krynetskaya, M G 
Ivanovskaya, M Vasseur and Z A Shabarova, Nucleic Acids Research 1993; 
21:5403-5407, Luebke K J and P B Dervan, Nucleic Acids Research 1992; 
20:3005-3009), and site-specific cleavage of DNA has been accomplished 
using oligonucleotides linked to reactive chemicals or non-specific 
nucleases (Lin S-B, K R Blake, P S Miller Biochemistry 1989; 28:1054-1061, 
Strobel, S A, L A Doucette-Stamm, L Riba, D E Housman, P B Dervan, Science 
1991; 254:1639-1642, Francois J-C, T Saison-Behmoaras, C Barbier, M 
Chassignol, N T Thuong, C Helene, Proc. Natl. Acad. Sci. USA 1989; 
86:9702-9706, Pei D, D R Corey, P G Schultz, Proc. Natl. Acad. Sci. USA 
1990; 87:9858-9862). Non-protein enzymes have also been used to manipulate 
DNA, as ribozymes have mediated both the cleavage and ligation of DNA 
(Tsang J, G F Joyce, Biochemistry 1994; 19:5966-5973, Cuenoud B, J W 
Szostak, Nature 1995; 375:611-614). 
Nucleotide analogs have been used in a variety of functions, and 
template-directed ligation could be mediated by adaptors with 
single-stranded ends containing universal nucleotides or discriminatory 
nucleotide analogues (Loakes D, D M Brown, Nucleic Acids Research 1994; 
22:4039-4043, Nichols R, P C Andrews, P Zhang, D E Bergstrom, Nature 1994; 
369:492-493). In addition, modified nucleotides other than methylated 
nucleotides have been found that block recognition by restriction 
endonucleases, and can be incorporated through primer-directed DNA 
synthesis (Huang L-H, C M Farnet, K C Ehrlich, M Ehrlich, Nucleic Acids 
Research 1982; 10:1579-1591, Seela F, W Herdering, A Kehne Helvetica 
Chimica Acta 1987; 70:1649-1660, and Seela F, A Roling, Nucleosides and 
Nucleotides 1991; 10:715-717). 
Technology now exists for the generation of a thousand distinct DNA 
segments at one time using the polymerase chain reaction (PCR), thus 
allowing the concurrent generation of a thousand DNA template precursors. 
Development of technology for template precursor generation is facilitated 
by present methods for the concurrent generation of multiple 
oligonucleotides, as oligonucleotides serve as primers for template 
precursor generation through DNA amplification in vitro (Caviana Pease A, 
Solas D, Sullivan E J, Cronin M T, Holmes C P, Fodor S P A, Proc Natl Acad 
Sci USA 1994; 91:5022-5026). Micro-chip based technology will allow the 
amplification of over 10,000 distinct DNA segments, each containing 
several hundred base pairs of DNA (Shoffner M A, J Cheng, G E Hvichia, L J 
Kricka, P Wilding, Nucleic Acids Research 1996; 24:375-379, and J Cheng, 
Shoffner M A, G E Hvichia, L J Kricka, P Wilding, Nucleic Acids Research 
1996; 24:380-385). This will allow a large portion of the human genome of 
an individual to be sorted on a biochip. Rapid technical progress in DNA 
sample generation creates a need for technology that can rapidly and 
accurately sequence arrayed samples of DNA in parallel. This invention 
addresses the need for technology that can sequence thousands of distinct 
DNA samples in parallel. 
Technology for generating double-stranded template-precursors via PCR, and 
for the fluorometric assessment of thousands of locations on a chip, will 
allow the sequencing of several thousand PCR products simultaneously using 
this invention, allowing large amounts of DNA to be sequenced using 
repetitive incubations in simple reagents. The template precursors can be 
bound to a silicon chip or contained in a matrix of chambers, so that 
cycles of adaptor ligation, template-directed DNA polymerization for 
amplification or sequencing, and cutting can be carried out on numerous 
templates in parallel. 
Technology that has been developed for the simultaneous assessment of 
thousands of locations on a chip will facilitate the simultaneous 
sequencing of these templates. For example, a microchip has been designed 
for the quantitative detection of DNA labeled with fluorescent, 
chemiluminescent or radioactive reporter groups (Eggers M, M Hogan, R K 
Reich, J Lamture, D Ehrlich, M Hollis, B Kosicki, T Powdrill, K Beattie, S 
Smith, R Varma, R Gangadharan, A Mallik, B Burke and D Wallace, 
BioTechniques 1994; 17:516-524). This microchip consists of a charged 
coupled device (CCD) detector that quantitatively detects and images the 
distribution of labeled DNA near spatially addressable pixels. DNA has 
been deposited onto a silicon wafer with a micro-jet using DNA with an 
amine modified 5' end, which is linked to the SiO2 surface by secondary 
amine formation. This immobilized DNA is on an SiO2 wafer overlying the 
pixels of the charged coupled device. A prototype 420.times.420 pixel 
device has been developed that can analyze 176,400 samples in parallel, 
enabling the detection of thousands of label incorporation events on a 
square centimeter chip (Eggers M, M Hogan, R K Reich, J Lamture, D 
Ehrlich, M Hollis, B Kosicki, T Powdrill, K Beattie, S Smith, R Varnia, R 
Gangadharan, A Mallik, B Burke and D Wallace, BioTechniques 1994; 
17:516-524). 
Technology that will further enhance the utility of the present invention 
include hybridization based approaches for sorting genomic DNA (as opposed 
to sequencing by hybridization) into unique restriction fragments, which 
can then be amplified at their addresses using a single set of PCR primers 
(Chetverin A B, F R Kramer, BioTechnology 1994; 12:1093-1099). In the 
future, it will be possible to apply the present invention to the 
sequencing of large portions of genomes for which there is no prior 
sequence information without cloning in vivo (e.g., in E. coli). New 
innovative hybridization based strategies have been proposed that use 
oligonucleotide arrays to sort restriction endonuclease generated 
fragments on the basis of their unique sequences. In one strategy, genomic 
DNA undergoes complete restriction endonuclease digestion. This is 
followed by ligation of the DNA ends to adaptors. These restriction 
fragments are sorted on a hybridization array of oligonucleotides through 
annealing to the adaptor sequence as well to unique adjacent sequences in 
the DNA fragments. This is followed by a ligation step that requires 
perfect complementarity of the unique sequence adjacent to the adaptor, 
resulting in sorting of the restriction fragments into unique addresses on 
the biochip. An additional step repeats this strategy using the opposite 
end of each fragment. These sorted fragments can then be PCR amplified in 
situ using a single set of primers that anneal to the adaptor sequences 
(Chetverin A B, F R Kramer, BioTechnology 1994; 12:1093-1099). Integrating 
this hybridization-based technology into the present method will allow the 
sequencing of genomes using a single set of PCR primers without prior 
sequence information. 
An area of technology development that can also be useful to the 
application of the proposed method is oligonucleotide synthesis from the 
5' to 3' direction (Coassin P J, J B Rampal, R S Matson International 
Workshop on Sequencing by Hybridization (Woodlands, Tex.) 1993; Report 8). 
This will allow amplifying primers to be manufactured on a chip. These 
bound primers could be used to amplify PCR products, as it has recently 
been confirmed that a primer can mediate PCR amplification while bound to 
a solid immobile matrix (Kohsaka H, D A Carson, Journal of Clinical 
Laboratory Analysis 1994; 8:452-455). 
Kits 
A variety of kits are provided for carrying out different embodiments of 
the invention. Generally, kits of the invention include adaptors tailored 
for the enzyme, e.g., a class IIS restriction endonuclease, and the 
detection scheme of the particular embodiment. Kits further include the 
enzyme reagents, the ligation reagents, PCR amplification reagents, and 
instructions for practicing the particular embodiment of the invention. In 
embodiments employing natural protein endonucleases and ligases, ligase 
buffers and endonuclease buffers may be included. In some cases, these 
buffers may be identical. Such kits may also include a methylase and its 
reaction buffer. Preferably, kits also include a solid phase support, e.g. 
magnetic beads, for anchoring target DNA segments. In one preferred kit, 
labeled ddNTP's are provided. In another preferred kit, fluorescently 
labeled probes are provided such that probes corresponding to different 
terminal nucleotides of probe or the target polynucleotide carry distinct 
spectrally resolvable fluorescent dyes. As used herein, "spectrally 
resolvable" means that the dyes may be distinguished on basis of their 
spectral characteristics, particularly fluorescence emission wavelength, 
under conditions of operation. Thus, the identity of the one or more 
terminal nucleotides would be correlated to a distinct color, or perhaps 
ratio of intensifies at different wavelengths. More preferably, four such 
probes are provided that allow a one-to-one correspondence between each of 
four spectrally resolvable fluorescent dyes and the four possible terminal 
nucleotides on a target DNA segment. Sets of spectrally resolvable dyes 
are disclosed in U.S. Pat. No. 4,855,225 and 5,188,934; International 
application PCT/US90/05565; and Lee et al., Nucleic Acids Research 
20:2471-2483 (1992). 
Automation of Iterative and Regenerative DNA Sequencing 
The foregoing sequencing steps, being iterative, may be automated and 
applied in parallel to an arbitrary number of separate samples. Such 
automation permits the sequencing method to generate a large amount of 
sequence information, and this information is further enhanced by the 
subinterval or adjacency order existing between the products of successive 
steps, as well as in a multiplex scheme, the immobilized spatial locations 
in which sequencing occurs. 
FIG. 8 shows a schematic outline of the overall architecture of a system 
100 for automating sequencing according to the present invention, which is 
preferably implemented by a processing apparatus 20 which operates on 
support arrays 10 such as microtiter plates or specially fabricated chip 
arrays that consist of an array of wells, chambers or surface 
immobilization positions each capable of holding a DNA sample at a 
localized site. Device 20 performs four general types of operations in 
parallel on the DNA segments in the support array 10, and these are shown 
schematically as separate classes of processes arrayed in stations or 
functional groupings 30, 40, 50, 60 around the central device 20. 
As shown, the four basic processes involve the addition of reagents 30, 
washing, separating or preparation steps 40, reading the labeled segments 
at 50, or incubation and amplification steps at 60. These are 
schematically illustrated as four separate workstations through which the 
support array 10 is shuttled or moved, but are preferably implemented with 
varying degrees of integration into the basic array handler 20. Thus, for 
example, the array 10 may stay in position on a stage to which the 
necessary conduits or manifolds are attached for addition of the reagents 
and washing of the samples, and which may be heated or cooled in cycles to 
incubate and amplify all materials on the support at once. Similarly, for 
reading, a charge couple device may be carried with appropriate optics by 
the device 20 to read the labeled material in each sample well between 
successive steps, or may be integrated into a cover plate or the structure 
of the sample support. In either case, each of these subunits or accessory 
portions of the system operates under control of a common controller 70 
which coordinates the movement, heating, provision of reagents and reading 
of the various steps so that the readout of nucleotide labels by the 
reading section 50 is stored and recorded for the DNA samples at each 
location on the array 10. 
As noted above, each of the DNA segments which are to be analyzed, which 
may, for example, be PCR products or vector inserts, is immobilized so 
that it resides at a unique address on the chip or support 10, and several 
hundred to thousands of DNA segments are distributed on the chip. They 
simultaneously undergo a series of incubation that result in the 
accumulation of sequence information. A reagent may be delivered, for 
example, by a robotically carried comb or pipette array, or preferably by 
bulk or flow-through addition of the reagent. Separate reagents in their 
respective buffers are represented by the jar in the left hand portion of 
the diagram and these are passed to the support array 10 by automated 
control in the order for performing the sequencing chemistry described 
herein. Sequencing occurs either following template-directed adaptor 
ligation (as described for Embodiments 1 and 2 in relation to FIGS. 1 and 
2 herein) or following template-directed polymerization (as described in 
relation to FIGS. 3 and 4). Simultaneous retrieval of sequence information 
from several thousand templates following template-directed incorporation 
of a label, is then done by reader 50. Reading can be accomplished 
concurrently using a charge coupled device, which is illustrated on the 
top of FIG. 8, or may be performed in a slower scanning fashion by 
stepping the array past a line of scintillation or other detectors. By 
operating with a support array in which the DNA segments are immobilized 
in a small area and volume, a relatively strong signal is obtained free of 
the spreading and cross-reading losses inherent in gel sequencing or 
migration-dependent methods. 
As described elsewhere herein, the method preferably includes a 
regeneration step. Illustratively, following the adaptor ligation step, an 
aliquot from each address undergoes PCR amplification in order to 
regenerate a template precursor for the next sequencing cycle. The 
appropriate primer sets and PCR mix are applied and the array undergoes a 
number of incubations. Preferably the device 20 has a heated stage with a 
Peltier cooler to accurately and quickly cycle the array through the 
required amplification regimen, or the array may pass to a separate 
processing chamber, e.g. an air oven thermal cycler of conventional type, 
for PCR amplification as illustrated on the bottom of the diagram. 
Following incubation with a reagent or PCR amplification, the DNA segments 
are frequently magnetically pelleted and washed to remove the reagent and 
any byproducts prior to a subsequent step. The magnet and wash buffer are 
illustrated by device processes or subassembly 40 on the right hand 
portion of FIG. 8. 
Once the necessary set of adapters and primers for cutting and 
amplification sets have been determined, the process steps are 
straightforward, and well-defined nucleotide determinations are achieved 
with small amounts of sample. The support arrays may thus carry a large 
number of sites. A chip or group of chips with 90,000 defined addresses 
will for example, allow the amplification of 90,000 DNA segments using 
PCR. Simultaneous amplification of a large number of samples may be done 
with a robotic thermal cycler using the approach of Meier-Ewert S, E 
Maier, A Ahmadi, J Curtis, H Lehrach. An automated approach to generating 
expressed sequence catalogues. Nature 1993; 361: 375-376 and Drmanac S, R 
Drmanac. Processing of cDNA and genomic kilobase-size clones for massive 
screening, mapping, and sequencing by hybridization. BioTechniques 1994; 
17: 328-336, as applied to PCR. The invention also contemplates that the 
support be a microchip, in which case the teachings of PCR amplification 
on a microchip by several investigators are modified to include multiplex 
PCR amplification features for carrying out the methods described here. 
See, for example Wilding P, M A Shoffer, L J Kricka. PCR in a silicon 
microstructure. Clinical Chemistry 1994; 40: 1815-1818; Shouffner M A, J 
Cheng, G E Hvichia, L J Kricka, P Wilding. Chip PCR. I. Surface 
passivation of microfabricated silicon-glass chips for PCR. Nucleic Acid 
Research 1996; 24: 375-379; Cheng J, M A Shoffier, G E Hvichia, L J 
Kricka, P Wilding. Chip PCR II Investigation of different PCR 
amplification systems in microfabricated silicon-glass chips. Nucleic Acid 
Research 1996; 24: 380-385; Burns M A, C H Mastrangelo, T S Sammarco, F P 
Man, J R Webster, B N Johnson, B Foerster, D Jones, Y Fields, A R Kaiser, 
D T Burke. Microfabricated structures for integrated DNA analysis. Proc. 
Natl. Acad. Sci. USA 1996; 93: 5556-5561. 
Automated sequencing is described below for a chip with 90,000 addresses 
using a protocol for Embodiment 1. One of the primers in each PCR 
amplification is biotinylated, allowing these products to be bound to 
magnetic streptavidin. The opposite primer contains the recognition domain 
for FokI restriction endonuclease. If FokI is used as the restriction 
endonuclease, and sequencing is done in intervals of nine nucleotides, 
nine initial templates are generated for each of 10,000 DNA regions to be 
sequenced. This is accomplished by using primers with offset FokI 
restriction endonuclease recognition domains, as described extensively 
elsewhere herein. In the case where the DNA samples to be sequenced are 
vector inserts, primers are generated that anneal to the vector, so that 
only a few primers need to be synthesized to sequence the 90,000 DNA 
segments. 
Following PCR amplification, the DNA segments are bound to magnetic 
streptavidin and magnetically pelleted, washed, and incubated with FokI in 
the corresponding buffer at 37.degree. C., resulting in generation of the 
initial templates. After magnetic pelleting and washing, the 90,000 
initial templates are incubated with a DNA ligase and the four sequencing 
adaptors, each with a unique label. Following magnetic pelleting and 
washing step to remove unligated adaptors, the ligated adaptor at each 
address is identified, for example with an automated reader using a charge 
coupled device. This is done in one embodiment by imaging the support 
array onto a CCD, and using automated analysis of the image pixels to 
threshold and read the luminescent labels, or by the approach described in 
Eggers M, M Hogan, R K Reich, J Lamture, D Ehrlich, M Hollis, B Kosicki, T 
Powdrill, K Beattie, S Smith, R Varma, R Gangadharan, A Mallik, B Burke, D 
Wallace. A microchip for quantitative detection of molecules utilizing 
luminescent and radioisotope reporter groups. BioTechniques 1994; 17: 
516-525 or Lamture J B, K L Beattie, B E Burke, M D Eggers, D J Ehrich, R 
Fowler, M A Hollis, B B Kosicki, R K Reich, S R Smith, R S Varma, M E 
Hogan. Direct detection of nucleic acid hybridization on the surface of a 
charged coupled device. Nucleic Acid Research 1994; 22: 2121-2125. 
Following reading of the labels, new template-precursors are regenerated by 
PCR amplification, bound to magnetic streptavidin, magnetically pelleted, 
washed, and cut with FokI, generating a new set of templates corresponding 
to the previous set of templates but with each strand shortened by nine 
nucleotides when compared to the prior corresponding template. 
PCR amplification is preferably carried in such a way as to limit "noise." 
This may be accomplished by amplifying only a small portion of each 
ligation mixture to prevent successive exponential PCR amplifications from 
generating an accumulation of products during successive sequencing 
cycles. Obtaining a small aliquot from each ligation mixture for PCR 
amplification is performed in an automated fashion by device 20, and this 
can be accomplished by one of several techniques: removal or retention of 
an aliquot of the ligation mixture. 
Removal of an aliquot for PCR amplification may be done by use of a 
dispersible solid phase, such as magnetic streptavidin. In a microtiter 
plate embodiment a subassembly such as a spotting robot that uses a pin 
transfer device may be used to transfer a small aliquot from each site on 
the microtiter plates as reported in the above-cited Meir-Ewert et al. 
article. When using a chip, a small aliquot can be removed by using an 
analogous hedgehog comb device as reported in Rosenthal A, O Coutelle, M 
Craxton. Large-scale production of DNA sequencing templates by microtitre 
format PCR. Nucleic Acid Research 1993; 21: 173-174, or by using a blotter 
to retain a small portion from each of the sample sites, followed by 
washing out of the remaining contents. PCR amplification is then performed 
using these retained aliquots as the templates. Other methods for 
retaining a small aliquot can be implemented such as a low intensity 
magnetic separation, or by using a chip with chambers shaped or positioned 
in relation to the flow path to retain a small aliquot by mechanical means 
when supernatant is removed (e.g. with a lip). 
Alternatively, to prevent the accumulation of PCR product during successive 
sequencing cycles, the automated device may be operated to retain only a 
small amount of each PCR product for subsequent steps. This can be done by 
using a streptavidin coated manifold as reported in Lagerkvist A, J 
Stewart, M Lagerstrom-Fermer, U Landegren. Manifold sequencing: Efficient 
processing of large sets of sequencing reactions. Proc. Natl. Acad. Sci. 
USA 1994; 91: 2245-2249 and inserting the manifold into the amplification 
mixture to bind a small proportion of the biotinylated PCR products. In 
this case, the manifold-bound DNA segments are then moved to and dipped 
into individual reagents in subsequent steps, rinsing the manifold with 
wash buffer between steps, so that while PCR amplification occurs in the 
chip, other steps are carried using DNA segments that are bound to the 
manifold. 
Removal or retention of an aliquot may also be effected by using a 
cleavable linkage, e.g. a chemically- or photo-cleavable linkage arm such 
as reported in Dawson B A, T Herman, J. Lough: Affinity isolation of 
transcriptionally active murine erythroleukemia cell DNA using a cleavable 
biotinylated nucleotide analog. Journal of Biological Chemistry 1989; 264: 
12830-12837, and Olejnik J, E Krzymanska-Olejnik, K J Rothschild: 
Photocleavable biotin phosphoramidite for 5 '-end-labeling, affinity 
purification and phosphorylation of synthetic oligonucleotides. Nucleic 
Acids Research 1996; 24: 361-366. In this case the cleavable linkage is 
employed for a portion, e.g. a small fraction, of the linkages used to 
attach the ligated DNA to the solid support or matrix. Cleavage then 
releases only the cleavably-bound DNA, permitting removal of a controlled 
portion of the DNA products. The PCR process may also be controlled by 
rendering much of the DNA product inaccessible to primer anealing and 
extension, for example by binding the DNA to a non-dispersible solid 
matrix or by pelleting a dispersible matrix. This takes advantage of the 
observation that immobilization of a nucleic acid component during PCR 
amplification reduces the efficiency of DNA amplification during solid 
phase PCR. Kohsaka H, D A Carson. Solid Phase Polymerase chain reaction. 
Journal of Clinical Laboratory Analysis 1994;8:452-455. 
FIG. 8 illustrates the reagent supply section 30 of the device to also 
contain DNA polymerase and ddNTPs. These have not been mentioned in the 
above description, but are used in the sequencing methods of Embodiments 3 
and 4 described above with relation to FIGS. 3 and 4, using labeled 
ddNTPs. In the method of FIG. 3, the automated apparatus is operated so 
that following FokI digestion, magnetic binding, and washing, the DNA 
templates are incubated with a DNA polymerase and the four nucleotide 
terminators, each with a unique label. Following magnetic binding and 
washing, the incorporated label at each address is identified using the 
charge coupled device or other detector and, as before, the readings are 
passed as ordered information to the microprocessor data handler to note 
the additional nucleotide or nucleotides read at each site. Then, an 
adaptor is ligated to each of the templates. This is followed by PCR 
amplification which regenerates the next set of template precursors for 
the next sequencing cycle. 
The above described automated process is highly efficient. By using unique 
adaptors and corresponding amplification primers during each sequencing 
cycle, about twenty sequencing cycles can be carried out, resulting in the 
sequencing of 180 nucleotides, of which typically at least 160 nucleotides 
will lie outside the primer in the end being sequenced. Thus, providing 
these DNA segments do not contain an internal FokI recognition domain, the 
above-described steps will generate 1,600,000 nucleotides of new sequence 
from a single 100.times.100 well chip. Since the FokI recognition domain 
has a five bp recognition domain, it is predicted to occur approximately 
every 1000 bp (4.sup.5 =1024) in random sequence. If the average size of 
each amplified fragment lying between the amplifying primers is 300 bp, 
then about 30% of the DNA segments to be sequenced will contain an 
internal FokI site and will not be successfully sequenced using only this 
simple protocol. Thus, in DNA sequences with a random distribution of 
equal numbers of GGATG nucleotides, about 70% of the fragments will be 
successfully sequenced, resulting in the sequencing of approximately 
1,120,000 nucleotides rather than 1,600,000. 
This processing obstacle imposed by pre-existing FokI recognition domains 
may be addressed by hemi-methylating these recognition domains. The 
methods described in FIGS. 1 and 3 do not provide for the hemi-methylation 
of those FokI recognition domains that lie outside the adaptor encoded 
domain. Prior studies such as Looney M C, L S Moran, W E Jack, G R 
Feehery, J S Benner, B E Slatko, G G Wilson. Nucleotide sequence of the 
FokI restriction-modification system: Separate strand-specificity domains 
in the methyltransferase. Gene 1989; 80: 193-208 have shown that 
hemimethylation of the FokI recognition domain prevents cutting from being 
mediated by these domains. However, since each strand of the FokI 
recognition domain contains all four nucleotides, the PCR based method 
described by Padgett and Sorge in Padgett K A, J A Sorge. Creating 
seamless junctions independent of restriction sites in PCR cloning. Gene 
1996; 168: 31-35 cannot be used to hemi-methylate such internal sites. 
Rather, when carrying out the invention with FokI, hemi-methylation 
requires the use of the method of Han and Rutter described in Han J, 
Rutter W J. .lambda.gt22S, a phage expression vector for the directional 
cloning of cDNA by the use of a single restriction enzyme SfiI. Nucleic 
Acids Res 1988; 16: 11837 as noted above. 
The method is thus augmented by the following step: Following PCR 
amplification, binding to streptavidin and magnetic pelleting, the 
non-biotinylated strand is removed by denaturation and magnetic pelleting, 
followed by washing to remove reagents and primers. Since FokI cutting 
requires a double-stranded recognition domain, as reported by Podhajska A 
J, W Szybalski. Conversion of the Fok I endonuclease to a universal 
restriction enzyme: Cleavage of phage M13mp7 DNA at predetermined sites. 
Gene 1985; 40: 175-182, this site is recreated, and the internal FokI 
sites are hemi-methylated, by using a primer containing the FokI 
recognition domain. This primer is complementary to the lower stand of the 
ligated sequencing adaptor up to the degenerate or universal nucleotides 
e.g., through the adenine moiety in the FokI recognition domain, as shown, 
and polymerization occurs using four nucleotides except that 
N6-methyl-dATP substituted for dATP. This process thus regenerates the 
adaptor encoded FokI recognition domain and hemimethylates those 
recognition domains that lie internal to the sequencing adaptor encoded 
domain. The DNA segments, once hemi-methylated, are then sequenced by the 
automated steps described above. 
The invention contemplates a number of practical implementations of novel 
chip-based support arrays for carrying out the described steps in an 
automated manner. 
Chips that house 50,000 DNA segments can be generated by microfabrication 
of microchambers using photolithography following the approaches and 
teachings of Wilding P, M A Shoffner, L J Kricka. PCR in a silicon 
microstructure. Clinical Chemistry 1994; 40: 1815-1818; of Kikuchi Y, K 
Sato, H Ohki, T Kaneko. Optically accessible microchannels formed in a 
single-crystal silicon substrate for studies of blood rheology. 
Microvascular Research 1992; 44: 226-240; of Woolley A T, R A Mathies. 
Ultra-high-speed DNA fragment separations using microfabricated capillary 
array electrophoresis chips. Proc. Natl. Acad. Sci. USA 1994; 91: 
11348-11352; of Baxter G T, L J Bousse, T D Dawes, J M Libby, D N Modlin, 
J C Owicki, J W Parce. Microfabrication in silicon microphysiometry. Clin. 
Chem. 1994; 40: 1800-1804; of Kricka L J, X Ji, O Nozaki, P Wilding. 
Imaging of chemiluminescent reactions in mesoscale silicon-glass 
microstructures. J. Biolumin. 1994; 9: 135-138; or may be fabricated using 
molded or etched polymers as described by Matson R S, J Rampal, S L Jr. 
Pentoney, P D Anderson, P Coassin. Biopolymer synthesis on polypropylene 
supports: Oligonucleotide arrays. Analytical Biochemistry 1995; 
224:110-116. Alternatively, chip addresses may be separated by hydrophobic 
borders which may, for example, be implemented with conventional sample 
cell construction techniques or formed by processes of lithography and 
chemical treatment. Movement of the reagents to and from this chip can be 
done using pumps as reported in Burns M A, C H Mastrangelo, T S Sammarco, 
F P Man, J R Webster, B N Johnson, B Foerster, D Jones, Y Fields, A R 
Kaiser, D T Burke. Microfabricated structures-for integrated DNA analysis. 
Proc. Natl. Acad. Sci. USA 1996; 93: 5556-5561 and in Wilding P, J 
Pfahler, H H Bau, J N Zemel, L J Kricka. Manipulation and flow of 
biological fluids in straight channels micromachined in silicon. Clinical 
Chemistry 1994; 40: 43-47. Alternatively, fluids may be brought to the 
sites by centrifugal force. 
In this case the overall requirements for conduits, valves and wash-out 
passages may be substantially reduced, as it is only necessary to supply 
each reagent or solution to a central position communicating with the 
array. The array itself may mount in a shallow tray or cover assembly 
which effectively channels the flow to the array sites. In general, the 
sequencing method of the invention does not require the transfer of small 
amounts of liquids through capillaries, and therefore avoids many of the 
technological obstacles resulting from shearing forces encountered in low 
diameter capillary flow, as reported in Wilding P, J Pfahler, H H Bau, J N 
Zemel, L J Kricka. Manipulation and flow of biological fluids in straight 
channels micromachined in silicon. Clinical Chemistry 1994; 40: 43-47. 
FIG. 9 shows an embodiment of a system 100 in which movement of reagents 
onto chips is effected by centrifugal force. In this device, the chips 10' 
are on a turntable. Reagents are placed closer to the center of the 
turntable, and rotating the turntable drives the reagents radially outward 
directly to one or more chips. Centrifugal force also allows reagents to 
be removed from chips. A chip or chip holder itself is preferably 
configured for flow-through operation to simplify and enhance the removal 
of reagents (see, e.g., Beattie K L, W G Beattie, L Meng, S L Turner, R 
Coral-Vazquez, D D Smith, P M McIntyre, D D Dao. Advances in genosensor 
research. Clinical Chemistry 1995; 41: 700-706). 
In the device 110, illustratively set up for the processes described 
herein, nine support arrays 11a, 11b, . . . 11i are located around a 
rotating stage with each communicating at a radially innermost corner with 
a corresponding flow supply conduit 12a, 12b, . . . 12i. Outlets (not 
shown) may be to a common drain. Thus each support array in this device 
embodiment may receive a separate set of reagents. For example, the nine 
arrays may be initially loaded with identical DNA samples in each 
respective well, and then all samples in an array processed to produce 
templates offset by a fixed x, with x={1,2 . . . 9} different for each 
array. Once the nine sets of templates on the corresponding supports have 
been created, running the sequencing process steps of the present method 
then produces a continuous nucleotide sequence for each of the initial 
segments. 
When performing the amplification steps, during incubations, the magnetic 
streptavidin bound DNA can be suspended by shaking or by magnetic 
oscillation as described in the Product information on MixSep.sup.c. 
Sigris Research, Inc. Brea, Calif. To retain a small portion of the 
magnetic particles prior to the addition of PCR reagents and PCR 
amplification, the magnetic pelleting can be adjusted electrically. In the 
chip embodiment, PCR thermal cycling is very efficient, since heat 
transfer occurs rapidly over short distances. The thermal cycler can be a 
Peltier heater-cooler device built into the stage, a set of fixed 
temperature plates or baths which are successively placed in thermal 
contact with the chips, or an air oven (see, for example, Meier-Ewert S, E 
Maier, A Ahmadi, J Curtis, H Lehrach. An automated approach to generating 
expressed sequence catalogues. Nature 1993; 361: 375-376; Drmanac S, R 
Drmanac. Processing of cDNA and genomic kilobase-size clones for massive 
screening, mapping, and sequencing by hybridization. BioTechniques 1994; 
17: 328-336; Wilding P, M A Shoffner, L J Kricka. PCR in a silicon 
microstructure. Clinical Chemistry 1994; 40: 1815-1818; and Shouffner M A, 
J Cheng, G E Hvichia, L J Kricka, P Wilding. Chip PCR. I. Surface 
passivation of microfabricated silicon-glass chips for PCR. Nucleic Acid 
Research 1996; 24: 375-379. Reading the identity of incorporated label can 
be carried out using a charge coupled device, as described above, or using 
a fluorescent microscope, fiber-optic detectors, biosensors, gas phase 
ionization detector, or a phosphorimager as described in Kinjo M, R 
Rigler. Ultrasensitive hybridization analysis using fluorescence 
correlation spectroscopy. Nucleic Acid Research 1995; 23: 1795-1799; Mauro 
J M, L K Cao, L M Kondracki, S E Walz, J R Campbell. Fiber-optic 
fluorometric sensing of polymerase chain reaction-amplified DNA using an 
immobilized DNA capture protein. Analytical Biochemistry 1996; 235: 61-72; 
Nilsson P, B Persson, M Uhlen, P Nygren. Real-time monitoring of DNA 
manipulations using biosensor technology. Analytical Biochemistry 1995; 
224: 400-408; Eggers M, D Ehrlich. A review of microfabricated devices for 
gene-based diagnostics. Hematologic Pathology 1995; 9: 1-15. 
Even without special biochip microfabrication, the methods of the present 
invention are advantageously implemented in a device that operates in a 
microtiter plate format. In this case the construction of the 
subassemblies for the scintillation counting of multi-well microtiter 
plates and for the automated picking of colonies into the wells, as well 
as the necessary reagent introduction and thermal cycling to amplify DNA 
simultaneously in multiple multi-well microtiter plates, allows the 
simultaneous amplification, treatment and reading of the array of samples. 
Indeed, with prior art subassemblies handling 120 plates, each with 384 
wells, 46,080 samples may be processed simultaneously. Therefore, the 
sequencing protocol estimated to sequence 160 nucleotides in a clone 
insert would sequence simultaneously 204,800 nucleotides from 1280 clones 
using a single 120 plate thermal cycler, 384 well scintillation counter, 
one radiolabel, a 384 pin transfer device (e.g., a hedgehog comb) and a 
robotic pipetter. 46,080 wells/9 initial templates=5120 samples; 5120/4 
ligations=1280 samples (clones). 1280 clones .times. 160 
nucleotides/clone=204,800 nucleotides!. (Meier-Ewert S, E Maier, A Ahmadi, 
J Curtis, H Lehrach. Nature 1993; 3631:375-376.) 
With the foregoing overview of the organization of a method and apparatus 
for large scale or multiplex processing of collections of segments, a 
detailed description will now be given of several embodiments of the 
sequencing method as applied to a single segment. 
This invention is further illustrated by the following Exemplification 
which should not be construed as limiting. The contents of all references 
and published patents and patent applications cited throughout the 
application are hereby incorporated by reference. 
Exemplification 
Experimental Strategy 
The present invention allows one to sequence numerous DNA segments in 
parallel without running a gel. It is an iterative method that allows one 
to sequence DNA in fixed intervals of greater than one nucleotide, and 
provides a means for regenerating the desired DNA segment following each 
iterative cycle. This is accomplished by the iterative application of a 
DNA ligase and an enzyme, e.g., a class-IIS restriction endonuclease, to 
generate templates for DNA sequencing. One simple schematic is outlined 
below. 
##STR2## 
In each cycle, adaptor ligation to one end of the DNA segment is followed 
by class-IIS restriction endonuclease cutting. The recognition domain of 
the class-IIS restriction endonuclease is encoded by the ligated adaptor, 
allowing restriction endonuclease digestion to trim the DNA segment, 
generating a new overhang sequence. One or both strands of an adaptor can 
be ligated, or one or both ends of a single-strand hairpin adaptor can be 
ligated. Also, one strand of an adaptor can be ligated followed by 
hybridization, without ligation of the complementary strand, to generate a 
double-stranded recognition domain. Iterative cycles generate a series of 
single-strand overhangs, each constituting a DNA template. The 
single-stranded overhangs are separated by fixed intervals that are 
limited by the distance between the recognition domain and the cut site in 
the cleavage domain for the class-IIS restriction endonuclease encoded by 
the ligated adaptor. This method exploits the separation of the cleavage 
domain and the recognition domain of class-IIS restriction endonucleases 
by allowing the sequencing in strides limited only by the distance between 
the recognition domain and the cleavage domain cut sites, distinguishing 
it from other iterative approaches. Since each DNA template is a short 
single-stranded region attached to double-stranded DNA, these 
single-strands have little opportunity to form secondary structures, 
providing a considerable advantage over competing methods. 
The overhang generated after each cycle constitutes a DNA template that is 
sequenced in one of a variety of ways. One way uses template-directed DNA 
ligation to discriminate between nucleotides at the ligation junction, 
allowing this ligation to generate sequence information. This is 
illustrated below: 
##STR3## 
Successful ligation requires that an adaptor's single-stranded end be 
complementary to the double-stranded DNA's single-stranded overhang 
sequence at the ligation junction. Four adaptors (or adaptor subsets) are 
used during each ligation, with each of the four adaptors differing at the 
nucleotide positioned to undergo ligation at the template-directed 
ligation junction. Ligation to one of the four adaptors and identification 
of that adaptor allows identification of the nucleotide at the ligation 
junction, thus generating sequence information. Sequencing can be 
accomplished by fluorometry using adaptors tagged with distinct 
fluorescent labels. This is followed by class-IIS restriction endonuclease 
mediated end trimming of the DNA using the recognition domain encoded by 
the ligated adaptor. This recognition domain is positioned so that 
cleavage results in the removal of nucleotides from each strand of the 
DNA, creating a new template for subsequent template-directed ligation to 
one of four adaptors or adaptor subsets. This strategy can use an enzyme, 
e.g., a class II-S restriction endonuclease, that generates either a 5' or 
a 3' overhang sequence, as either type of overhang can serve as a template 
for template-directed ligation. 
Another approach uses template-directed polymerization instead of 
template-directed ligation to sequence DNA. In this case, adaptor ligation 
can be template-directed but is not used to discriminate between 
nucleotides at the ligation junction. Sequencing occurs through a separate 
template-directed DNA polymerization step. In order to use 
template-directed polymerization to sequence the overhang sequence, the 
overhang must be a 5' overhang, since template-directed polymerization 
requires a recessed 3' end. A simple schematic of this approach is 
outlined below. 
##STR4## 
Ligation can be template-directed, occurring using an adaptor with a 
promiscuous nucleotide or nucleotides (degenerate or universal) at the 
ligation junction, so that this ligation is not used to discriminate 
between nucleotides at the ligation junction, and therefore does not 
generate sequence information. Ligation of the adaptor is followed by 
class-IIS restriction endonuclease trimming, generating a 5' overhang 
sequence. The 5' overhang has a recessed 3' end, forming a substrate for 
template-directed DNA polymerization. Template-directed polymerization 
occurs in the presence of each of the four labeled nucleotide terminators 
(e.g. ddNTPs). These nucleotide terminators can each have distinct 
fluorescent tags, so that following incorporation of one of these labeled 
nucleotide terminators, a fluorometer can identify the incorporated 
nucleotide (Prober J M, Trainor G L, Dam R J, Hobbs F W, Robertson C W, 
Zagursky R J, Cocuzza A J, Jensen M A, Baumeister K., Science 1987; 
238:336-341). Iterative cycles of adaptor ligation and IIS cutting create 
new templates for sequencing by template-directed polymerization. 
One obstacle inherent in iterative methods that generate a product is that 
even if the constituent enzymatic steps approach 100% completion, 
incompletely processed products can accumulate to significant levels. For 
example, during oligonucleotide synthesis of a 70-mer, requiring 69 
couplings, a 99% coupling efficiency results in only 50% of the generated 
oligonucleotides being full length (0.99.sup.69 =0.50). The present 
invention eliminates this problem by allowing one to sequence in intervals 
of greater than one nucleotide. For example, the FokI recognition domain 
is separated from its cleavage domain by nine nucleotides. Using a FokI 
based protocol, single-strand overhangs can be generated in each cycle 
that are separated by nine nucleotide long intervals over time and space, 
so that five cycles will allow one to span 45 nucleotides, instead of just 
five nucleotides using an iterative method that sequences consecutive 
nucleotides (e.g. the base addition DNA sequencing scheme). This is termed 
striding, as it covers a considerable stretch of DNA with few iterative 
steps. Sequencing single nucleotides in intervals of greater than one 
nucleotide requires the sequencing of the nucleotides that fall within 
each interval. One sequencing method generates DNA templates separated by 
intervals of nine nucleotides, and sequences a single nucleotide in each 
template, by making nine initial templates for each DNA segment being 
sequenced, such that sequencing these nine initial templates will sequence 
nine adjacent nucleotides. The nine initial templates can be generated by 
ligating one end of each DNA segment to be sequenced to nine distinct 
adaptors in nine separate ligations, each adaptor containing a FokI 
recognition domain, with these domains offset from each other by one base 
pair when comparing adjacently positioned recognition domains. In one 
embodiment, the DNA segment to be sequenced is generated by PCR 
amplification, and offset recognition domains are incorporated during PCR 
amplification by encoding the recognition domain into one of the 
amplifying primers according to the method of Mullis K, Faloona F, Scharf 
S, Saiki R, Horn G, Erlich H., Cold Spring Harbor Symposia on Quantitative 
Biology, Cold Spring Harbor Laboratory, LI:263-273. When the DNA samples 
to be sequenced are vector inserts, as in a genomic or cDNA library, a set 
of initial template precursors can be generated for each DNA insert to be 
sequenced using a single set of initial adaptors. For example, following 
digestion with a restriction endonuclease that cuts the vector adjacent to 
each insert, offset recognition domains can be appended to each of the 
numerous vector inserts through ligation to each of the initial adaptors. 
This can be followed by PCR, to seal nicks and retrieve the product. An 
alternative approach is to use PCR alone to generate offset recognition 
domains. For example, when sequencing DNA libraries, primers can be 
designed to anneal to a vector sequence immediately flanking each insert. 
Once this set of DNA segments with offset (i.e., staggered) recognition 
domains is generated for each DNA segment to be sequenced, these DNA 
segments can be sequenced concurrently, so that the number of steps 
necessary to sequence a contiguous stretch of DNA in the original DNA 
segment is markedly reduced. Using any of the above approaches, only a few 
primers must be made to sequence numerous vector inserts. Furthermore, 
each of the nine products can have a uniquely positioned recognition 
domain, so that digestion with FokI cleaves both strands of each DNA 
segment and generates a set of nine overhang sequences positioned as a 
staggered array separated by one base pair. Generating several initial DNA 
templates for each DNA segment to be sequenced diminishes the number of 
successive steps necessary to sequence a given stretch of DNA, and 
therefore significantly diminishes the accumulation of background signal 
when sequencing over a given span of DNA. 
In order to regenerate the product of interest following each cycle of 
restriction endonuclease digestion and adaptor ligation, an additional 
step is designed. Specifically, this invention uses adaptor ligation 
during each sequencing cycle. These ligated adaptors can differ during 
each cycle (or every several cycles), allowing the product generated 
following each cycle of restriction endonuclease digestion and 
template-directed ligation to have a unique end created by the ligated 
adaptor. This unique end can generate a primer annealing site during PCR, 
such that PCR can amplify the desired product over a million fold 
following each adaptor ligation step (Saiki R K, D H Gelfand, S Stoffel, S 
J Scharf, R Higuchi, G T Horn, K B Mullis, H A Erlich, Science 1988; 
239:487-491). Nucleic acid amplification in vitro can be exponential, as 
is usually done, or linear, in which one primer undergoes one or more 
cycles of primer extension, followed by its removal and cycles of single 
primer extension using the opposite primer. This in vitro amplification 
step replenishes the desired product (some product is inevitably lost in 
prior steps), and prevents uncut products or unligated products from 
generating background signal. It also regenerates the template precursor 
by eliminating base mismatches, nicks, and displaced ends lying between 
the recognition domain and the cleavage domain following adaptor ligation. 
Thus, cutting efficiencies need not approach 100%; this method allows one 
to use lower concentrations of restriction endonuclease that preferably 
cut with very high specificity (&gt;99.9%) for the canonical recognition 
domain (Fuchs R, R Blakesley, Methods in Enzymology 1983; 100:3-38). 
Furthermore, this method works well even when DNA ligation is inefficient, 
as when ligating fragments with a single nucleotide overhang, because the 
desired template precursor can be readily amplified over one million fold 
using PCR amplification. Also, following fill-in with labeled ddNTPs, even 
if the label interferes with ligation, only a fraction of those filled in 
would need to be labeled, as product regeneration through amplification in 
vitro does not require a large proportion of the filled-in product to 
undergo efficient ligation. The remaining product could either not undergo 
fill-in (in the presence of low numbers of labelled ddNTPs) or undergo 
fill-in in the presence of unlabelled ddNPTs (along with labelled ddNPTs). 
When using nucleic acid amplification in vitro to re-generate each 
template-precursor, the adaptor does not need to have a double-stranded 
recognition domain, as the recognition can be encoded by an adaptor 
containing only a single-strand of the recognition domain, with the 
double-stranded recognition domain generated during the nucleic acid 
amplification in vitro. 
In one embodiment, recognition domains for the class-IIS restriction 
endonuclease used to generate the DNA templates that occur in the original 
DNA segment (internal to the ligated adaptor), are methylated or otherwise 
blocked to prevent cutting mediated by these internal domains. Blocking of 
internal recognition domains can be accomplished by treatment with the 
corresponding methylase (Fok I methylase for Fok I restriction 
endonuclease (Kita K, H Kotani, H Sugisaki, M Takanami, J Biol Chem 
1989;264:5751-5756, Looney M C, L S Moran, W E Jack, G R Feehery, J S 
Benner, B E Slatko, G G Wilson, Gene 1989; 80:193-208), prior to adaptor 
ligation. This prevents cutting mediated by these internal recognition 
domains, without preventing cleavage directed by the ligated adaptor 
(whose recognition domain is not methylated). 
Hemi-methylation of these internal recognition domains can be carried out 
using the strategy of Han and Rutter or using the PCR-based strategy of 
Padgett and Sorge, as described in more detail herein (Han J. Rutter W J. 
Nucleic Acids Res 1988; 16:11837, Padgett K A, J A Sorge, Gene 1996; 
168:31-35). Each strategy hemi-methylates, and effectively blocks, 
internal recognition domains without methylating the primer-encoded 
recognition domain. The method of Padgett and Sorge cannot be used if each 
strand of the chosen recognition domain contains all four nucleotides, 
because PCR amplification cannot be carried out with selective methylation 
of those recognition domains that lie outside of the primer encoded 
recognition domain, as the strand antisense to the primer's recognition 
domain will be hemi-methylated during PCR. The method described by Han and 
Rutter can hemi-methylate the internal recognition domains regardless of 
the nucleotide composition of each strand of the recognition domain, and 
it can be incorporated into a linear amplification step. 
The PCR-based method of Padgett and Sorge has the advantage of allowing the 
simultaneous exponential amplification of the product of interest along 
with hemi-methylation of the internal recognition domains. This is 
accomplished by amplification with a methylated nucleotide that does not 
lie within the sequence antisense to the recognition domain sequence in 
the amplifying primer, and can be carried out using ligated adaptors and 
amplifying primers that vary during each cycle (or every several cycles) 
as described. In this case, however, the 3' end of each amplifying primer 
must encode at least a portion of the restriction endonuclease recognition 
domain of the class-IIS restriction endonuclease used to trim the DNA 
segment. This may diminish the specificity of the PCR amplification for 
the product of interest, as these shared 3' ends may result in some 
amplification of uncut DNA products. The strategy of Han and Rutter can be 
modified to linearly amplify the product of interest, while simultaneously 
hemi-methylating the internal recognition domains. This can be carried out 
by iterative primer extensions using the primer encoding at least a 
portion of the recognition domain, with a methylated nucleotide 
substituting for its normal counterpart, before or after reiterative 
primer extensions with the opposite primer using the four normal dNTPs. 
Any of the above strategies for hemi-methylating internal recognition 
domains can be carried following in vitro amplification of the product of 
interest, and such prior in vitro amplification could occur through PCR or 
a related method, such as strand displacement amplification (Walker G T, M 
S Fraiser, J L Schram, M C Little, J G Nadeau, D P Malinowski Nucleic 
Acids Research 1992; 20:1691-1696). Such prior DNA amplification in vitro 
need not have a portion of the recognition domain incorporated into any of 
the amplifying primers, allowing exquisite specificity during product 
regeneration. 
EXAMPLE 1 
Demonstration of Interval Sequencing Mediated by Class-IIS Restriction 
Endonuclease Generated 5' Overhangs and Template-Directed Ligation 
Using a FokI based strategy, single nucleotides separated by intervals of 
nine nucleotides were sequenced using simple reagents and a scintillation 
counter. The initial template precursor was a 93 bp PCR product containing 
a portion of the Cystic Fibrosis Transmembrane Conductance Regulator gene 
that had been amplified directly from human genomic DNA. Sequencing was 
accomplished by template-directed ligation using six sequencing cycles. 
Following sequencing of the first nucleotide, five additional nucleotides 
were sequenced at nine nucleotide intervals, so that the sequencing 
covered a span of 46 nucleotides (1+(5.times.9)=46). The non-biotinylated 
primer used to generate the template precursor contained a recognition 
domain for FokI. The opposite primer had a biotinylated 5' end, and was 
used to bind the template precursor to magnetic streptavidin beads. Use of 
magnetic streptavidin beads allowed enzymatic reactions to occur in 
solution, and facilitated removal of a small aliquot for each PCR 
amplification step during the sequencing cycles. During the sequencing 
cycles, only two sets of adaptors were used, and each unique PCR 
amplifying primer used during the sequencing cycles was identical to the 
upper strand of the previously used adaptor, so that these unique 
amplifying primers contained the FokI recognition domain in their 3' ends, 
minimizing the number of oligonucleotides synthesized. In this protocol, 
identification of a nucleotide during each sequencing cycle took place 
using four ligation reactions (for the single template precursor). In each 
ligation, all four adaptors were present, with the 3' end of a different 
one of the four adaptors in each ligation tagged with .sup.35 S. 
Quantitation of retained .sup.35 S radiolabel was carried out using a 
scintillation counter, and a dominant signal for the correct nucleotide 
was clearly detected during each cycle. The details are outlined below: 
Sequencing Adaptor Generation 
Adaptor set #1 (lower strands of this adaptor set are shown in the box 
below) was generated as follows: 6.3 .mu.l of the lower strand of the 
first three of the four adaptors (100 pmole/.mu.l) were added, in three 
separate reactions (one for each oligonucleotide) to 4.4 .mu.l H.sub.2 O, 
3.3 .mu.l 5.times. Terminal deoxynucleotidyl transferase buffer (500 mM 
cacodylate buffer, pH 6.8, 5 mM CoCl.sub.2, 0.5 mM DTT); 1.3 .mu.l 
Terminal deoxynucleotidyl transferase (20 U/.mu.l; Promega, Madison Wis.) 
and 1.0 .mu.l .sup.35 S!ddATP (12.5 .mu.Ci/.mu.l). The final 
oligonucleotide was processed as described above, except that half amounts 
were used. All of the samples were incubated at 37.degree. C. for one hour 
followed by heat inactivation at 70.degree. C. for 10 minutes, resulting 
in a final volume of 16.3 .mu.l for the first three labeled 
oligonucleotides, and a final volume of 8.2 .mu.l for the final labeled 
oligonucleotide (with the 5' G). 
5'P-CNNNCATCCGACCCAGGCGTGCG (SEQ ID NO:1) or 
5'P-ANNNCATCCGACCCAGGCGTGCG (SEQ ID NO:2) 
or 5'P-TNNNCATCCGACCCAGGCGTGCG (SEQ ID NO:3) or 
5'P-GNNNCATCCGACCCAGGCGTGCG (SEQ ID NO:4); only the 5' end varies between 
these four oligonucleotides, and this nucleotide is underlined; the FokI 
recognition sequence is in bold type; N represents nucleotides with 4-fold 
degeneracy. 
The 16.3 .mu.l of each of the first three labeled oligonucleotides were 
separately added to 2.5 .mu.l 10.times. T.sub.4 DNA Ligase buffer (660 mM 
Tris-HCl, 50 mM MgCl.sub.2, 10 mM dithioerythritol, 10 mM ATP, pH 7.5) and 
to 6.2 .mu.l of the upper strand of the sequencing adaptor (100 
pmole/.mu.l): 
5'-CGCACGCCTGGGTCGGATG (SEQ ID NO:5); the FokI recognition sequence is in 
bold type. 
The last labeled oligonucleotide (with the 5' G) was processed as described 
above, except in half amounts, resulting in a final volume of 25 .mu.l for 
each of the first three adaptors and 12.5 .mu.l for the final adaptor. 
Non-radiolabeled counterparts to the above four adaptors were generated by 
adding 20.0 .mu.l (100 pmole/.mu.l) of each of the first three lower 
strands, separately to 20.0 .mu.l (100 pmole/.mu.l) of the upper strand, 
8.0 .mu.l of 10.times. T.sub.4 DNA Ligase buffer and 32 .mu.l H.sub.2 O, 
for a final volume of 80 .mu.l, and 10.0 .mu.l (100 pmole/.mu.l) of the 
final lower strand (with the 5' G) was added to half amounts of the above 
constituents, for a final volume of 40 .mu.l. Each of the eight sets of 
adaptors (four radiolabeled and four non-radiolabeled) were incubated at 
93.degree. C. for 30 seconds followed by annealing at 25.degree. C. for 5 
minutes. The radiolabeled final adaptor (with the 5' G) was added to 12.5 
.mu.l H.sub.2 O, to bring the final volume to 25 .mu.l, like the other 
radiolabeled adaptors, and the 40 .mu.l of the non-radiolabeled final 
adaptor was added to 40 .mu.l H.sub.2 O, to bring the final volume to 80 
.mu.l, like the other non-radiolabeled adaptors. Each adaptor with a 5' G 
was at half the concentration of the other adaptors based on ligation data 
from preliminary experiments. 
Each radiolabeled adaptor was added to 25 .mu.l of the non-radiolabeled 
adaptors with the other three 5' ends. This resulted in four adaptor #1 
mixes, each with one radiolabeled adaptor and the remaining three 
non-radiolabeled adaptors. Using four ligation mixtures allows one to 
sequence nucleotides using a single label and a simple detection apparatus 
(e.g. a scintillation counter). 
Adaptor set #2 was made the same way as adaptor set #1, except that the 
four oligonucleotides for the lower strands of the adaptors were: 
5'P-CNNNCATCCTCTGGGCTGCACGGG (SEQ ID NO:6) or 
5'P-ANNNCATCCTCTGGGCTGCACGGG (SEQ ID NO:7) or 
5'P-TNNNCATCCTCTGGGCTGCACGGG (SEQ ID NO:8) or 
5'P-GNNNCATCCTCTGGGCTGCACGGG (SEQ ID NO:9); 
only the 5' end varies between each of these four oligonucleotides, and 
this nucleotide is underlined; the FokI recognition sequence is in bold 
type; N represents nucleotides with 4-fold degeneracy. 
and the oligonucleotide for the upper strand of the adaptors was: 
5'-CCCGTGCAGCCCAGAGGATG (SEQ ID NO:10); the FokI recognition sequence is in 
bold type. 
Initial Sequencing Template Generation 
PCR amplification of a 93 bp initial template precursor from human genomic 
DNA was carried out using primers A and B (shown in the box below) as 
follows: 200 ng human genomic DNA (Promega, Madison Wis.) in 2.0 .mu.l was 
placed with 41.6 .mu.l H.sub.2 O, 6.0 .mu.l 10.times. buffer (100 mM 
Tris-HCl pH 8.3, 1.0M KCl, 0.5% Tween 20, 50% Glycerol), 4.0 .mu.l 
containing 5.0 mM each dNTP (100 mM stock (Boehringer Mannheim, 
Indianapolis, Ind.) diluted in H.sub.2 O), 1.0 .mu.l Primer A (25 
pmole/.mu.l), 1.0 .mu.l Primer B (25 pmole/.mu.l), 4.4 .mu.l 25 mM 
Mg(OAc).sub.2, in each of four microcentrifuge tubes. A wax bead was added 
(Perkin Elmer, Foster City, Calif.) and the tubes were heated to 
80.degree. C. for 3 minutes and then cooled to 25.degree. C. An upper 
layer of reagents consisting of 35.0 .mu.l H.sub.2 O, 4.0 .mu.l 10.times. 
buffer and 1.0 .mu.l rTth DNA Polymerase (2.5 .mu.l; Perkin Elmer) was 
placed on top of each wax bead, and the four tubes underwent an initial 
denaturation step at 94.degree. C. for 1 minute followed by 30 thermal 
cycles using the following parameters (94.degree. C. for 30 seconds, 
50.degree. C. for 30 seconds), a final extension at 72.degree. C. for 7 
minutes, and a 4.degree. C. soak. 
Primer A: GTTTTCCTGGATGATGCCCTGGC (SEQ ID NO:11); mismatch to genomic DNA 
underlined; FokI recognition sequence in bold type. Primer B: 5' 
Biotin-CATGCTTTGATGACGCTTCTGTATC (SEQ ID NO:12); the biotinylated 5' end 
was generated during oligonucleotide synthesis using a biotin 
phosphoramidite (Glenn Research, Sterling Va.). 
The samples were combined, and 360 .mu.l of this product was incubated with 
4.0 .mu.l Exonuclease I (20 U/.mu.l; Epicentre, Madison Wis.) at 
37.degree. C. for 30 minutes, followed by heat inactivation at 80.degree. 
C. for 15 minutes. The sample was purified by glass bead extraction using 
Mermaid (BIO101, La Jolla, Calif.) and was suspended in 90 .mu.l TE (10.0 
mM Tris-HCl pH 8.0, 1.0 mM EDTA). Eighty .mu.l of this product was 
digested with 5.0 .mu.l FokI (3 U/.mu.l; Boehringer Mannheim) in the 
manufacturer's 1.times. buffer in a total volume of 100 .mu.l at 
37.degree. C. for 1 hour followed by heat inactivation at 65.degree. C. 
for 15 minutes. 87.5 .mu.l of this product was mixed with 90 .mu.l of 
washed magnetic streptavidin beads in 2.times. binding-wash buffer 
(prepared from 150 .mu.l Dynabeads M-280 Streptavidin, Dynal, Oslo Norway, 
as directed by the manufacturer), incubated for 1 hour at room temperature 
(23.degree. C.) with mixing to disperse the magnetic beads, magnetically 
pelleted (Dynal Magnetic Pellet Concentrator-E), washed three times in 
binding-wash buffer, and resuspended in 50 .mu.l TE. 
Adaptor Ligation 
The template underwent ligation separately to each of the four adaptor 
mixes in adaptor set #1 as follows: 12.5 .mu.l of the template was added 
to 10 .mu.l of each adaptor mix, 17.5 .mu.l H.sub.2 O, 5.0 .mu.l 10.times. 
T.sub.4 DNA Ligase buffer, and 5.0 .mu.l T.sub.4 DNA Ligase (1.0 U/.mu.l; 
Boehringer Mannheim, Indianapolis Ind.) and incubated at 23.degree. C. for 
1 hour with mixing every 15 minutes. Then, the mixture was magnetically 
pelleted, the supernatant removed, and the pellets were washed three times 
in binding-wash buffer and then were resuspended in 50 .mu.l TE. 
Scintillation Counting 
Forty .mu.l each of the four ligated samples were added to 2.5 ml of 
scintillation fluid (Beckman Ready Gel, Beckman Instruments, Fullerton, 
Calif.) in a scintillation vial and underwent scintillation counting using 
a Beckman LS 1801 scintillation counter. 
PCR Amplification 
One .mu.l from each ligation (from the 10 .mu.l remaining that did not 
undergo scintillation counting) underwent PCR amplification as was done in 
generating the initial template precursor, except that 42.6 .mu.l H.sub.2 
O was used (instead of 41.6 .mu.l) and the upper strand of sequencing 
adaptor set #1 was used as the PCR primer in place of Primer A. 
Second Sequencing Cycle 
The steps were identical to the first sequencing cycle, except that the 
adaptor set used for adaptor ligation was adaptor set #2, and the upper 
strand of sequencing adaptor set #2 was used as a PCR primer instead of 
the upper strand of sequencing adaptor set #1. 
Third Sequencing Cycle 
The steps were identical to the second sequencing cycle, except that the 
adaptor set used for adaptor ligation was adaptor set #1, and the upper 
strand of sequencing adaptor set #1 was used as a PCR primer instead of 
the upper strand of sequencing adaptor set #2. 
Subsequent Sequencing Cycles 
Following the third sequencing cycle, the second sequencing cycle was 
repeated, and following this second sequencing cycle, the third sequencing 
cycle was repeated, and following this third sequencing cycle, the second 
sequencing cycle was repeated through the scintillation counting step. 
Sequencing Results 
The FokI recognition domain is positioned in each ligated adaptor so that 
one nucleotide was sequenced at 9 nucleotide intervals. The initial 
template precursor is shown below, along with its FokI recognition domain 
(bold type). Underlined sequences are the original amplifying primers 
(Primer A and Primer B). The cut sites for this recognition domain, as 
well as subsequent cut sites directed by ligated adaptors, are shown by 
dissecting lines. Cleavage generates a single-strand overhang that 
constitutes a template, and the nucleotide sequenced at each interval is 
shown by a numbered asterisk, the number identifying the sequencing cycle 
for sequencing the nucleotide. 
##STR5## 
The scintillation counts for each of the four adaptors at each sequencing 
interval (identified by sequencing cycle) is shown below. The highest 
counts are in bold type. Counts for the correct nucleotide were four fold 
greater than background (counts for any other nucleotide) in the first 
five cycles and greater than twice background in the final cycle (cycle 
6). 
__________________________________________________________________________ 
Sequencing Cycle Number 
1 2 3 4 5 6 
__________________________________________________________________________ 
Template nucleotide 
A A T T G T 
at ligation junction 
Predicted 5' end of 
T T A A C A 
adaptor undergoing 
ligation 
Scintillation 
G 662 
1,504 
1,625 
6,793 
1,441 
1,779 
counts for 
A 2,568 
1,618 
68,007 
34,753 
3,335 
14,397 
adaptors 
T 32,917 
32,563 
5,797 
3,934 
14,787 
2,962 
(identified by 
C 1,703 
988 
1,704 
1,745 
67,233 
5,304 
.sup.35 S labelled 3' 
end) 
__________________________________________________________________________ 
EXAMPLE 1B 
Demonstration of Interval Sequencing Mediated by Class-IIS Restriction 
Endonuclease Generated 5' Overhangs and Template-Directed Ligation 
Using a FokI based strategy, single nucleotides separated by intervals of 
nine nucleotides were sequenced using simple reagents and a scintillation 
counter. The initial template precursor was a 93 bp PCR product containing 
a portion of the Cystic Fibrosis Transmembrane Conductance Regulator gene 
that had been amplified directly from human genomic DNA. Sequencing was 
accomplished by template-directed ligation using three sequencing cycles. 
Following sequencing of the first nucleotide, two additional nucleotides 
were sequenced at nine nucleotide intervals, so that the sequencing 
covered a span of 19 nucleotides (1+(2.times.9)=19). The non-biotinylated 
primer used to generate the template precursor contained a recognition 
domain for FokI. The opposite primer had a biotinylated 5' end, and was 
used to bind the template precursor to magnetic streptavidin beads. Use of 
magnetic streptavidin beads allowed enzymatic reactions to occur in 
solution, and facilitated removal of a small aliquot for each PCR 
amplification step during the sequencing cycles. During the sequencing 
cycles, only two sets of adaptors were used, and each unique PCR 
amplifying primer used during the sequencing cycles was identical to the 
upper strand of the previously used adaptor. In this test protocol, 
identification of a nucleotide during each sequencing cycle took place 
using four ligation reactions (for the single template precursor). In each 
ligation, all four adaptors were present, with the 3' end of a different 
one of the four adaptors in each ligation tagged with .sup.32 P. 
Quantitation of retained .sup.32 P radiolabel was carried out using a 
scintillation counter, and a dominant signal for the correct nucleotide 
was clearly detected during each cycle. The details are outlined below: 
Sequencing Adaptor Generation 
Adaptor set #1 (lower strands of this adaptor set are shown in the box 
below) was generated as follows: 20.0 .mu.l of the lower strand of the 
four adaptors (100 pmole/.mu.l) were added, in four separate reactions 
(one for each oligonucleotide) to 12.5 .mu.l H.sub.2 O, 12.0 .mu.l 
5.times. Terminal deoxynucleotidyl transferase buffer (500 mM cacodylate 
buffer, pH 6.8, 5 mM CoCl.sub.2, 0.5 mM DTT), 3.0 .mu.l Terminal 
deoxynucleotidyl transferase (20 U/.mu.l; Promega, Madison, Wis.) and 12.5 
.mu.l .sup.32 P!dATP (10.0 .mu.Ci/.mu.l). All of the samples were 
incubated at 37.degree. C. for one hour followed by heat inactivation at 
70.degree. C. for 10 minutes. Unincorporated .sup.32 P!dATP was removed 
from each tube using a Qiagen nucleotide removal column (Qiagen, 
Chatsworth Calif.) and each oligonucleotide was eluted in 50 .mu.l TE. 
5'P-CNNNCATCCGACCCAGGCGTGCG (SEQ ID NO:13) or 
5'P-ANNNCATCCGACCCAGGCGTGCG (SEQ ID NO:14) or 
5'P-TNNNCATCCGACCCAGGCGTGCG (SEQ ID NO:15) or 
5'P-GNNNCATCCGACCCAGGCGTGCG (SEQ ID NO: 16); 
only the 5' end varies between these four oligonucleotides, and this 
nucleotide is underlined; the FokI recognition sequence is in bold type; N 
represents nucleotides with 4-fold degeneracy. 
15.8 .mu.l of each of the first three labeled oligonucleotides were 
separately added to 2.5 .mu.l 10.times. T.sub.4 DNA Ligase buffer (660 mM 
Tris-HCl, 50 mM MgCl.sub.2, 10 mM dithioerythritol, 10 mM ATP, pH 7.5), 
0.5 .mu.l H.sub.2 O and to 6.2 .mu.l of the upper strand of the sequencing 
adaptor (100 pmole/.mu.l): 
5'-CGCACGCCTGGGTCGGATG (SEQ ID NO:17); the FokI recognition sequence is in 
bold type. 
The last labeled oligonucleotide (with the 5' G) was processed as described 
above, except in half amounts, resulting in a final volume of 25 .mu.l for 
each of the first three adaptors and 12.5 .mu.l for the final adaptor. 
Non-radiolabeled counterparts to the above four adaptors were generated by 
adding 20.0 .mu.l (100 pmole/.mu.l) of each of the first three lower 
strands, separately to 20.0 .mu.l (100 pmole/.mu.l) of the upper strand, 
8.0 .mu.l of 10.times. T.sub.4 DNA Ligase buffer and 32 .mu.l H.sub.2 O, 
for a final volume of 80 .mu.l, and 10.0 .mu.l (100 pmole/.mu.l) of the 
final lower strand (with the 5' G) was added to half amounts of the above 
constituents, for a final volume of 40 .mu.l. Each of the eight sets of 
adaptors (four radiolabeled and four non-radiolabeled) were incubated at 
93.degree. C. for 30 seconds followed by annealing at 25.degree. C. for 5 
minutes. The radiolabeled final adaptor (with the 5' G) was added to 12.5 
.mu.l H.sub.2 O, to bring the final volume to 25.mu.l, like the other 
radiolabeled adaptors, and the 40 .mu.l of the non-radiolabeled final 
adaptor was added to 40 .mu.l H.sub.2 O, to bring the final volume to 80 
.mu.l, like the other non-radiolabeled adaptors. Each adaptor with a 5' G 
was at half the concentration of the other adaptors based on ligation data 
from preliminary experiments. 
Each radiolabeled adaptor was added to 25 .mu.l of the non-radiolabeled 
adaptors with the other three 5' ends. This resulted in four adaptor #1 
mixes, each with one radiolabeled adaptor and the remaining three 
non-radiolabeled adaptors. Using four ligation mixtures allows one to 
sequence nucleotides using a single label and a simple detection apparatus 
(e.g. a scintillation counter). 
Adaptor set #2 was made the same way as adaptor set #1, except that the 
four oligonucleotides for the lower strands of the adaptors were: 
5'P-CNNNCATCCTCTGGGCTGCACGGG (SEQ ID NO:18) or 
5'P-ANNNCATCCTCTGGGCTGCACGGG (SEQ ID NO:19) or 
5'P-TNNNCATCCTCTGGGCTGCACGGG (SEQ ID NO:20) or 
5'P-GNNNCATCCTCTGGGCTGCACGGG (SEQ ID NO:21); 
only the 5' end varies between each of these four oligonucleotides, and 
this nucleotide is underlined; the FokI recognition sequence is in bold 
type; N represents nucleotides with 4-fold degeneracy. 
and the oligonucleotide for the upper strand of the adaptors was: 
5'-CCCGTGCAGCCCAGAGGATG (SEQ ID NO:22); the FokI recognition sequence is in 
bold type. 
Initial Sequencing Template Generation 
PCR amplification of a 93 bp initial template precursor from human genomic 
DNA was carried out as described in Example 1. 
The samples were combined and mixed with 400 .mu.l of washed magnetic 
streptavidin beads in 2.times. binding-wash buffer (prepared from 140 
.mu.l Dynabeads M-280 Streptavidin, Dynal, Oslo Norway, as directed by the 
manufacturer), incubated for 1 hour at room temperature (23.degree. C.) 
with mixing to disperse the magnetic beads, magnetically pelleted (Dynal 
Magnetic Pellet Concentrator-E), washed three times in binding-wash 
buffer, and resuspended in 100 .mu.l H.sub.2 O. This product was digested 
with 7.0 .mu.l FokI (3 U/.mu.l; Boehringer Mannheim) in the manufacturer's 
1.times. buffer in a total volume of 150 .mu.l at 37.degree. C. for 1 
hour, with mixing every 15 minutes, magnetically pelleted, washed three 
times in binding-wash buffer, and the template was suspended in 50 .mu.l 
H.sub.2 O. 
Adaptor Ligation 
The template underwent ligation separately to each of the four adaptor 
mixes in adaptor set #1 as follows: 12.5 .mu.l of the template was added 
to 10 .mu.l of each adaptor mix, 18.5 .mu.l H.sub.2 O, 4.0 .mu.l 10.times. 
T.sub.4 DNA Ligase buffer, and 5.0 .mu.l T.sub.4 DNA Ligase (1.0 U/.mu.l; 
Boehringer Mannheim, Indianapolis Ind.) and incubated at 23.degree. C. for 
1 hour with mixing every 15 minutes. Then, the mixture was magnetically 
pelleted, the pellets were washed three times in binding-wash buffer and 
then were resuspended in 50 .mu.l TE (10.0 mM Tris-HCl pH 8.0, 1.0 mM 
EDTA). 
Scintillation Counting 
Forty .mu.l each of the four ligated samples were added to 2.5 ml of 
scintillation fluid (Beckman Ready Gel, Beckman Instruments, Fullerton 
Calif.) in a scintillation vial and underwent scintillation counting using 
a Beckman LS 1801 scintillation counter. 
PCR Amplification 
One .mu.l from each ligation (from the 10 .mu.l remaining that did not 
undergo scintillation counting) underwent PCR amplification as was done in 
generating the initial template precursor, except that 42.6 .mu.l H.sub.2 
O was used (instead of 41.6 .mu.l) and the upper strand of sequencing 
adaptor set #1 was used as the PCR primer in place of Primer A. 
Second Sequencing Cycle 
The steps were identical to the first sequencing cycle, except that the 
adaptor set used for adaptor ligation was adaptor set #2, and the upper 
strand of sequencing adaptor set #2 was used as a PCR primer instead of 
the upper strand of sequencing adaptor set #1. 
Third Sequencing Cycle 
The template precursor that had been amplified in the second sequencing 
cycle underwent binding to magnetic streptavidin, FolkI digestion, adaptor 
ligation, and scintillation counting as was done in the second sequencing 
cycle, except that the adaptor set used for adaptor ligation was adaptor 
set #1. 
Sequencing Results 
The FokI recognition domain is positioned in each ligated adaptor so that 
one nucleotide was sequenced at 9 nucleotide intervals. The scintillation 
counts for each of the four adaptors at each sequencing interval 
(identified by sequencing cycle) is shown below. The highest counts are in 
bold type. The second adaptor set did not label as efficiently as the 
first adaptor set. Counts for the correct nucleotide were &gt;12 fold greater 
than background (counts for any other nucleotide) in the first three 
cycles. Counts for the correct nucleotide were dominant for cycles 4 and 
5, but were less than 2-fold over background. 
______________________________________ 
Sequencing Cycle Number 
1 2 3 4 5 
______________________________________ 
Template nucleotide 
A A T T G 
at ligation junction 
Predicted 5' end of 
T T A A C 
adaptor undergoing 
ligation 
Scintillation 
G 712 329 1,337 2,420 1,597 
counts for 
A 1,933 344 40,284 
3,169 11,394 
adaptors T 25,568 6,769 3,105 1,404 7,307 
(identified by 
C 1,007 366 1,330 242 21,178 
.sup.32 p labelled 3' 
end) 
______________________________________ 
EXAMPLE 2 
Demonstration of Interval Sequencing Mediated by Class-IIS Restriction 
Endonuclease Generated 3' overhangs and Template-Directed Ligation 
A BseRI based protocol was used to sequence single nucleotides separated by 
intervals of eight nucleotides using a scintillation counter. The initial 
template precursor was a 103 bp PCR product containing a portion of the 
Cystic Fibrosis Transmembrane Conductance Regulator gene that had been 
amplified directly from human genomic DNA. Sequencing was accomplished by 
template-directed ligation using three sequencing cycles, and covered a 
span of 17 nucleotides (1+(2.times.8)=17). The non-biotinylated primer 
used to generate the template precursor contained a recognition domain for 
BseRI. The opposite primer had a biotinylated 5' end, and was used to bind 
the template precursor to magnetic streptavidin beads. During the 
sequencing cycles, only two sets of adaptors were used, and each unique 
PCR amplifying primer used during the sequencing cycles was identical to 
the upper strand of the previously used adaptor, except it did not have 
the final two nucleotides on the 3' end, so that these unique amplifying 
primers contained the BseRI recognition domain in their 3' ends ensuring 
sufficient length for efficient priming when using these adaptors. In this 
test protocol, identification of a nucleotide during each sequencing cycle 
took place using four ligation reactions (for the single template 
precursor). In each ligation, all four adaptors were present, with the 5' 
end of a different one of the four adaptors in each ligation tagged with 
.sup.32 P. Quantitation of retained .sup.32 P radiolabel was carried out 
using a scintillation counter. Signal for the correct nucleotide was four 
fold greater than background in each of the three cycles. The details are 
outlined below: 
Sequencing Adaptor Generation 
Adaptor set #1 (upper strands of this adaptor set are shown in the box 
below) was generated as follows: 4.0 .mu.l of the upper strand of the four 
adaptors (100 pmole/.mu.l) were added, in four separate reactions (one for 
each oligonucleotide) to 5.0 .mu.l H.sub.2 O, 16.0 .mu.l 10.times. 
Polynucleotide Kinase buffer (700 mM Tris-HCl (pH 7.6), 100 mM MgCl.sub.2, 
50 mM dithiothreitol), 10.0 .mu.l T.sub.4 Polynucleotide Kinase (10 
U/.mu.l; New England BioLabs, Beverly, Mass.) and 125.0 .mu.l .sup.32 
P!ATP (2.0 .mu.Ci/.mu.l). All of the samples were incubated at 37.degree. 
C. for one hour followed by heat inactivation at 65.degree. C. for 20 
minutes. Unincorporated .sup.32 P!ATP was removed from each tube using a 
Qiagen nucleotide removal column (Qiagen, Chatsworth, Calif.) and each 
oligonucleotide was eluted in 50 .mu.l TE. 
5' CGCACGGCTGGGTCGGAGGAGNC (SEQ ID NO:23) or 
5' CGCACGGCTGGGTCGGAGGAGNA (SEQ ID NO:24)or 
5' CGCACGGCTGGGTCGGAGGAGNT (SEQ ID NO:25)or 
5' CGCACGGCTGGGTCGGAGGAGNG (SEQ ID NO:26); 
only the 3' end varies between each oligonucleotide, and this nucleotide is 
underlined; the BseRI recognition sequence is in bold type; N represents 
nucleotides with 4-fold degeneracy. 
The four labeled oligonucleotides (8 pmole/.mu.l) were separately added to 
an equal volume of the lower strand of the adaptor 
(CTCCTCCGACCCAGCCGTGCG (SEQ ID NO:27); the BseRI recognition sequence is in 
bold type. 
suspended in 2.times. T.sub.4 DNA Ligase buffer (8 pmole/.mu.l). 
Non-radiolabeled counterparts to the above four adaptors were generated as 
follows: Unlabeled upper strands of the adaptors (8 pmole/.mu.l) were 
added, separately, to an equal volume of the lower strand of the adaptor 
suspended in 2.times. T.sub.4 DNA Ligase buffer (8 pmole/.mu.l). Each of 
the eight sets of adaptors (four radiolabeled and four non-radiolabeled) 
were incubated at 93.degree. C. for 30 seconds followed by annealing at 
25.degree. C. for 5 minutes. Five .mu.l of each radiolabeled adaptor was 
added to 5 .mu.l of those non-radiolabeled adaptors with the other three 
3' ends. This resulted in four adaptor #1 mixes, each with one 
radiolabeled adaptor and the remaining three non-radiolabeled adaptors. 
Adaptor set #2 was made the same way as adaptor set #1, except that the 
four oligonucleotides for the upper strands of the adaptors were: 
5' GGTGCGCCAGTCCAGCGAGGAGNC (SEQ ID NO:28)or 
5' GGTGCGCCAGTCCAGCGAGGAGNA (SEQ ID NO:29)or 
5' GGTGCGCCAGTCCAGCGAGGAGNT (SEQ ID NO:30)or 
5' GGTGCGCCAGTCCAGCGAGGAGNG (SEQ ID NO:31); 
only the 3' end varies between each oligonucleotide, and this nucleotide is 
underlined; the BseRI recognition sequence is in bold type; N represents 
nucleotides with 4-fold degeneracy. 
The oligonucleotide for the lower strand of the adaptors was: 
(CTCCTCGCTGGACTGGCGCACC (SEQ ID NO:32); the BseRI recognition sequence is 
in bold type. 
Initial Sequencing Template Generation 
PCR amplification of a 103 bp initial template precursor from human genomic 
DNA was carried out as in Example 1, except that Primer A had the 
following sequence: 
5' TCTGTTCTCAGTTTTCCTGGATGAGGAGTGGCACC (SEQ ID NO:33); mismatches to 
genomic DNA underlined; BseRI recognition sequence in bold type. 
The samples were combined, and the 400 .mu.l was digested with 5.0 .mu.l 
BseRI (4 U/.mu.l; New England BioLabs) in the manufacturer's 1.times. 
buffer in a total volume of 460 .mu.l at 37.degree. C. for 1 hour followed 
by heat inactivation at 65.degree. C. for 20 minutes. This product was 
mixed with 460 .mu.l of washed magnetic streptavidin beads (140 .mu.l 
Dynabeads washed and then suspended in 2.times. binding-wash buffer 
following the manufacturer's instructions), incubated for 1 hour at room 
temperature (23.degree. C.) with mixing to disperse the magnetic beads, 
magnetically pelleted (Dynal Magnetic Pellet Concentrator-E), washed three 
times in binding-wash buffer, and resuspended in 50 .mu.l TE. 
Adaptor Ligation 
The template underwent ligation separately to each of the four adaptor 
mixes in adaptor set #1 as follows: 12.5 .mu.l of the template was added 
to 20 .mu.l of each adaptor mix, 9.5 .mu.l H.sub.2 O, 3.0 .mu.l 10.times. 
T.sub.4 DNA Ligase buffer, and 5.0 .mu.l T.sub.4 DNA Ligase (1.0 U/.mu.l; 
Boehringer Mannheim, Indianapolis, Ind.) and incubated at 23.degree. C. 
for 1 hour with mixing every 15 minutes. Then, the mixture was 
magnetically pelleted, and the pellets were washed three times in 
binding-wash buffer and then were resuspended in 50 .mu.l TE. 
Scintillation Counting 
Twenty five .mu.l of each of the four ligated samples was added to 2.5 ml 
of scintillation fluid (Beckman Ready Gel) in a scintillation vial and 
underwent scintillation counting using a Beckman LS 1801 scintillation 
counter. 
PCR Amplification 
One .mu.l from each ligation (of the 10 .mu.l remaining that did not 
undergo scintillation counting) underwent PCR amplification as was done in 
generating the initial template precursor, except that 42.6 .mu.l H.sub.2 
O was used (instead of 41.6 .mu.l) and 
5' CGCACGGCTGGGTCGGAGGAG (SEQ ID NO:34); BseRI recognition sequence is in 
bold type. 
was used as the PCR primer in place of Primer A. 
Second Sequencing Cycle 
The steps were identical to the first sequencing cycle, except that the 
adaptor set used for adaptor ligation was adaptor set #2, and 
5' GGTGCGCCAGTCCAGCGAGGAG (SEQ ID NO:35); BseRI recognition sequence is in 
bold type. was used as the PCR primer replacing primer A. 
Third Sequencing Cycle 
The template precursor that had been amplified in the second sequencing 
cycle underwent BseRI digestion, binding to magnetic streptavidin, adaptor 
ligation and scintillation counting as was done in the second sequencing 
cycle, except that the adaptor set used for adaptor ligation was adaptor 
set #1. 
Sequencing Results 
The BseRI recognition domain is positioned in each ligated adaptor so that 
one nucleotide was sequenced at 8 nucleotide intervals. The initial 
template precursor is shown below, along with its BseRI recognition domain 
(bold type). Underlined sequences are the original amplifying primers 
(Primer A and Primer B). The cut sites for this recognition domain, as 
well as subsequent cut sites directed by ligated adaptors, are shown by 
dissecting lines. Cleavage generates a single-strand overhang that 
constitutes a template, and the nucleotide sequenced at each interval is 
shown by a numbered asterisk, the number identifying the sequencing cycle 
for sequencing the nucleotide. 
##STR6## 
The scintillation counts for each of the four adaptors at each sequencing 
interval (identified by sequencing cycle) is shown below. The highest 
counts are in bold type. Signal for the correct nucleotide was four fold 
greater than background in each of the three cycles. 
______________________________________ 
Sequencing Cycle Number 
1 2 3 
______________________________________ 
Template nucleotide 
A T A 
at ligation junction 
Predicted 3' end of 
T A T 
adaptor undergoing 
ligation 
Scintillation 
G 146,170 111,660 
100,550 
counts for A 130,570 507,140 
32,023 
adaptors T 1,290,660 83,787 
668,140 
(identified by 
C 209,660 95,120 
51,515 
phophorylated 
5' end) 
______________________________________ 
This invention was also tested to see whether it could detect a 
heterozygote for the cystic fibrosis delta 508 mutation. In this carrier, 
one would expect the third cycle to detect both an A and a C (ligation of 
adaptors with a 3' T or G). In this test, all adaptors with a 3' G were at 
half the concentration used previously, since the adaptors with a 3' G 
tended to give higher background counts, and following the sequencing of 
the initial template, templates were diluted 1:10 prior to PCR 
amplification. The results are shown below: 
______________________________________ 
Sequencing Cycle Number 
1 2 3 
______________________________________ 
Template nucleotide 
A T A and C 
at ligation junction 
Predicted 3' end of 
T A T and G 
adaptor undergoing 
ligation 
Scintillation 
G 38,430 42,824 102,340 
counts for A 77,540 198,350 
10,968 
adaptors T 598,840 40,092 110,640 
(identified by 
C 125,320 47,620 21,430 
phophorylated 
5' end) 
______________________________________ 
The heterozygote was clearly detected with counts four fold higher for each 
of the two predicted nucleotides over the background counts for the other 
nucleotides. 
EXAMPLE 3 
Demonstration of Interval Sequencing Template Generation Mediated by 
Class-IIS Restriction Endonuclease Generated 5' overhangs, 
Template-Directed Polymerization and Adaptor Ligation 
A FokI based protocol was used to generate a series of templates separated 
by intervals of nine nucleotides. The initial template precursor was the 
identical 93 bp PCR product that was used as the initial template 
precursor in Example 1. During the sequencing cycles, only two adaptors 
were used, and each unique PCR amplifying primer used during the 
sequencing cycles was identical to the upper strand of the previously used 
adaptor. In this test protocol, sequencing was simulated by the 
incorporation of a ddNTP into the template during five sequencing cycles, 
and successful trimming of the template was confirmed by acrylamide gel 
resolution of the PCR products constituting the template precursors during 
each simulated sequencing cycle. The template was trimmed as predicted 
over the five sequencing cycles. The details are given below: 
Sequencing Adaptor Generation 
Adaptor #1 was generated as follows: 30 .mu.l of the lower strand of 
adaptor #1 (100 pmole/.mu.l ): 
5' NNNCATCCGACCCAGGCGTGCG (SEQ ID NO:36); the FokI recognition sequence is 
in bold type; N represents nucleotides with 4-fold degeneracy. 
and 30 .mu.l of the upper strand of adaptor #1 (100 pmole/.mu.l): 
5' CGCACGCCTGGGTCGGATG (SEQ ID NO:37); the FokI recognition sequence is in 
bold type. 
were added to 12 .mu.l H.sub.2 O and to 8.0 .mu.l 10.times. T.sub.4 DNA 
Ligase buffer. The adaptor was incubated at 93.degree. C. for 30 seconds 
followed by annealing at 25.degree. C. for 5 minutes. 
Adaptor #2 was made the same way as adaptor set #1, except that the 
oligonucleotide for the lower strand of adaptor #2 was: 
5' NNNCATCCTCTGGGCTGCACGGG (SEQ ID NO:38); the FokI recognition sequence is 
in bold type; N represents nucleotides with 4-fold degeneracy. 
and the oligonucleotide for the upper strand of the adaptors was: 
5' CCCGTGCAGCCCAGAGGATG (SEQ ID NO:39); the FokI recognition sequence is in 
bold type. 
Initial Sequencing Template Generation 
PCR amplification of a 93 bp initial template precursor from human genomic 
DNA was carried out as described in Example 1, except that only 100 .mu.l 
(one tube) was amplified. Following PCR amplification, 50 .mu.l was 
removed to be run on a acrylamide gel later. The remaining 50 .mu.l was 
mixed with 100 .mu.l of washed magnetic streptavidin beads (16 .mu.l 
Dynabeads M-280 Streptavidin washed and suspended in 2.times. binding-wash 
buffer) and 50 .mu.l H.sub.2 O, incubated for 1 hour at 23.degree. C. with 
mixing, magnetically pelleted, washed three times in binding-wash buffer, 
and resuspended in 50 .mu.l H.sub.2 O. This product was digested with 1.0 
.mu.l FokI (3 U/.mu.l) with mixing every 15 minutes in the 1.times. 
restriction endonuclease buffer in a total volume of 100 .mu.l at 
37.degree. C. for 1 hour, magnetically pelleted, washed three times in 
binding-wash buffer, and resuspended in 25 .mu.l H.sub.2 O. 
Template Directed Polymerization Using Nucleotide Terminators 
This product was added to 10 .mu.l of each ddNTP (500 .mu.M each), 14 .mu.l 
H.sub.2 O, 20 .mu.l 5.times. Sequenase buffer, and 1.0 .mu.l Sequenase 
(Amersham) and was incubated at 23.degree. C. for 20 minutes with mixing 
every 10 minutes. The mixture was magnetically pelleted, washed three 
times in binding-wash buffer and suspended in 25 .mu.l TE. 
Adaptor Ligation 
The template (following simulated sequencing by ddNTP fill-in) underwent 
ligation to adaptor #1 as follows: 25 .mu.l of the template was added to 
10 .mu.l of adaptor #1, 6.0 .mu.l H.sub.2 O, 4.0 .mu.l 10.times. T.sub.4 
DNA Ligase buffer, and 5.0 .mu.l T.sub.4 DNA Ligase (1.0 U/.mu.l) and 
incubated at 23.degree. C. for 1 hour with mixing every 15 minutes. Then, 
the mixture was magnetically pelleted, washed three times in binding-wash 
buffer, and suspended in 50 .mu.l TE. 
PCR Amplification 
1 .mu.l from the ligation underwent PCR amplification as was done in 
generating the initial template precursor, except that 42.6 .mu.l H.sub.2 
O was used (instead of 41.6 .mu.l) and the upper strand of adaptor #1 was 
used as the PCR primer in place of Primer A. 
Second Sequencing Cycle 
The steps were identical to the first sequencing cycle, except that the 
adaptor used for adaptor ligation was adaptor #2, and the upper strand of 
adaptor #2 was used as a PCR primer instead of the upper strand of adaptor 
#1. 
Third Sequencing Cycle 
Identical to the second sequencing cycle, except that the adaptor used for 
adaptor ligation was adaptor #1, and the upper strand of adaptor #1 was 
used as a PCR primer instead of the upper strand of adaptor #2. 
Subsequent Sequencing Cycles 
Following the third sequencing cycle, the second sequencing cycle was 
repeated, and following this second sequencing cycle, the third sequencing 
cycle was repeated. 
Results 
Following each PCR amplification, generating the template precursors, 50 
.mu.l were removed and were later run on a acrylamide gel, as shown in 
FIG. 5. Following the sequencing cycles 1-5, the template precursor was 
trimmed as predicted, with high specificity in the first four sequencing 
cycles, and some extraneous product in the template -precursor following 
the fifth sequencing cycle. 
EXAMPLE 3B 
Demonstration of Interval Sequencing Mediated by Class-IIS Restriction 
Endonuclease Generated 5' overhangs, Template-Directed Polymerization and 
Adaptor Ligation 
This example is essentially the same as Example 3, except that during each 
template-directed polymerization with ddNTPs, a .sup.33 P labeled ddNTP 
was substituted for its corresponding normal ddNTP, in four separate 
template-directed polymerizations, each with a single and different 
radiolabeled ddNTP. Then, an aliquot from each of these reactions 
underwent scintillation counting. 
Sequencing Adaptor Generation 
Sequencing adaptor generation was carried out as described in Example 3. 
Initial Sequencing Template Generation 
PCR amplification of the initial template precursor from human genomic DNA 
was carried out as described in Example 3, except that two tubes were 
amplified (200 .mu.l). Following PCR amplification, the entire PCR product 
was bound to 200 .mu.l of washed magnetic streptavidin beads (64 .mu.l 
Dynabeads M-280 Streptavidin washed and suspended in 2.times. binding-wash 
buffer), incubated for 1 hour at 23.degree. C. with mixing, magnetically 
pelleted, washed three times in binding-wash buffer, and resuspended in 
100 .mu.l H.sub.2 O. This product was digested with 4.0 .mu.l FokI (3 
U/.mu.l) in the corresponding 1.times. restriction endonuclease buffer in 
a total volume of 150 .mu.l at 37.degree. C. for 1 hour with mixing every 
15 minutes, magnetically pelleted, washed three times in binding-wash 
buffer, and resuspended in 100 .mu.l H.sub.2 O. 
Template Directed Polymerization using Nucleotide Terminators 
25 .mu.l underwent four separate template directed polymerizations using 
ddNTPs, each exactly as was done in Example 3, except a different three 
non-radiolabeled ddNTPs were added in each reaction, with the fourth ddNTP 
being 5.0 .mu.l of the corresponding .sup.33 PddNTP (0.45 .mu.Ci/.mu.l; 
Amersham). Also, 19 .mu.l H.sub.2 O were used instead of 14 .mu.l H.sub.2 
O, and 3 U of Sequence (1.2 .mu.l of a 1:5 dilution in 1.times. Sequenase 
buffer) were used instead of 1 .mu.l of undiluted Sequenase (13 U/.mu.l). 
Following incubation for 20 minutes at 23.degree. C. with mixing every 10 
minutes, each mixture was magnetically pelleted, washed three times in 
binding-wash buffer and suspended in 50 .mu.l H.sub.2 O. 
Scintillation Counting 
40 .mu.l underwent scintillation counting as described in Example 1. 
Adaptor Ligation 
The remaining 10 .mu.l of each of the four samples were combined, and 
underwent adaptor ligation as in Example 3, except that 10 .mu.l of 
10.times. ligase buffer and 35 .mu.l H.sub.2 O were used, resulting in a 
final volume of 100 .mu.l, and following ligation, magnetic pelleting and 
washing, the pellet was suspended in 25 .mu.l TE. 
PCR Amplification 
One .mu.l from the ligation underwent PCR amplification in each of two 
tubes as was done in generating the initial template precursor, except 
that 42.6 .mu.l H.sub.2 O was used (instead of 41.6 .mu.l) and the upper 
strand of adaptor #1 was used as the PCR primer in place of Primer A. 
Second Sequencing Cycle 
The steps were identical to the first sequencing cycle, except that the 
adaptor used for adaptor ligation was adaptor #2, and the upper strand of 
adaptor #2 was used as a PCR primer instead of the upper strand of adaptor 
#1. 
Third Sequencing Cycle 
Identical to the second sequencing cycle, except that the adaptor used for 
adaptor ligation was adaptor #1, and the upper strand of adaptor #1 was 
used as a PCR primer instead of the upper strand of adaptor #2. 
Subsequent Sequencing Cycles 
Following the third sequencing cycle, the second sequencing cycle was 
repeated, and following this second sequencing cycle, the third sequencing 
cycle was repeated through the scintillation counting step. 
Sequencing Results 
The scintillation counts at each sequencing interval (identified by 
sequencing cycle) are shown below. The highest counts are in bold type. 
Counts for the correct nucleotide were greater than 3.50 fold greater than 
background (counts for any other nucleotide) in each of the five cycles. 
__________________________________________________________________________ 
Sequencing Cycle Number 
1 2 3 4 5 
__________________________________________________________________________ 
Template nucleotide 
A A T T G 
adjacent to double- 
stranded domain 
Predicted ddNTP 
T T A A C 
incorporated by 
template-directed 
polymerization 
Scintillation 
G 51,444 
20,848 
74,217 261,280 
12,436 
counts for 
A 255,340 
58,063 
3,433,960 
2,805,872 
167,928 
incorporated 
T 897,960 
2,061,827 
9,434 43,309 
229,760 
.sup.33 P labelled 
C 13,124 
7,490 
7,877 18,042 
886,184 
ddNTPs 
__________________________________________________________________________ 
EXAMPLE 4 
This example demonstrates a method that uses restriction endonuclease 
digestion to selectively remove primer directed sequence from a PCR 
product, without using a free methylated nucleotide during PCR 
amplification. This demonstration is the first use of a PCR primer with a 
methylated recognition domain sequence designed to permit selective 
cleavage directed by the primer encoded end of a PCR product. In the 
context of the sequencing method of this invention, when generating 
initial sequencing templates, the ability to remove PCR primer encoded 
sequence and its complement at the end to be sequenced decreases the 
number of cycles necessary to sequence PCR product that lies beyond the 
primer. 
There is currently only one commercially available restriction 
endonuclease, Dpn I, that requires a methylated sequence for cutting. Dpn 
I recognizes the sequence GATC, where the A is methylated. Cutting by Dpn 
I generates a blunt end. The methylated A was incorporated into the primer 
sequence during routine oligonucleotide synthesis, as methyl A is 
commercially available as a phosphoramidite. PCR amplification occurred 
using regular non-methylated nucleotides, so no portion of any PCR 
product, apart from the methylated primer, was methylated. A 55 bp PCR 
product was amplified from the plasmid pUC19. This 55 bp PCR product and 
its 40 bp Dpn I digest product are illustrated in FIG. 6, and the 
denaturing acrylamide gel showing the original PCR product and its DpnI 
digestion product is shown in FIG. 7. 
PCR Product Generation with a Primer Encoded Hemi-Methylated Dpn I 
Recognition Domain 
PCR amplification of a 55 bp product from 4 ng of the plasmid pUC19 was 
carried out using 1.6 .mu.l rTth DNA Polymerase (2.5 U/.mu.l; Perkin 
Elmer) in a 1.times.Tth DNA polymerase buffer (20 mM Tricine pH 8.7, 85 mM 
KOAc, 8% glycerol, 2% (vol/vol) DMSO, 1.1 mM Mg(OAc).sub.2), and 200 .mu.M 
each dNTP with 25 pmoles of each of the primers shown in the box below, 
using the following parameters: 94.degree. C. for 1 minute followed by 30 
thermal cycles (94.degree. C. for 30 seconds, 45.degree. C. for 30 
seconds), a final extension at 72.degree. C. for 7 minutes, and a 
4.degree. C. soak. 
Primer A: 5'CCATCCGTAAGATGATCTTCTG (SEQ ID NO:40); mismatches to pUC19 DNA 
underlined; DpnI recognition sequence in bold type. The A was methylated, 
and was incorporated during oligonucleotide synthesis using a methylated 
phosphoramidite (Glenn Research). Primer B: 5'CTCAGAATGACTTGGTTG (SEQ ID 
NO:41). 
Digestion with DpnI 
33 .mu.l of this product was digested with 1.0 .mu.l or 5.0 .mu.l DpnI (20 
U/.mu.l; New England BioLabs) in the manufacturer's 1.times. buffer in a 
total volume of 40 .mu.l at 37.degree. C. for 1 hour. The initial PCR 
product and its DpnI cut portions were each run on a denaturing acrylamide 
gel, as shown in FIG. 7. Dpn I cut the PCR end to very near completion 
(FIG. 7). In this example, the DpnI site was created near the 3' end of 
the primer, and incorporating this recognition domain required two 
mismatches to the original template. This illustrates that Dpn I, with its 
short 4 bp recognition domain, can be readily incorporated near the 3' end 
of a primer without preventing PCR amplification. For the sequencing of 
inserts cloned in a vector insert, the recognition domain can be placed in 
the immediate 3' end of the amplifying primer, because its nucleotide 
sequence can be encoded in the vector adjacent to the inserts to be 
sequenced. Following digestion with DpnI, an end is generated that can be 
ligated to the initial adaptors with offset recognition domains for the 
class-IIS restriction endonuclease used in sequencing the insert. 
Equivalents 
Those skilled in the art will be able to recognize, or be able to ascertain 
using no more than routine experimentation, numerous equivalents to the 
specific procedures described herein. Such equivalents are considered to 
be within the scope of this invention and are covered by the following 
claims. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 41 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
CNNNCATCCGACCCAGGCGTGCG23 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
ANNNCATCCGACCCAGGCGTGCG23 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
TNNNCATCCGACCCAGGCGTGCG23 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
GNNNCATCCGACCCAGGCGTGCG23 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
CGCACGCCTGGGTCGGATG19 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
CNNNCATCCTCTGGGCTGCACGGG24 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
ANNNCATCCTCTGGGCTGCACGGG24 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
TNNNCATCCTCTGGGCTGCACGGG24 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
GNNNCATCCTCTGGGCTGCACGGG24 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
CCCGTGCAGCCCAGAGGATG20 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
GTTTTCCTGGATGATGCCCTGGC23 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
CATGCTTTGATGACGCTTCTGTATC25 
(2) INFORMATION FOR SEQ ID NO:13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
CNNNCATCCGACCCAGGCGTGCG23 
(2) INFORMATION FOR SEQ ID NO:14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
ANNNCATCCGACCCAGGCGTGCG23 
(2) INFORMATION FOR SEQ ID NO:15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
TNNNCATCCGACCCAGGCGTGCG23 
(2) INFORMATION FOR SEQ ID NO:16: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
GNNNCATCCGACCCAGGCGTGCG23 
(2) INFORMATION FOR SEQ ID NO:17: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
CGCACGCCTGGGTCGGATG19 
(2) INFORMATION FOR SEQ ID NO:18: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
CNNNCATCCTCTGGGCTGCACGGG24 
(2) INFORMATION FOR SEQ ID NO:19: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
ANNNCATCCTCTGGGCTGCACGGG24 
(2) INFORMATION FOR SEQ ID NO:20: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 
TNNNCATCCTCTGGGCTGCACGGG24 
(2) INFORMATION FOR SEQ ID NO:21: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 
GNNNCATCCTCTGGGCTGCACGGG24 
(2) INFORMATION FOR SEQ ID NO:22: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: 
CCCGTGCAGCCCAGAGGATG20 
(2) INFORMATION FOR SEQ ID NO:23: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: 
CGCACGGCTGGGTCGGAGGAGNC23 
(2) INFORMATION FOR SEQ ID NO:24: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: 
CGCACGGCTGGGTCGGAGGAGNA23 
(2) INFORMATION FOR SEQ ID NO:25: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: 
CGCACGGCTGGGTCGGAGGAGNT23 
(2) INFORMATION FOR SEQ ID NO:26: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: 
CGCACGGCTGGGTCGGAGGAGNG23 
(2) INFORMATION FOR SEQ ID NO:27: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: 
CTCCTCCGACCCAGCCGTGCG21 
(2) INFORMATION FOR SEQ ID NO:28: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: 
GGTGCGCCAGTCCAGCGAGGAGNC24 
(2) INFORMATION FOR SEQ ID NO:29: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: 
GGTGCGCCAGTCCAGCGAGGAGNA24 
(2) INFORMATION FOR SEQ ID NO:30: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: 
GGTGCGCCAGTCCAGCGAGGAGNT24 
(2) INFORMATION FOR SEQ ID NO:31: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: 
GGTGCGCCAGTCCAGCGAGGAGNG24 
(2) INFORMATION FOR SEQ ID NO:32: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: 
CTCCTCGCTGGACTGGCGCACC22 
(2) INFORMATION FOR SEQ ID NO:33: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 35 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: 
TCTGTTCTCAGTTTTCCTGGATGAGGAGTGGCACC35 
(2) INFORMATION FOR SEQ ID NO:34: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: 
CGCACGGCTGGGTCGGAGGAG21 
(2) INFORMATION FOR SEQ ID NO:35: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: 
GGTGCGCCAGTCCAGCGAGGAG22 
(2) INFORMATION FOR SEQ ID NO:36: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: 
NNNCATCCGACCCAGGCGTGCG22 
(2) INFORMATION FOR SEQ ID NO:37: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: 
CGCACGCCTGGGTCGGATG19 
(2) INFORMATION FOR SEQ ID NO:38: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: 
NNNCATCCTCTGGGCTGCACGGG23 
(2) INFORMATION FOR SEQ ID NO:39: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: 
CCCGTGCAGCCCAGAGGATG20 
(2) INFORMATION FOR SEQ ID NO:40: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40: 
CCATCCGTAAGATGATCTTCTG22 
(2) INFORMATION FOR SEQ ID NO:41: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: 
CTCAGAATGACTTGGTTG18 
__________________________________________________________________________