The present invention is directed to a process for amplifying and detecting any target nucleic acid sequence contained in a nucleic acid or mixture thereof and for assembling large polynucleotides from component polynucleotides, each involving generating concatemers formed by PCR amplification of overlapping fragments.

TECHNICAL FIELD 
The present invention relates generally to the field of recombinant DNA 
technology and, more particularly, to improved methods for producing 
amplified heterogeneous populations of polynucleotides from limited 
quantities of DNA or RNA or other nucleic acids. The invention provides 
compositions and methods for a chain reaction amplification of a target 
polynucleotide species using a thermostable polymerase or other suitable 
polynucleotide polymerase compatible with the method. 
BACKGROUND 
Selective amplification of polynucleotides represents a major research goal 
of molecular biology, with particular importance in diagnostic and 
forensic applications, as well as for general manipulations of genetic 
materials and laboratory reagents. 
The polymerase chain reaction (PCR) is a method by which a specific 
polynucleotide sequence can be amplified in vitro. PCR is an extremely 
powerful technique for amplifying specific polynucleotide sequences, 
including genomic DNA, single-stranded cDNA, and mRNA among others. As 
described in U.S. Pat. Nos. 4,683,202, 4,683,195, and 4,800,159 (which are 
incorporated herein by reference), PCR typically comprises treating 
separate complementary strands of a target nucleic acid with two 
oligonucleotide primers to form complementary primer extension products on 
both strands that act as templates for synthesizing copies of the desired 
nucleic acid sequences. By repeating the separation and synthesis steps in 
an automated system, essentially exponential duplication of the target 
sequences can be achieved. 
A number of variations of the basic PCR methodology have been described. 
U.S. Pat. No. 5,066,584 discloses a method wherein single stranded DNA can 
be generated by the polymerase chain reaction using two oligonucleotide 
primers, one present in a limiting concentration. U.S. Pat. No. 5,340,728 
discloses an improved method for performing a nested polymerase chain 
reaction (PCR) amplification of a targeted piece of DNA, wherein by 
controlling the annealing times and concentration of both the outer and 
the inner set of primers according to the method disclosed, highly 
specific and efficient amplification of a targeted piece of DNA can be 
achieved without depletion or removal of the outer primers from the 
reaction mixture vessel. U.S. Pat. No. 5,286,632 discloses recombination 
PCR (RPCR) wherein PCR is used with at least two primer species to add 
double-stranded homologous ends to DNA such that the homologous ends 
undergo in vivo recombination following transfection of host cells. 
Horton et al. (1989) Gene 77: 61, discloses a method for making chimeric 
genes using PCR to generate overlapping homologous regions. In the Horton 
method, fragments of different genes that are to form the chimeric gene 
are generated in separate polymerase chain reactions. The primers used in 
these separate reactions are designed so that the ends of the different 
products of the separate reactions contain complementary sequences. When 
these separately produced PCR products are mixed, denatured and 
reannealed, the strands having matching sequences at their 3'-ends overlap 
and act as primers for each other. Extension of this overlap by DNA 
polymerase produces a molecule in which the original sequences are spliced 
together to form the chimeric gene. 
Silver and Keerikatte (1989) J. Virol. 63: 1924 describe another variation 
of the standard PCR approach (which requires oligonucleotide primers 
complementary to both ends of the segment to be amplified) to allow 
amplification of DNA flanked on only one side by a region of known DNA 
sequence. This technique requires the presence of a known restriction site 
within the known DNA sequence and a similar site within the unknown 
flanking DNA sequence which is to be amplified. After restriction and 
recircularization, the recircularized fragment is restricted at an unique 
site between the two primers and the resulting linearized fragment is used 
as a template for PCR amplification. 
Triglia et al. (1988) Nucl.Acids Res. 16: 8186, describe an approach which 
requires the inversion of the sequence of interest by circularization and 
re-opening at a site distinct from the one of interest, and is called 
"inverted PCR." A fragment is first created in which two unknown sequences 
flank on either side a region of known DNA sequence. The fragment is then 
circularized and cleaved with an unique restriction endonuclease which 
only cuts within the known DNA sequence creating a new fragment containing 
all of the DNA of the original fragment but which is then inverted with 
regions of known sequence flanking the region of unknown sequence. This 
fragment is then utilized as a PCR substrate to amplify the unknown 
sequence. 
Vallette et al. (1989) Nucl.Acids Res. 17: 723 disclose using PCR in a 
specific approach which involves using a supercoiled plasmid DNA as a 
template for PCR and a primer bearing a mutated sequence which is 
incorporated into the amplified product. Using this method, DNA sequences 
may be inserted only at the 5'-end of the DNA molecule which one wishes to 
alter. Mole et al. (1989) Nucl.Acids Res. 17: 3319, used PCR to create 
deletions within existing expression plasmids. However, PCR was performed 
around the entire plasmid (containing the fragment to be deleted) from 
primers whose 5'-ends defined the region to be deleted. Self-ligation of 
the PCR product recircularized the plasmid. 
U.S. Pat. No. 5,279,952 discloses a method for using PCR to generate 
mutations (e.g., deletions) and chimeric genes by forming head-to-tail 
concatemers of a known starting sequence and employing at least two PCR 
primers to amplify a DNA segment which is altered as compared to the known 
starting sequence. 
Jones and Howard (1990) BioTechniques 8: 178, report a site-specific 
mutagenesis method using PCR, termed recombinant circle PCR (RCPCR). In 
RCPCR, separate PCR amplifications (typically two) of a known 
polynucleotide generate products that, when combined, denatured, and 
annealed, form double-stranded DNA with discrete, cohesive single-stranded 
ends designed so that they may anneal and form circles of DNA. 
Oliner et al. (1993) Nucl. Acids. Res. 21: 5192, report a method for 
engineering PCR products to contain terminal sequences identical to 
sequences at the two ends of a linearized vector such that co-transfection 
of the PCR product and linearized vector into a recombination-competent 
host cell results in formation of a covalently linked vector containing 
the PCR product, thus avoiding the need for in vitro ligation. 
In spite of such recent advances, including PCR and its various 
modifications noted above, there exists a need for improved methods of 
identifying and cloning polynucleotides, for accurate in vitro 
amplification of selected polynucleotides, and for facile assembly of 
polynucleotides from a mixture of component oligonucleotides or 
polynucleotides without necessitating the use of DNA ligase. In 
particular, there is a need for a PCR amplification method which can be 
performed with (1) only a single primer species, or (2) with multiple 
overlapping polynucleotide fragments (or oligonucleotides) in the absence 
of a conventional PCR primer, and which can result in formation of an 
amplified product which can be a concatemer and/or which can be a 
covalently-closed circle. The present invention fulfills these and other 
needs. 
The references discussed herein are provided solely for their disclosure 
prior to the filing date of the present application. Nothing herein is to 
be construed as an admission that the inventors are not entitled to 
antedate such disclosure by virtue of prior invention. All publications 
cited are incorporated herein by reference. 
SUMMARY OF THE INVENTION 
A basis of the present invention is the use of polymerase-mediated chain 
extension, such as for example PCR, in combination with at least two 
polynucleotides having complementary ends which can anneal whereby at 
least one of said polynucleotides has a free 3'-hydroxyl capable of 
polynucleotide chain elongation by a DNA polymerase, such as a 
thermostable polymerase (e.g., Thermus acruaticus (Taq) polymerase or 
Thermococcus litoralis (Vent.TM., New England Biolabs) polymerase or TthI 
polymerase (Perkin-Elmer). Although the method can be practiced using PCR, 
in some embodiments either a single primer species or no primer whatsoever 
is required, and hence PCR is not a necessary component of the general 
method. 
In one embodiment, a target polynucleotide is contacted with a "bivalent 
primer" typically comprising an oligonucleotide having two regions of 
complementarity to the target polynucleotide: (1) a first portion which is 
in the 5' portion of the primer and which is substantially complementary 
to a sequence in the 5' portion of the sequence to be amplified (target 
sequence) in the target polynucleotide, and (2) a second portion which is 
in the 3' portion of the primer and which is substantially complementary 
to a sequence in the 3' portion of the sequence to be amplified (target 
sequence) in the target polynucleotide. The contacting is performed under 
conditions suitable for hybridization of the bivalent primer to the target 
polynucleotide, most often following thermal denaturation of the target 
polynucleotide if it is initially present as a double-stranded form. The 
target polynucleotide may be substantially homogeneous or may be present 
in a mixture of polynucleotide species (e.g., in a genome, biological 
sample, or mixture of synthetic polynucleotides). Subsequent or 
concomitant with the contacting of the bivalent primer to the target 
polynucleotide, a polynucleotide polymerase, such as a thermostable DNA 
polymerase, catalyzes, under suitable reaction conditions, polynucleotide 
synthesis (chain elongation) primed from the 3'-hydroxyl of the annealed 
bivalent primer to form a strand complementary to the target sequence, 
thereby forming a nascent complementary strand. Following completion of 
the nascent complementary strand spanning the target sequence, the target 
polynucleotide and the nascent strand are denatured, typically by 
elevation of temperature, and allowed to reanneal, typically by reduction 
of temperature, with another molecule of the bivalent primer species or 
with a complementary strand of a target polynucleotide or an amplified 
copy thereof. The denatured nascent strand species following the first 
elongation cycle will contain a copy of the target sequence and has a 
terminal repeat of its 5'-terminal sequence at the 3' terminus, resulting 
from the bivalent primer, and wherein the terminal repeat is of sufficient 
length to support annealing under PCR conditions to an overlapping 
complementary strand in a head-to-tail arrangement (see, FIG. 1). 
Following reannealing, the described polymerase 
elongation/denaturation/reannealing cycle is repeated from 1 to about 100 
times as desired, resulting in formation of amplified product which 
comprises head-to-tail concatemers of the target sequence. The concatemers 
typically increase in length as the number of amplification cycles 
increase and as the amount of bivalent primer decreases. Following 
amplification forming concatameric head-to-tail repeats of the target 
sequence, the concatemer(s) can optionally be resolved, such as (1) by 
cleaving with a restriction endonuclease which cuts within (or at the 
termini of) the concatemeric unit(s), (2) by homologous recombination 
between concatemer units to form covalently closed circles, or (3) by 
cleavage with a restriction endonuclease followed by ligation with DNA 
ligase to form covalently closed circles and/or by direct transformation 
into host cells for in vivo ligation. 
Often, a target polynucleotide sequence which is amplified as described 
above will form amplification intermediates in the form of cyclized DNA or 
spiral DNA (see, FIG. 2), as a result of the 3' terminus of an overlapped 
nascent strand annealing to the 3' terminus of an overlapped complementary 
strand forming a cyclized structure similar to a gapped circle; the 
cyclized structure has a strand with an extendable 3'-hydroxyl which can 
be extended with a DNA polymerase substantially lacking exonuclease 
activity (e.g., a thermostable polymerase such as Vent (exo.sup.-).TM. or 
Klenow fragment, etc.) in a rolling circle format whereby the leading 
terminus of the nascent strand continually displaces the lagging portion 
of the nascent strand (see, FIG. 2) producing a concatemeric single strand 
emanating from the rolling circle intermediate. Most often, such rolling 
circle intermediates will form under dilute conditions more favorable to 
intramolecular cyclization of overlapped strands. Once a rolling circle 
intermediate is established, the template need not be denatured in order 
to continue amplification of the target sequence as in conventional PCR, 
thus avoiding the necessity of multiple thermal cycles of PCR to denature 
template (and the resultant time loss needed for heating and cooling). 
Often, however, the template is repeatedly denatured, annealed, and 
extended with polymerase in the presence of ribonucleotide or 
deoxyribonucleotides under suitable reaction conditions. 
Furthermore, whether the method generates a rolling circle intermediate or 
linear concatemers, an advantage of the method is that it requires a 
substantially reduced amount of primer (bivalent primer) as compared to 
conventional PCR, since following the initial cycle(s) an increasing 
percentage of the priming of nascent strand synthesis is primed from 
3'-hydroxyl groups of the amplified strands, rather than from the 
oligonucleotide primer(s). In the case of a rolling circle intermediate, 
theoretically only a single bivalent primer molecule is necessary to 
generate the rolling circle which then can produce multiple concatenated 
copies by rolling circle-style polymerase catalysis using a polymerase 
capable of strand displacement of the lagging edge of the nascent strand 
as replication proceeds around the cyclized template. 
In an embodiment, a product polynucleotide is assembled from a plurality of 
component polynucleotides by formation of overlapped strands of 
alternating polarity and having substantially complementary termini (see, 
FIG. 3). This method employs a series of overlapping substantially 
complementary termini to determine the linear order of component sequences 
in the final product. Concomitant with or subsequent to formation of the 
overlapped strands of the component polynucleotides in a reaction, a 
polynucleotide polymerase (e.g., a thermostable DNA polymerase) under 
suitable reaction conditions catalyzes strand elongation from the 
3'-hydroxyl portions of the overlapped (annealed) joints, filling in the 
portion between joints and processively displacing or processively 
degrading exonucleolytically the 5' termini of downstream component 
strands of the same polarity as the nascent strand elongates. After a 
cycle of chain elongation forming substantially double-stranded 
polynucleotides, the reaction conditions are altered (typically by 
increasing the temperature) to effect denaturation of the double-stranded 
polynucleotides, followed by altering the reaction conditions to permit 
reannealing of complementary strands or portions thereof (i.e., 
overlapping termini) to form molecules having overlapped termini (joints), 
and chain elongation by a polynucleotide polymerase under suitable 
reaction conditions catalyzes strand elongation from the 3'-hydroxyl 
portions of the overlapped (annealed) joints, as in the first cycle. One 
to about 100 cycles of denaturation/annealing/polymerization can be 
performed to generate a product comprising the component polynucleotide 
sequences covalently linked in linear order according to the order of the 
overlapping joints. In this embodiment, a product polynucleotide can be 
constructed from a plurality of smaller component polynucleotides 
(typically oligonucleotides) and enables assembly of a variety of products 
with alternate substitutable polynucleotide components at a given position 
serving as structural "alleles" (see, FIG. 4). The component 
polynucleotides are often provided in single-strand form, but may 
initially be present in double-strand form and be denatured (typically by 
elevated temperature) for the assembly of the product by PCR 
amplification. Substantially any type of product polynucleotide can be 
assembled in this way, including cloning and expression vectors, viral 
genomes, gene therapy vectors, genes (including chimeric genes), 
polynucleotides encoding peptide libraries, protein libraries, vector 
libraries, viral libraries, and the like. In a variation, one or more of 
the component polynucleotides represents a site-directed mutation or 
variable-sequence kernel. In a variation, PCR employing a low-fidelity 
polymerase is used to introduce additional sequence variation into the 
product polynucleotide(s) during amplification cycles. The method can be 
used to produce a library of sequence-variant product polynucleotides, if 
desired. 
In an embodiment of the invention, very long distance PCR is provided, 
wherein PCR or other suitable amplification method is used to generate, in 
a single reaction or in parallel reactions which are subsequently pooled, 
a set of overlapping large DNA fragments which can be denatured and 
annealed to form very large (e.g., greater than 25 to 50 kilobases) DNA 
structures composed of overlapped single strands of DNA having alternating 
polarity with each overlapped joint providing an extendable 3'-hydroxyl 
group for forming phosphodiester bonds catalyzed by a polynucleotide 
polymerase in the presence of free ribonucleotide or deoxyribonucleotides. 
Typically, the method comprises forming at least three overlapping 
polynucleotides, wherein the 3' terminus of a first single-stranded 
polynucleotide is substantially complementary to the 3' terminus of a 
second single-stranded polynucleotide of the opposite polarity, and 
wherein the 5' terminus of said second single-stranded polynucleotide is 
substantially complementary to the 3' terminus of a third single-stranded 
polynucleotide having polarity identical to said first single-stranded 
polynucleotide, thereby generating an overlapped structure capable of 
chain elongation by a suitable polymerase to generate a double-stranded 
product spanning the three initial overlapped polynucleotides. With such a 
method, polynucleotides of 50 kb to 100 kb or more can be generated by a 
facile amplification method capable of generating amplification products 
much longer than is possible with conventional long-range PCR methods. The 
method can comprise parallel processing PCR reactions, wherein a plurality 
of primer sets are employed in a single reaction or multiple reactions 
which are subsequently pooled, each primer sets priming the PCR 
amplification of a polynucleotide sequence which comprises terminal 
sequences which are complementary to terminal sequences in at least one 
other amplification product produced by a different primer set, thus 
generating a set of overlapping PCR products with which a large product 
spanning the entire set of PCR products is generated by end-complementary 
polymerase reaction. 
In some embodiments of the invention, the polynucleotides product(s) 
generated thereby are labelled, such as with radioisotopic, biotinyl, or 
fluorescent label moieties, by incorporation of labelled ribonucleotide or 
deoxyribonucleotides or the like into nascent polynucleotide by 
polymerase-mediated catalysis. 
The invention also provides kits comprising a bivalent primer 
polynucleotide and/or a plurality of component polynucleotides and 
instructions for use describing the present end-complementary 
amplification method disclosed herein. Frequently, a polynucleotide 
polymerase, such as a thermostable DNA polymerase (Taq or Vent.TM. 
polymerase) is also present in the kit. Optionally, one or more target 
polynucleotides may be provided in the kit, such as for calibration and/or 
for use as a positive control to verify correct performance of the kit. 
In an embodiment, the invention provides a method termed continuous 
multiplex amplification which affords amplification of a plurality of 
initially unlinked polynucleotide species at substantially comparable 
amplification rates by forming a linked amplification product wherein the 
plurality of initially unlinked polynucleotide sequences are linked by 
end-complementary amplification. An amplification unit, termed an 
amplicon, comprising at least one copy of each member of the plurality of 
initially unlinked polynucleotide species is formed by one or more cycles 
of end-complementary amplification. From one to about 100, typically three 
to 35, amplification cycles can be conducted and result in formation of a 
population of linked amplification products, which can comprise 
concatemers of said amplicon. The amplification products can be linear or 
circular, as desired, based on appropriate selection of the bivalent 
primers. In a variation, the amplification product is cleaved with a 
nucleolytic agent, such as a restriction enzyme which cuts at least one 
restriction site present in the amplicon, DNase, nuclease S1, bleomycin, 
ionizing radiation, or the like or by other suitable cleaving means. 
A further understanding of the nature and advantages of the invention will 
become apparent by reference to the remaining portions of the 
specification and drawings.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
Unless defined otherwise, all technical and scientific terms used herein 
have the same meaning as commonly understood by one of ordinary skill in 
the art to which this invention belongs. Although any methods and 
materials similar or equivalent to those described herein can be used in 
the practice or testing of the present invention, the preferred methods 
and materials are described. For purposes of the present invention, the 
following terms are defined below. 
Definitions 
Unless specified otherwise, the conventional notation used herein portrays 
polynucleotides as follows: the lefthand end of single-stranded 
polynucleotide sequences is the 5' end; the lefthand direction of 
double-stranded polynucleotide sequences is referred to as the 5' 
direction. The direction of 5' to 3' addition of nascent RNA transcripts 
is referred to as the transcription direction; sequence regions on the DNA 
strand having the same sequence as the RNA and which are 5' to the 5' end 
of the RNA transcript are referred to as "upstream sequences"; sequence 
regions on the DNA strand having the same sequence as the RNA and which 
are 3' to the 3' end of the coding RNA transcript are referred to as 
"downstream sequences". 
As used herein, the term "polynucleotide" refers to a polymer composed of a 
multiplicity of nucleotide units (ribonucleotide or deoxyribonucleotide or 
related structural variants) linked via phosphodiester bonds. A 
polynucleotide can be of substantially any length, typically from about 10 
nucleotides to about 1.times.10.sup.9 nucleotides or larger. As used 
herein, an "oligonucleotide" is defined as a polynucleotide of from 6 to 
100 nucleotides in length. Thus, an oligonucleotide is a subset of 
polynucleotides. 
The term "naturally-occurring" as used herein as applied to an object 
refers to the fact that an object can be found in nature. For example, a 
polypeptide or polynucleotide sequence that is present in an organism 
(including viruses) that can be isolated from a source in nature and which 
has not been intentionally modified by man in the laboratory is 
naturally-occurring. Generally, the term naturally-occurring refers to an 
object as present in a non-pathological (undiseased) individual, such as 
would be typical for the species. 
The term "corresponds to" is used herein to mean that a polynucleotide 
sequence is homologous (i.e., is identical, not strictly evolutionarily 
related) to all or a portion of a reference polynucleotide sequence. In 
contradistinction, the term "complementary to" is used herein to mean that 
the complementary sequence is homologous to all or a portion of a 
reference polynucleotide sequence. For illustration, the nucleotide 
sequence "TATAC" corresponds to a reference sequence "TATAC" and is 
complementary to a reference sequence "GTATA". 
The following terms are used to describe the sequence relationships between 
two or more polynucleotides: "reference sequence", "comparison window", 
"sequence identity", "percentage of sequence identity", and "substantial 
identity". A "reference sequence" is a defined sequence used as a basis 
for a sequence comparison; a reference sequence may be a subset of a 
larger sequence, for example, as a segment of a full-length cDNA or gene 
sequence given in a sequence listing, or may comprise a complete cDNA or 
gene sequence. Generally, a reference sequence is at least 12 nucleotides 
in length, frequently at least 15 to 18 nucleotides in length, and often 
at least 25 nucleotides in length. Since two polynucleotides may each (1) 
comprise a sequence (i.e., a portion of the complete polynucleotide 
sequence) that is similar between the two polynucleotides, and (2) may 
further comprise a sequence that is divergent between the two 
polynucleotides, sequence comparisons between two (or more) 
polynucleotides are typically performed by comparing sequences of the two 
polynucleotides over a "comparison window" to identify and compare local 
regions of sequence similarity. 
A "comparison window", as used herein, refers to a conceptual segment of at 
least 12 contiguous nucleotide positions wherein a polynucleotide sequence 
may be compared to a reference sequence of at least 12 contiguous 
nucleotides and wherein the portion of the polynucleotide sequence in the 
comparison window may comprise additions or deletions (i.e., gaps) of 20 
percent or less as compared to the reference sequence (which does not 
comprise additions or deletions) for optimal alignment of the two 
sequences. Optimal alignment of sequences for aligning a comparison window 
may be conducted by the local homology algorithm of Smith and Waterman 
(1981) Adv. Appl. Math. 2: 482, by the homology alignment algorithm of 
Needleman and Wunsch (1970) J. Mol. Biol. 48: 443, by the search for 
similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 
(U.S.A.) 85: 2444, by computerized implementations of these algorithms 
(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software 
Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, 
Wis.), or by inspection, and the best alignment (i.e., resulting in the 
highest percentage of homology over the comparison window) generated by 
the various methods is selected. 
The term "sequence identity" means that two polynucleotide sequences are 
identical (i.e., on a nucleotide-by-nucleotide basis) over the window of 
comparison. The term "percentage of sequence identity" is calculated by 
comparing two optimally aligned sequences over the window of comparison, 
determining the number of positions at which the identical nucleic acid 
base (e.g., A, T, C, G, U, or I) occurs in both sequences to yield the 
number of matched positions, dividing the number of matched positions by 
the total number of positions in the window of comparison (i.e., the 
window size), and multiplying the result by 100 to yield the percentage of 
sequence identity. The terms "substantial identity" as used herein denotes 
a characteristic of a polynucleotide sequence, wherein the polynucleotide 
comprises a sequence that has at least 80 percent sequence identity, 
preferably at least 85 percent identity and often 90 to 95 percent 
sequence identity, more usually at least 99 percent sequence identity as 
compared to a reference sequence over a comparison window of at least 20 
nucleotide positions, frequently over a window of at least 25-50 
nucleotides, wherein the percentage of sequence identity is calculated by 
comparing the reference sequence to the polynucleotide sequence which may 
include deletions or additions which total 20 percent or less of the 
reference sequence over the window of comparison. The reference sequence 
may be a subset of a larger sequence. 
The primers herein are selected to be substantially complementary to the 
different strands of each specific sequence to be amplified. The primers 
must be sufficiently complementary to hybridize with their respective 
strands. Therefore, the primer sequence need not reflect the exact 
sequence of the template. For example, a non-complementary nucleotide 
fragment may be attached to the 5' end of the primer, with the remainder 
of the primer sequence being complementary to the strand. Alternatively, 
noncomplementary bases or longer sequences can be interspersed into the 
primer, provided that the primer sequence has sufficient complementarity 
with the sequence of the strand to be amplified to hybridize therewith and 
thereby form a template for synthesis of the extension product of the 
other primer. 
As used herein, a "bivalent primer" is a polynucleotide having two regions 
of complementarity to a predetermined target polynucleotide: (1) a first 
portion which is in the 5' portion of the bivalent primer and which is 
substantially complementary to a sequence in the 5' portion of the 
sequence to be amplified (target sequence) in the target polynucleotide, 
and (2) a second portion which is in the 3' portion of the primer and 
which is substantially complementary to a sequence in the 3' portion of 
the sequence to be amplified (target sequence) in the target 
polynucleotide. The portion of the bivalent primer which is substantially 
complementary to a sequence in the 3' portion of the sequence to be 
amplified (target sequence) is sufficiently long and sufficiently 
complementary to the target sequence to anneal under the reaction 
conditions and serve as an extendable primer for the polymerase to 
catalyze chain elongation. Similarly, the portion of the bivalent primer 
which is substantially complementary to a sequence in the 5' portion of 
the sequence to be amplified (target sequence) is sufficiently long and 
sufficiently complementary to the target sequence to anneal under the 
reaction conditions and serve as an extendable primer for the polymerase 
to catalyze chain elongation. Practitioners in the art will select at 
their discretion the specific structure of the bivalent primer(s) to be 
used in view of the necessity for annealing to the target. Typically, the 
portions of the bivalent primer which is substantially complementary to a 
sequence in the 5' and 3' portions of the sequence to be amplified (target 
sequence) are each at least 12 to 15 nucleotides in length, often 18 to 20 
nucleotides in length, and are preferably 100 percent identical to the 
complement of the annealing portion of the target sequence. Often, 
bivalent primers of the invention are oligonucleotides. 
The term "primer" as used herein refers to an oligonucleotide whether 
occurring naturally as in a purified restriction digest or produced 
synthetically, which is capable of acting as a point of initiation of 
synthesis when placed under conditions in which synthesis of a primer 
extension product which is complementary to a nucleic acid strand is 
induced, i.e., in the presence of nucleotides and an agent for 
polymerization such as DNA polymerase and at a suitable temperature and 
pH. The primer is preferably single-stranded for maximum efficiency in 
amplification, but may alternatively be double stranded. If double 
stranded, the primer is first treated to separate its strands before being 
used to prepare extension products. Preferably, the primer is an 
oligodeoxyribonucleotide. The primer must be sufficiently long to prime 
the synthesis of extension products in the presence of the agent for 
polymerization. The exact lengths of the primers will depend on many 
factors, including temperature and source of primers. For example, 
depending on the complexity of the target sequence, the oligonucleotide 
primer typically contains 15-25 or more nucleotides, although it may 
contain fewer nucleotides. Short primer molecules generally require cooler 
temperatures to form sufficiently stable hybrid complexes with template. 
In some embodiments, the primers can be large polynucleotides, such as 
from about 200 nucleotides to several kilobases or more. 
As used herein, "suitable reaction conditions" are those conditions 
suitable for conducting PCR amplification using conventional reagents. 
Such conditions are known or readily established by those of skill in the 
art, and can be exemplified by the reaction conditions used in U.S. Pat. 
Nos. 4,683,202, 4,683,195, and 4,800,159, which are incorporated herein by 
reference. As one example and not to limit the invention, suitable 
reaction conditions can comprise: 0.2 mM each dNTP, 2.2 mM MgCl.sub.2, 50 
mM KCl, 10 mM Tris-HCl pH9.0, 0.1% Triton X-100. 
As used herein the term "physiological conditions" refers to temperature, 
pH, ionic strength, viscosity, and like biochemical parameters which are 
compatible with a viable organism, and/or which typically exist 
intracellularly in a viable cultured yeast cell or mammalian cell. For 
example, the intracellular conditions in a yeast cell grown under typical 
laboratory culture conditions are physiological conditions. Suitable in 
vitro reaction conditions for PCR and many polynucleotide enzymatic 
reactions and manipulations are generally physiological conditions. In 
general, in vitro physiological conditions comprise 50-200 mM NaCl or KCl, 
pH 6.5-8.5, 20.degree.-45.degree. C. and 0.001-10 mM divalent cation 
(e.g., Mg.sup.++, Ca.sup.++); preferably about 150 mM NaCl or KCl, pH 
7.2-7.6, 5 mM divalent cation, and often include 0.01-1.0 percent 
nonspecific protein (e.g., BSA). A non-ionic detergent (Tween, NP-40, 
Triton X-100) can often be present, usually at about 0.001 to 2%, 
typically 0.05-0.2% (v/v). Particular aqueous conditions may be selected 
by the practitioner according to conventional methods. For general 
guidance, the following buffered aqueous conditions may be applicable: 
10-250 mM NaCl, 5-50 mM Tris HCl, pH 5-8, with optional addition of 
divalent cation(s) and/or metal chelators and/or nonionic detergents 
and/or membrane fractions and/or antifoam agents and/or scintillants. 
As used herein, the terms "label" or "labeled" refers to incorporation of a 
detectable marker, e.g., by incorporation of a radiolabeled nucleotide or 
incorporation of nucleotide having biotinyl moieties that can be detected 
by marked avidin (e.g., streptavidin containing a fluorescent marker or 
enzymatic activity that can be detected by optical or calorimetric 
methods). Various methods of labeling polynucleotides are known in the art 
and may be used. Examples of labels include, but are not limited to, the 
following: radioisotopes (e.g., .sup.3 H, .sup.14 C, .sup.35 S, .sup.125 
I, .sup.131 I), fluorescent labels (e.g., FITC, rhodamine, lanthanide 
phosphors), enzymatic labels (e.g., horseradish peroxidase, 
.beta.-galactosidase, luciferase, alkaline phosphatase), biotinyl groups, 
and the like. In some embodiments, labels are attached by spacer arms of 
various lengths to reduce potential steric hindrance. 
As used herein,, "substantially pure" means an object species is the 
predominant species present (i.e., on a molar basis it is more abundant 
than any other individual macromolecular species in the composition), and 
preferably a substantially purified fraction is a composition wherein the 
object species comprises at least about 50 percent (on a molar basis) of 
all macromolecular species-present. Generally, a substantially pure 
composition will comprise more than about 80 to 90 percent of all 
macromolecular species present in the composition. Most preferably, the 
object species is purified to essential homogeneity (contaminant species 
cannot be detected in the composition by conventional detection methods) 
wherein the composition consists essentially of a single macromolecular 
species. Solvent species, small molecules (&lt;500 Daltons), and elemental 
ion species are not considered macromolecular species. 
The term "recombinant" used herein refers to macromolecules produced by 
recombinant DNA techniques wherein the gene coding for a polypeptide is 
cloned by known recombinant DNA technology. For example, an amplified or 
assembled product polynucleotide may be inserted into a suitable DNA 
vector, such as a bacterial plasmid, and the plasmid used to transform a 
suitable host. The gene is then expressed in the host to produce the 
recombinant protein. The transformed host may be prokaryotic or 
eukaryotic, including mammalian, yeast, Aspergillus and insect cells. One 
preferred embodiment employs bacterial cells as the host. Alternatively, 
the product polynucleotide may serve a non-coding function (e.g., 
promoter, origin of replication, ribosome-binding site, etc.). 
Generally, the nomenclature used hereafter and many of the laboratory 
procedures in cell culture, molecular genetics, and nucleic acid chemistry 
and hybridization described below are those well known and commonly 
employed in the art. Standard techniques are used for recombinant nucleic 
acid methods, polynucleotide synthesis, in vitro polypeptide synthesis, 
and the like and microbial culture and transformation (e.g., 
electroporation). Generally enzymatic reactions and purification steps are 
performed according to the manufacturer's specifications. The techniques 
and procedures are generally performed according. to conventional methods 
in the art and various general references (see, generally, Sambrook et al. 
Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor 
Laboratory Press, Cold Spring Harbor, N.Y.; each of which is incorporated 
herein by reference) which are provided throughout this document. The 
procedures therein are believed to be well known in the art and are 
provided for the convenience of the reader. All the information contained 
therein is incorporated herein by reference. 
Oligonucleotides can be synthesized on an Applied Bio Systems 
oligonucleotide synthesizer according to specifications provided by the 
manufacturer. 
Methods for PCR amplification are described in the art (PCR Technology: 
Principles and Applications for DNA Amplification ed. H A Erlich, Stockton 
Press, New York, N.Y. (1989); PCR Protocols: A Guide to Methods and 
Applications, eds. Innis, Gelfland, Snisky, and White, Academic Press, San 
Diego, Calif. (1990); Mattila et al. (1991) Nucleic Acids Res. 19: 4967; 
Eckert, K. A. and Kunkel, T. A. (1991) PCR Methods and Applications 1: 17; 
and U.S. Pat. Nos. 4,683,202 and 4,965,188, each of which are incorporated 
herein by reference) and exemplified hereinbelow. 
Overview 
A basis of the present invention is the use of polymerase in combination 
with at least two polynucleotides having complementary ends which can 
anneal whereby at least one of said polynucleotides has a free 3'-hydroxyl 
capable of polynucleotide chain elongation by a DNA polymerase, such as a 
thermostable polymerase (e.g., Thermus aquaticus (Taq) polymerase or 
Thermococcus litoralis (Vent.TM.) polymerase. In an embodiment, the method 
is performed using PCR, typically with multiple cycles of heat 
denaturation and DNA synthesis. However, there are several variations of 
the basic method of end-complementary polymerase reaction which are 
exemplified hereinbelow and which shall be evident to the skilled artisan 
in view of the present specification. Some variations do not require 
primers and/or sequential cycles of thermal denaturation. 
In embodiments where the product size increases with the number of 
denaturation, annealing, and extension cycles (e.g., as the mean length of 
concatemers increase), it is typically advantageous to increase the 
denaturation temperature, and optionally increase the reannealing time, 
for subsequent cycles. Such conditions are readily optimized by the 
practitioner using pilot reactions to establish a calibration curve for 
any particular embodiment. 
Single-Primer Amplification 
A target polynucleotide is contacted with a bivalent primer typically 
comprising an oligonucleotide having two regions of complementarity to the 
target polynucleotide: (1) a first portion which is in the 5' portion of 
the primer and which is substantially complementary to a sequence in the 
5' portion of the sequence to be amplified (target sequence) in the target 
polynucleotide, and (2) a second portion which is in the 3' portion of the 
primer and which is substantially complementary to a sequence in the 3' 
portion of the sequence to be amplified (target sequence) in the target 
polynucleotide. The contacting is performed under conditions suitable for 
hybridization of the bivalent primer to the target polynucleotide for 
polymerase-mediated chain elongation, most often following thermal 
denaturation of the target polynucleotide if it is initially present as a 
double-stranded form. 
The first portion of the bivalent primer which is in the 5' portion of the 
primer and which is substantially complementary to a sequence in the 5' 
portion of the sequence to be amplified (target sequence) in the target 
polynucleotide is typically at least 12 nucleotides in length, often at 
least 15 nucleotides in length, frequently at least 18 nucleotides in 
length, and is commonly 20 to 25 or more nucleotides in length, but 
usually does not exceed 10,000 nucleotides in length and is frequently 
less than 50 to 500 nucleotides in length. The first portion of the 
bivalent primer is substantially identical to the complement of a sequence 
at the 3' end of the target sequence, however there may be additional 
terminal nucleotides of the first portion of the bivalent primer which are 
substantially non-identical to a target sequence or its complement. Such 
terminal nucleotides must be substantially non-interfering so that their 
presence does not significantly inhibit the capability of the bivalent 
primer to selectively anneal to the target sequence and initiate chain 
elongation under suitable reaction conditions in the presence of 
polymerase. Although the first portion of the bivalent primer is 
substantially identical to the complement of a sequence at the 5' end of 
the target sequence, it need not be exactly identical; often a sequence 
identity of at least 80 percent is sufficient, typically at least 90 
percent sequence identity is present, and preferably at least 95 percent 
or 100 percent sequence identity is present. As the length of the 
complementary sequence increases, typically the percentage of sequence 
identity necessary for specific annealing decreases within certain limits 
(pp. 399-407, in Berger and Kimmel, Methods in Enzymology Volume 152. 
Guide to Molecular Cloning Techniques (1987), Academic Press, Inc., San 
Diego, Calif., which is incorporated herein by reference). 
The second portion of the bivalent primer which is in the 3' portion of the 
primer and which is substantially complementary to a sequence in the 3' 
portion of the sequence to be amplified (target sequence) in the target 
polynucleotide is typically at least 12 nucleotides in length, often at 
least 15 nucleotides in length, frequently at least 18 nucleotides in 
length, and is commonly 20 to 25 or more nucleotides in length, but 
usually does not exceed 10,000 nucleotides in length and is frequently 
less than 50 to 500 nucleotides in length. The second portion of the 
bivalent primer is substantially identical to the complement of a sequence 
at the 3' end of the target sequence, however there may be additional 
terminal nucleotides of the first portion of the bivalent primer which are 
substantially non-identical to a target sequence or its complement. Such 
terminal nucleotides must be substantially non-interfering so that their 
presence does not significantly inhibit the capability of the bivalent 
primer to selectively anneal to the target sequence and initiate chain 
elongation under suitable reaction conditions in the presence of 
polymerase. Although the second portion of the bivalent primer is 
substantially identical to the complement of a sequence at the 3' end of 
the target sequence, it need not be exactly identical; often a sequence 
identity of at least 80 percent is sufficient, typically at least 90 
percent sequence identity is present, and preferably at least 95 percent 
or 100 percent sequence identity is present. In some embodiments, sequence 
identity of less than so percent is practicable, but the amount of 
sequence identity and length of overlap for the joints is determined by 
the discretion of the practitioner. 
The amount of sequence identity necessary for any given application will 
vary depending on several factors including: (1) complexity of the 
population of polynucleotides in which the target polynucleotide(s) is/are 
present, (2) temperature and ionic strength, (3) sequence composition of 
the target sequence, (4) length of sequence identity, and (5) size of the 
primer. Practitioners will select bivalent primers having a first portion 
with sufficient sequence identity and length to serve as selective 
amplification primers which specifically hybridize to the desired target 
polynucleotide(s). Specific hybridization is the formation of hybrids 
between a primer polynucleotide and a target polynucleotide, wherein the 
primer polynucleotide preferentially hybridizes to the target DNA such 
that, for example, at least one discrete band can be identified on a gel 
of amplification products obtained from amplification of genomic DNA 
prepared from eukaryotic cells that contain (or are spiked with) the 
target polynucleotide sequence. In some instances, a target sequence may 
be present in more than one target polynucleotide species (e.g., a 
particular target sequence may occur in multiple members of a gene family 
or in a known repetitive sequence). It is evident that optimal 
hybridization conditions will vary depending upon the sequence composition 
and length(s) of the targeting polynucleotide(s) and target(s), and the 
experimental method selected by the practitioner. Various guidelines may 
be used to select appropriate primer sequences and hybridization 
conditions (see, Maniatis et al., Molecular Cloning: A Laboratory Manual 
(1989), 2nd Ed., Cold Spring Harbor, N.Y.; Berger and Kimmel, Methods in 
Enzymology. Volume 152, Guide to Molecular Cloning Techniques (1987), 
Academic Press, Inc., San Diego, Calif.; PCR Protocols: A Guide to Methods 
and Applications, eds. Innis, Gelfland, Snisky, and White, Academic Press, 
San Diego, Calif. (1990); Benton W D and Davis R W (1977) Science 196: 
180; Goodspeed et al. (1989) Gene 76: 1; Dunn et al. (1989) J. Biol. Chem. 
264: 13057 which are incorporated herein by reference. 
The target polynucleotide may be substantially homogeneous or may be 
present in a mixture of polynucleotide species (e.g., in a genome, 
biological sample, or mixture of synthetic polynucleotides). Subsequent or 
concomitant with the contacting of the bivalent primer to the target 
polynucleotide, a polynucleotide polymerase, such as a thermostable DNA 
polymerase, e.g., Taq polymerase, TThI polymerase (Perkin Elmer) or 
Vent.TM. (New England Biolabs, Beverly, Mass.), catalyzes, under suitable 
reaction conditions, polynucleotide synthesis (chain elongation) primed 
from the 3'-hydroxyl of the annealed bivalent primer to form a strand 
complementary to the target sequence, thereby forming a nascent 
complementary strand. Following completion of the nascent complementary 
strand spanning the target sequence, the target polynucleotide and the 
nascent strand are denatured, typically by elevation of temperature, and 
allowed to reanneal, typically by reduction of temperature, with another 
molecule of the bivalent primer species or with a complementary strand of 
a target polynucleotide or an amplified copy thereof. The denatured 
nascent strand species following the first elongation cycle will contain a 
copy of the target sequence and has a terminal repeat of its 5'-terminal 
sequence at the 3' terminus, resulting from the bivalent primer, and 
wherein the terminal repeat is of sufficient length to support annealing 
under PCR conditions to an overlapping complementary strand in a 
head-to-tail arrangement (see, FIG. 1). Following reannealing, the 
described polymerase elongation/denaturation/reannealing cycle is repeated 
from 1 to about 100 times as desired, resulting in formation of amplified 
product which comprises head-to-tail concatemers of the target sequence. 
The concatemers typically increase in length as the number of 
amplification cycles increase and as the amount of bivalent primer 
decreases. Following amplification forming concatameric head-to-tail 
repeats of the target sequence, the concatemer(s) can optionally be 
resolved, such as (1) by cleaving with a restriction endonuclease which 
cuts within (or at the termini of) the concatemeric unit(s), (2) by 
homologous recombination between concatemer units to form covalently 
closed circles, or (3) by cleavage with a restriction endonuclease 
followed by ligation with DNA ligase to form covalently closed circles 
and/or by direct transformation into host cells for in vivo ligation. 
By this method, a single primer (bivalent primer) is used to amplify a 
target polynucleotide sequence having a predetermined 5' terminal sequence 
and a predetermined 3' terminal sequence. The predetermined 5' terminal 
sequence and a predetermined 3' terminal sequence may be contained 
internally within a larger polynucleotide; hence the use of the term 
"terminal" refers only to their terminality within the target sequence, 
not necessarily the complete target polynucleotide which may be a superset 
of the target sequence. 
Rollins Circle PCR Amplification 
Often, a target polynucleotide sequence which is amplified by the present 
method will form amplification intermediates in the form of cyclized DNA 
(see, FIG. 2), as a result of the 3' terminus of an overlapped nascent 
strand annealing to the 3' terminus of an overlapped complementary strand 
forming a cyclized (circular) structure similar to a gapped circle. The 
cyclized structure has a strand with an extendable 3'-hydroxyl which can 
be extended with a DNA polymerase substantially lacking exonuclease 
activity (e.g., a thermostable polymerase such as Vent(exo.sup.-).TM. or 
Klenow fragment, etc.) in a rolling circle format whereby the leading 
terminus of the nascent strand continually displaces the lagging portion 
of the nascent strand (see, FIG. 2) producing a concatemeric single strand 
propagating from the rolling circle intermediate. Most often, such rolling 
circle intermediates will form under dilute conditions more favorable to 
intramolecular cyclization of overlapped strands than to formation of 
additional intermolecular overlaps. Once a rolling circle intermediate is 
established, the template need not be denatured in order to continue 
amplification of the target sequence as in conventional PCR, since the 
polymerase continues around the circle processively. Thus, the 
advantageous formation of the rolling circle intermediate in the present 
method avoids the necessity of multiple thermal cycles of PCR to 
repeatedly denature and renature the amplification template (and the 
resultant time loss needed for heating and cooling). 
Overlapped Assembly of Polynucleotides 
The present invention also provides for assembly of one or more product 
polynucleotide(s) from a plurality of component polynucleotides which have 
overlapping complementary sequence portions at their termini. The 
component polynucleotides are conveniently single-stranded 
oligonucleotides, but can include double-stranded polynucleotides (which 
are generally denatured with elevated temperature) and long 
single-stranded polynucleotides. 
A desired product polynucleotide (or polynucleotide library) is assembled 
from a plurality of component polynucleotides by formation of overlapped 
strands of alternating polarity and having substantially complementary 
termini (see, FIG. 3). This method employs a series of overlapping 
substantially complementary termini to determine the linear order of 
component sequences in the final product. Concomitant with or subsequent 
to formation of the overlapped strands of the component polynucleotides in 
a reaction, a polynucleotide polymerase (e.g., a thermostable DNA 
polymerase) under suitable reaction conditions catalyzes strand elongation 
from the 3'-hydroxyl portions of the overlapped (annealed) joints, filling 
in the portion between joints and processively displacing or processively 
degrading exonucleolytically the 5' termini of downstream component 
strands of the same polarity as the nascent strand elongates. After a 
cycle of chain elongation forming substantially double-stranded 
polynucleotides, the reaction conditions are altered (typically by 
increasing the temperature) to effect denaturation of the double-stranded 
polynucleotides, followed by altering the reaction conditions to permit 
reannealing of complementary strands or portions thereof (i.e., 
overlapping termini) to form molecules having overlapped termini (joints), 
and chain elongation by a polynucleotide polymerase under suitable 
reaction conditions catalyzes strand elongation from the 3'-hydroxyl 
portions of the overlapped (annealed) joints, as in the first cycle. One 
to about 100 cycles of denaturation/annealing/polymerization can be 
performed to generate a product comprising the component polynucleotide 
sequences covalently linked in linear order according to the order of the 
overlapping joints. In this embodiment, a product polynucleotide can be 
constructed from a plurality of smaller component polynucleotides 
(typically oligonucleotides) and enables assembly of a variety of products 
with alternate substitutable polynucleotide components at a given position 
serving as structural "alleles" (see, FIG. 4). The component 
polynucleotides are often provided in single-strand form, but may 
initially be present in double-strand form and be denatured (typically by 
elevated temperature) for the assembly of the product by PCR 
amplification. Substantially any type of product polynucleotide can be 
assembled in this way, including cloning and expression vectors, viral 
genomes, gene therapy vectors, genes (including chimeric genes), 
polynucleotides encoding peptide libraries, and the like. In a variation, 
one or more of the component polynucleotides represents a site-directed 
mutation or variable-sequence kernal. In a variation, PCR employing a 
low-fidelity polymerase is used to introduce additional sequence variation 
into the product polynucleotide(s) during amplification cycles. The method 
can be used to produce a library of sequence-variant product 
polynucleotides, if desired. 
Kits 
The invention also provides kits comprising a bivalent primer 
polynucleotide and/or a plurality of component polynucleotides and 
instructions for use describing the present end-complementary 
amplification method disclosed herein. Frequently, a polynucleotide 
polymerase, such as a thermostable DNA polymerase (Taq or Vent.TM. 
polymerase) is also present in the kit. Optionally, one or more target 
polynucleotides may be provided in the kit, such as for calibration and/or 
for use as a positive control to verify correct performance of the kit. 
General Aspects 
The target polynucleotides or component polynucleotides may be obtained 
from any source, for example, from plasmids such as pBR322, from cloned 
DNA or RNA, or from natural DNA or RNA from any source, including 
bacteria, yeast, viruses, and higher organisms such as plants or animals. 
DNA or RNA may be extracted from blood, tissue material such as chorionic 
villi or amniotic cells by a variety of techniques such as that described 
by Maniatis et al., Molecular Cloning: A Laboratory Manual, (New York: 
Cold Spring Harbor Laboratory, 1982), pp. 280-281. Alternatively, the 
polynucleotides may be produced by chemical synthesis by any of the 
art-recognized methods. 
Any specific nucleic acid sequence can be produced by the present process. 
It is only necessary that a sufficient number of bases at both ends of the 
sequence be known in sufficient detail so that a bivalent primers can be 
prepared which will hybridize to the desired sequence and at relative 
positions along the sequence such that an extension product initially 
synthesized from the bivalent primer, when it is separated from its 
template (complement), can anneal with a stand of the opposite polarity to 
form an overlapped joint of a head-to-tail concatemer and serve as a 
template for extension of the 3'-hydroxyl from each overlapped joint. The 
greater the knowledge about the bases at both ends of the sequence, the 
greater can be the specificity of the primer for the target nucleic acid 
sequence, and thus the greater the efficiency of the process. It will be 
understood that the word bivalent primer as used hereinafter may refer to 
more than one bivalent primer, particularly in the case where there is 
some ambiguity in the information regarding the terminal sequence(s) of 
the fragment to be amplified. For instance, in the case where a nucleic 
acid sequence is inferred from protein sequence information a collection 
of primers containing sequences representing all possible codon variations 
based on degeneracy of the genetic code will be used for each strand. 
The polynucleotide primers may be prepared using any suitable method, such 
as, for example, the phosphotriester and phosphodiester methods, or 
automated embodiments thereof. In one such automated embodiment 
diethylphosphoramidites are used as starting materials and may be 
synthesized as described by Beaucage et al. (1981) Tetrahedron Letters 22: 
1859. One method for synthesizing oligonucleotides on a modified solid 
support is described in U.S. Pat. No. 4,458,066. It is also possible to 
use a primer which has been isolated from a biological source (such as a 
restriction endonuclease digest or the like. 
The specific nucleic acid sequence is produced by using the target 
polynucleotide containing that sequence as a template. If the target 
polynucleotide contains two strands, it is necessary to separate the 
strands of the nucleic acid before it can be used as the template, either 
as a separate step or simultaneously with the synthesis of the primer 
extension products. This strand separation can be accomplished by any 
suitable denaturing method including physical, chemical or enzymatic 
means. One physical method of separating the strands of the polynucleotide 
involves heating the polynucleotide until it is substantially denatured. 
Typical heat denaturation may involve temperatures ranging from about 
80.degree. to 105.degree. C. for times ranging from about 10 seconds to 
about 10 minutes or more. Strand separation may also be induced by an 
enzyme from the class of enzymes known as helicases or the enzyme RecA, 
which has helicase activity and in the presence of rATP is known to 
denature DNA. The reaction conditions suitable for separating the strands 
of polynucleotides with helicases are described by Cold Spring Harbor 
Symposia on Quantitative Biology, Vol. XLIII "DNA: Replication and 
Recombination" (New York: Cold Spring Harbor Laboratory, 1978), B. Kuhn et 
al., "DNA Helicases", pp. 63-67, and techniques for using RecA are 
reviewed in C. Radding, Ann. Rev. Genetics, 16:405-37 (1982). 
PCR synthesis can be performed using any suitable method. Generally it 
occurs in a buffered aqueous solution, preferably at a pH of 7-9, most 
preferably about 8. The bivalent primer(s) is/are added in suitable 
amounts (molar ratio to target), typically less than conventional PCR 
methods because of the self-priming nature of the overlapped concatemers. 
The deoxyribonucleoside triphosphates dATP, dCTP, dGTP and TTP are also 
added to the synthesis mixture in adequate amounts and the resulting 
solution is heated to about 85.degree.-100.degree. C. for from about 1 to 
10 minutes, preferably from 1 to 4 minutes. After this heating period the 
solution is allowed to cool to from 20.degree.-40.degree. C., which is 
preferable for the primer hybridization. To the cooled mixture is added an 
agent for polymerization, and the reaction is allowed to occur under 
conditions known in the art. This synthesis reaction may occur at from 
room temperature up to a temperature above which the agent for 
polymerization no longer functions efficiently. Thus, for example, if DNA 
polymerase is used as the agent for polymerization, the temperature is 
generally no greater than about 45.degree. C. The agent for polymerization 
may be any compound or system which will function to accomplish the 
synthesis of primer extension products, including enzymes. Suitable 
enzymes for this purpose include, for example, E. coli DNA polymerase I, 
Klenow fragment of E. coli DNA polymerase I, T4 DNA polymerase, other 
available DNA polymerases, reverse transcriptase, and other enzymes, 
including heat-stable enzymes, which will facilitate combination of the 
nucleotides in the proper manner to form the primer extension products 
which are complementary to each nucleic acid strand. Generally, the 
synthesis will be initiated at the 3' end of the primer and proceed in the 
5' direction along the template strand, until synthesis terminates, 
producing molecules of different lengths. 
The newly synthesized strand and its complementary nucleic acid strand form 
a double-stranded molecule which is used in the succeeding steps of the 
process. In the next step, the strands of the double-stranded molecule are 
separated using any of the procedures described above to provide 
single-stranded molecules. 
The steps of strand separation and extension product synthesis can be 
repeated as often as needed to produce the desired quantity of the 
specific nucleic acid sequence. The amount of the specific nucleic acid 
sequence produced will accumulate in an exponential fashion and the 
average size of the product will also increase as the length of the 
concatemers increases with each cycle. 
The method herein may also be used to enable detection and/or 
characterization of specific nucleic acid sequences associated with 
infectious diseases, genetic disorders or cellular disorders such as 
cancer, e.g., oncogenes. Amplification is useful when the amount of 
nucleic acid available for analysis is very small, as, for example, in the 
prenatal diagnosis of sickle cell anemia using DNA obtained from fetal 
cells. 
Continuous Multiplex Amplification 
Continuous multiplex amplification can be used to amplify by any suitable 
amplification method, typically by PCR, a plurality of unlinked or 
distantly linked polynucleotide sequences. Certain genetic diagnostic 
tests require amplification of multiple segments (e.g., exons) of a gene. 
Each segment is typically amplified in a separate amplification reaction. 
Unfortunately, it is generally difficult or impossible to amplify each 
segment in approximately equimolar ratios due to differences in priming 
efficiency, length of extension, secondary structure, or other factors 
which affect amplification rate. In continuous multiplex amplification, 
the amplification reactions can be run together in a single reaction 
vessel using a common pool of reagents where the unlinked (or distantly 
linked) sequences become part of the same amplification product, which 
affords the substantially equimolar amplification of the unlinked (or 
distantly linked) sequences. An embodiment of the invention is illustrated 
schematically in FIGS. 9A-9C in the case of amplifying two unlinked 
sequences represented in the double-stranded polynucleotide fragments 
ABC/A'B'C' and DEF/D'E'F'. primers C'X'D', F'X'A', FYA, and CYD are added 
and annealed to the denatured polynucleotide fragments; the primer 
concentrations are typically lower than conventionally used for PCR 
primers. X and Y, and their complements X' and Y' are generally 
predetermined sequences which are selected to destabilize the 
primer:primer hybrids CYD/C'X'D' and FYA/F'X'A', such as by having the X 
and Y sequences (and their complements) lack substantial sequence 
identity. After extension with a polymerase, the following products and 
their complements result: ABCXD, DEFXA, FYABC, and CYDEF. A variety of 
hybrid combinations of product:product and primer:product can form and 
after another round of amplification a variety of amplification products 
result. Each of the pairs is capable of self-priming with its complement 
or with the complement of another fragment which has a complementary 
sequence. Through multiple cycles of amplification, the initial primer 
population becomes depleted and primarily extended products remain. These 
extended products will prime each other and generate increasingly longer 
amplification products which contain the initial unlinked (or distantly 
linked) sequences in equal amounts. After completion of amplification, 
several options can be pursued; the amplification product(s) can be used 
directly, the X and/or Y sequences can contain restriction sites 
(preferably unique sites) to allow digestion with the restrictino enzyme 
and, if desired, separation and/or purification of the two (or more) 
originally unlinked sequences. Alternatively, or in combination, 
transcription promoters (e.g., T3 and T7) can be included in X and/or Y 
sequences to facilitate transcription of the amplified sequences. FIGS. 
10A-10C show a linear format of the continuous multiplex amplification 
method. FIGS. 11A-11C show an embodiment of circular continuous multiplex 
amplification wherein bivalent primers contain T3 and T7 promoters and the 
functional promoter sequences are thereby introduced into the 
amplification product(s). 
The following examples are given to illustrate the invention, but are not 
to be limiting thereof. 
EXPERIMENTAL EXAMPLES 
The following examples are offered by way of example and not by way of 
limitation. Variations and alternate embodiments will be apparent to those 
of skill in the art. 
Example 1 
LacZ Alpha Gene Reassembly 
This example shows that small fragments having overlapping regions of 
homology can be amplified and reassembled by PCR amplification methods in 
the absence of any primer. 
1) Substrate Preparation 
The substrate for the reassembly reaction was the dsDNA polymerase chain 
reaction ("PCR") product of the wild-type LacZ alpha gene from pUC18. 
(Gene Bank No. XO2514) The primer sequences were 5'AAAGCGTCGATTTTTGTGAT3' 
(SEQ ID NO:1) and 5'ATGGGGTTCCGCGCACATTT3' (SEQ ID NO:2). The free primers 
were removed from the PCR product by Wizard PCR prep (Promega, Madison 
Wis.) according to the manufacturer's directions. The removal of the free 
primers was found to be important. 
2) DNAseI Digestion 
About 5 .mu.g of the DNA substrate was digested with 0.15 units of DNAseI 
(Sigma, St. Louis Mo.) in 100 .mu.l of (50 mM Tris-HCl pH 7.4, 1 mM 
MgCl.sub.2), for 10-20 minutes at room temperature. The digested DNA was 
run on a 2% low melting point agarose gel. Fragments of 10-70 basepairs 
(bp) were purified from the 2% low melting point agarose gels by 
electrophoresis onto DE81 ion exchange paper (Whatman, Hillsborough 
Oreg.). The DNA fragments were eluted from the paper with 1 M NaCl and 
ethanol precipitated. 
3) DNA Reassembly 
The purified fragments were resuspended at a concentration of 10-30 
ng/.mu.l in PCR Mix (0.2 mM each dNTP, 2.2 mM MgCl.sub.2, 50 mM KCl, 10 mM 
Tris-HCl pH 9.0, 0.1% Triton X-100, 0.3 .mu.l Taq DNA polymerase, 50 .mu.l 
total volume). No primers were added at this point. A reassembly program 
of 94.degree. C. for 60 seconds, 30-45 cycles of 94.degree. C. for 30 
seconds, 50.degree.-55.degree. C. for 30 seconds, 72.degree. C. for 30 
seconds! and 5 minutes at 72.degree. C. was used in an MJ Research 
(Watertown Mass.) PTC-150 thermocycler. The PCR reassembly of small 
fragments into larger sequences was followed by taking samples of the 
reaction after 25, 30, 35, 40, and 45 cycles of reassembly. 
Whereas the reassembly of 100-200 bp fragments can yield a single PCR 
product of the correct size, 10-50 base fragments typically yield some 
product of the correct size, as well as products of heterogeneous 
molecular weights. Most of this size heterogeneity appears to be due to 
single-stranded sequences at the ends of the products, since after 
restriction enzyme digestion a single band of the correct size is 
obtained. 
4) PCR with Primers 
After dilution of the reassembly product into the PCR Mix with 0.8 .mu.M of 
each of the above primers (SEQ ID Nos: 1 and 2) and about 15 cycles of 
PCR, each cycle consisting of (94.degree. C. for 30 seconds, 50.degree. C. 
for 30 seconds and 72.degree. C. for 30 seconds), a single product of the 
correct size was obtained. 
5) Cloning and Analysis 
The PCR product from step 4 above was digested with the terminal 
restriction enzymes BamHI and EcoO109 and gel purified as described above 
in step 2. The reassembled fragments were ligated into pUC18 digested with 
BamHI and EcoO109. E. coli were transformed with the ligation mixture 
under standard conditions as recommended by the manufacturer (Stratagene, 
San Diego Calif.) and plated on agar plates having 100 .mu.g/ml 
ampicillin, 0.004% X-gal and 2 mM IPTG. The resulting colonies having the 
HinDIII-NheI fragment which is diagnostic for the ++ recombinant were 
identified because they appeared blue. 
This Example illustrates that a 1.0 kb sequence carrying the LacZ alpha 
gene can be digested into 10-70 bp fragments, and that these gel purified 
10-70 bp fragments can be reassembled to a single product of the correct 
size, such that 84% (N=377) of the resulting colonies are LacZ.sup.+ 
(versus 94% without shuffling). This principal finding is extended 
substantially in the present invention to assemble component 
polynucleotides into product polynucleotides, and the component 
polynucleotides are not limited to randomly digested fragments of a 
naturally-occurring gene sequence. 
The DNA encoding the LacZ gene from the resulting LacZ.sup.- colonies was 
sequenced with a sequencing kit (United States Biochemical Co., Cleveland 
Ohio) according to the manufacturer's instructions and the genes were 
found to have point mutations due to the reassembly process (Table 1). 
11/12 types of substitutions were found, and no frameshifts. 
TABLE 1 
______________________________________ 
Mutations introduced by mutagenic shuffling 
Transitions 
Frequency Transversions 
Frequency 
______________________________________ 
G-- A 6 A-- T 1 
A-- G 4 A-- C 2 
C-- T 7 C-- A 1 
T-- C 3 C-- G 0 
G-- C 3 
G-- T 2 
T-- A 1 
T-- G 2 
______________________________________ 
A total of 4,437 bases of shuffled lacZ DNA were sequenced. 
The rate of point mutagenesis during DNA reassembly from 10-70 bp pieces 
was determined from DNA sequencing to be 0.7% (N=4,473), which is similar 
to error-prone PCR. Without being limited to any theory it is believed 
that the rate of point mutagenesis may be lower if larger fragments are 
used for the reassembly, or if a proofreading polymerase is added. 
When plasmid DNA from 14 of these point-mutated LacZ.sup.- colonies were 
combined and again reassembled/shuffled by the method described above, 34% 
(N=291) of the resulting colonies were LacZ.sup.+, and these colonies 
presumably arose by recombination of the DNA from different colonies. 
The expected rate of reversal of a single point mutation by error-prone 
PCR, assuming a mutagenesis rate of 0.7% (10), would be expected to be 
&lt;1%. 
Thus large DNA sequences can be reassembled from a random mixture of small 
fragments by a reaction that is surprisingly efficient and simple. One 
application of this technique is the recombination or shuffling of related 
sequences based on homology. A second application is the assembly of a 
large product polynucleotide by PCR amplification of component 
polynucleotides (oligonucleotides) having overlapping regions of homology 
to form annealed joints during PCR amplification. 
Example 2 
One-Step Circular Plasmid Assembly From Oligonucleotides 
This example demonstrates assembly of a 2.71 kb plasmid p182SfiI (Stemmer 
(1994) Nature 370: 389) which encodes the gene and promoter region for 
R-TEM1 .beta.-lactamase. A collection of 132 component oligonucleotides, 
each 40 bases in length, as well as one 56-mer and one 47-mer (see, FIG. 
5A-E) were synthesized and used to assemble the plasmid by 
end-complementary polymerase reaction (ECPR) employing the overlapping 
ends of the oligonucleotides. This collection of component polynucleotides 
collectively encode the plasmid p182SfiI. The plus strand and the minus 
strand were each initially directed by oligonucleotides 40 nucleotides 
long which, upon assembly, overlapped by 20 nucleotides (FIG. 6). The 
oligonucleotides were synthesized and 5'-phosphorylated simultaneously on 
a 96-well parallel-array DNA synthesizer using standard phosphoramidite 
chemistry. After cleavage from the solid support and deprotection, the 
dried down oligonucleotides were resuspended in distilled water and used 
without further purification. 
The oligonucleotides were diluted to a final concentration (all oligos 
combined) of 1 .mu.M (14 ng/.mu.l) in 20 .mu.l of GeneAMP XL PCR Mix 
(Perkin-Elmer, Branchburg, N.J.; 0.2 mM each dNTP, 2.2 mM MgCl.sub.2, 50 
mM KCl, 10 mM Tris-HCl pH 9.0, 0.1% Triton X-100). An aliquot of the 
reaction mix (A) was electrophoresed on an agarose gel (FIG. 7). The 
reaction was started with 5 .mu.l of a 50:1 (v/v) mixture of Taq 
polymerase (Promega, Madison, Wis.) and Pfu polymerase (Stratagene, La 
Jolla, Calif.) such that 1 unit of Taq and 0.02 unit of Pfu polymerase 
were added. The PCR program consisted of the following program: 40.degree. 
C. for 2 minutes, 72.degree. C. for 10 seconds, then 40 cycles of 
(94.degree. C. for 15 seconds, 40.degree. C. for 30 seconds, and 
72.degree. C. for 10 seconds+1 second/cycle!). An aliquot of the 
resulting reaction product (B) was electrophoresed on an agarose gel (FIG. 
7), the remainder was then diluted 3x with XL PCR Mix and enzyme and 
amplified with the following program: 25 cycles of (94.degree. C. for 15 
seconds, 40.degree. C. for 30 seconds, and 72.degree. C. for 45 seconds 
+1 second/cycle!). An aliquot of the resulting reaction product (C) was 
electrophoresed on an agarose gel (FIG. 7) and the remainder then diluted 
3x with XL PCR Mix and enzyme and amplified with the following program: 20 
cycles of (94.degree. C. for 15 seconds, 40.degree. C. for 30 seconds, and 
72.degree. C. for 70 seconds +1 second/cycle!). An aliquot of the 
resulting reaction product (D) was electrophoresed on an agarose gel (FIG. 
7). 
The an aliquot of the reaction product (D) was electrophoresed on an 
agarose gel, as was an aliquot which was digested with BamHI, which 
verified assembly of large DNA molecules consistent with formation of 
large concatemers which was resolved to unit length with BamHI digestion. 
The PCR product obtained by this method was shown to be concatemeric, and 
was resolvable by BamHI digestion into a single 2.7 kb band by agarose gel 
electrophoresis. 
Aliquots of reaction product (D) were digested with various restriction 
enzymes (shown in FIG. 7). FIG. 7 shows that the unique cutters EcoRI and 
BamHI liberated a 2.71 kb fragment consistent with the size of a complete 
2.71 kb plasmid p182SfiI (Stemmer (1994) Nature 370: 389, incorporated 
herein by reference). Furthermore, the digestion results with NcoI, SfiI, 
PstI, and BglII all yielded fragments consistent with the restriction map 
of the complete 2.71 kb plasmid p182SfiI. 
After digestion of the PCR product with BamHI, the 2.7 kb fragment was gel 
purified and ligated with ligase, and transformed into E. coli.and 
transformed into E. coli K-12. Tetracycline-resistant transformants were 
selected. 
This example demonstrates that the circular DNA assembly method allows for 
rapid and inexpensive construction of long DNA sequences, such as genes, 
gene libraries, plasmids, viral genomes, etc. The assembly method 
facilitates several mutagenesis approaches, such as point mutagenesis, 
combinatorial cassette mutagenesis, and doping, or mixing in other 
nucleotides during oligonucleotide synthesis. Deliberate modifications to 
the DNA sequence can be made simply by substituting one or more new oligos 
followed by reassembly. To reduce the rate of PCR mutagenesis during 
assembly, the addition of a proofreading polymerase can assure efficient 
long-read PCR reactions by combining high processivity with proofreading. 
Example 3 
Antibody Germline Assembly from Oligos With Rolling Circle Concatemeric 
Amplification 
A scFv antibody with germline sequences (V.sub.H 251 and V.sub.IA 25) was 
constructed from 19 oligonucleotides by cyclized assembly. The oligos were 
at 2-20 ng per .mu.l in PCR Mix. the program was 20 cycles of (94.degree. 
C. for 15s, 48.degree. C. for 30s, 72.degree. C. for 30+1s/cycle). The 
size of the product of this reaction was 200-500 bp. The PCR product was 
diluted 4-fold in PCR Mix and PCR was run for 24 cycles of (94.degree. C. 
for 15s, 55.degree. C. for 30s, 72.degree. C. for 30+8s/cycle), followed 
by one additional 3-fold dilution and 20 cycles of (94.degree. C. for 15s, 
55.degree. C. for 30s, 72.degree. C. for 30+8s/cycle). The product was &gt;50 
kilobases, and after digestion with SfiI and NotI resulted in a single DNA 
fragment of the correct size. 
FIG. 8 shows a schematic for end-complementary polymerase reaction (ECPR) 
in conjunction with parallel-processing PCR to amplify very large 
polynucleotides, such as those larger than can be amplified reliably by 
conventional PCR using only a single primer set. 
Example 4 
Plasmid Assembly With Rolling Circle Concatemeric Amplification 
pGJ103 is a 5.5 kilobase plasmid containing an intact ars operon (Ji and 
Silver (1992) Proc. Natl. Acad. Sci. (USA) 89: 9474). 
In one example, pGJ103 was digested with DNAseI into random 100-400 bp 
fragments which were reassembled by circular shuffling in PCR Mix with a 
program of 50 cycles of (94.degree. C. for 15s, 68.degree. C. for 
30s+8s/cycle), using three different concentrations of fragments. Each 
reassembly yielded a product of &gt;50 kb which was digested with BamHI to 
yield a single band of the correct (predicted) size, which was ligated, 
transformed into E. coli, and preplated on increasing levels of arsenate 
to select for up-mutants. 
Cells and Plasmids 
Plasmid pGJ103 is pUC19 derivative containing the 2.5 kb arsenic resistance 
operon from S. aureus plasmid pI258. E. coli strain TG1 was obtained 
commercially (Pharmacia, Tarrytown, N.J.). Sodium arsenate (Sigma) was 
used as a 2.5 M stock solution, neutralized to pH 7 with NaOH. Selection 
for increased arsenate resistance was performed at 35.degree. C. on agar 
plates with LB medium (Life Technologies) containing varying 
concentrations of arsenate. 
The 5.5 kb plasmid pGJ103 was fragmented by sonication into fragments of 
400-1500 bp, and reassembled by PCR using Perkin Elmer XL-PCR reagents 
with 10% PEG-6000, using PCR without added primers. The PCR program for 
the assembly was 90.degree. C. 30 s, than 60 cycles of: 94.degree. C. 20 
s, 40.degree.-45.degree. C. 30 s, 72.degree. C. 39s+1 s per cycle in a 
PTC-150 minicycler (MJ Research, Watertown, Mass.). The PCR process 
yielded plasmid multimers of about 15 to 40 kb in size, that were digested 
into 5.5 kb monomers with the restriction enzyme BamHI, which has a single 
unique site in plasmid pGJ103. The 5.5 kb plasmid monomer was purified 
from an agarose gel after electrophoresis, and after self-ligation, it was 
electroporated into electrocompetent E. coli TG1 cells. 
Arsenate Resistance Selection 
Transformed E. coli cells were plated on LB plates containing a range of 
concentrations of sodium and arsenate and incubated at 37.degree. C. for 
24 hrs., and at least 1,000 colonies from the plates with the highest 
arsenate levels were pooled by scraping the plates. The harvested cells 
were grown in liquid in the presence of the same concentration of arsenate 
as in the petri dish and a plasmid pool was prepared from this liquid 
culture. Rounds 2-4 were identical to round 1, except that the cells were 
plated at higher arsenate levels. 
Arsenate Resistance Quantification 
Induced inoculum cells of E. coli TG1(pGJ103), wild type ars operon, and 
TG1 with mutant pGJ103 plasmid pools were grown overnight at 37.degree. C. 
in 2 mM or 50 mM arsenate, respectively. Equal amounts of cells (by 
turbidity as OD600 nm) were on plates containing a range of concentrations 
of arsenate and grown for 18 hrs. at 37.degree. C. Cell growth was 
quantitated by resuspending the cells and measuring the OD600 nm. 
Arsenate Detoxification Assay 
The ability of E. coli constructs to detoxify arsenate was measured by 
intact cell arsenate reduction assay using radioactive 73As043-as 
substrate and separation of arsenate and arsenite of by thin layer 
chromatography, followed by quantitation in an Ambis radioactive counter. 
DNA Sequencing 
The sequence of the entire operon after selection was determined by dideoxy 
DNA sequencing using fluorescent terminating substrate and an ABI 
sequencer. 
Results and Discussion 
The wild type plasmid pGJ103 ars operon confers on E. coli strain TG1 
resistance to up to 4 mM arsenate when grown on LB plates at 37.degree. C. 
for 24 hrs. Selection round one, which was plated on 2, 4, 8, 16 and 32 mM 
arsenate, yielded about 2,000 colonies growing at 16 mM arsenate. 
Selection round two was plated 16, 32, 64 and 128 mM arsenate and yielded 
about 4,000 colonies growing at 64 mM arsenate. Round three was plated at 
64, 128 and 256 mM arsenate and yielded about 1,500 colonies at 128 mM 
arsenate, and round 4 was plated on 128, 256 and 512 mM arsenate. Colonies 
were harvested from the plates with 256 mM arsenate and replated on 200, 
300 and 400 mM arsenate. Single colonies from plates with 400 mM arsenate 
were grown in liquid culture with 400 mM arsenate and frozen at 
-70.degree. C., and used for further characterization. Resistance levels 
were increased by DNA shuffling to arsenate (as selected) and also to 
arsenite and antimony salts (FIG. 12), which are the two toxic oxyanions 
to which resistance requires the ArsB membrane transporter but not the 
ArsC arsenate reductase enzyme. In this growth experiment, done with the 
pool from three cycles of DNA shuffling (which retained good growth in LB 
broth), not only was growth clear about 100 mM As043-, but increased 
resistance to arsenite (As02-) and antimony (Sb03+) was clearly=20 shown. 
These results require mutational effects beyond those possibly limited to 
the arC gene, which affects resistance to arsenate alone. 
Chromosomal Integration 
Cells selected and grown at and above 128 mM arsenate resulted in smaller 
growth, lower cell growth yields, and in low and variable plasmid yields. 
Plasmids were isolated that had apparently lost the arsenate operon, and 
most cells showed a complete loss of plasmids. It appeared that the DNA 
shuffling plus selection for high arsenate resistance resulted in 
integration of the ars operon into the E. coli chromosome, since the ars 
operon could be recovered from chromosomal DNA of clones which had lost 
the entire plasmid by conventional PCR amplification with "upstream" and 
"downstream" oligonucleotide primers. 
Integration Mechanism 
The arsenate resistance operon of plasmid pGJ103 is flanked on both sides 
by 200 bp inverted homologous regions, which appear to be the terminal 
portions of site-specific recombinase genes. Attempts to recover the 
operon from the total cellular DNA of highly resistant cells by PCR showed 
that oligonucleotide primers near the inside ends of the recombinanse 
genes, immediately flanking the arsenate genes, yielded a PCR product of 
the correct size (2.0 kb) and with the expected restriction nuclease site 
pattern. However, primers located toward the middle or near the outside 
ends of the 200 bp homologous sequences did not yield predicted PCR 
products with the intact ars operon. Presumably, chromosomal integration 
was selected because the integrated operon somehow resulted in increased 
arsenate resistance and the homologous sequences at the ends of the ars 
operon facilitated chromosomal integration by recombination. 
Chromosomal ars Operon 
The chromosome of E. coli normally contains an arsenate resistance operon 
which is distantly related to the pI258 operon and results in a low level 
of arsenate resistance. The operon which was recovered from the chromosome 
of highly resistant cells by PVR was shown by restriction mapping and by 
DNA sequencing to be derived from the pI258 operon, and not from the E. 
coli K-12 chromosomal operon. 
Chromosomal Shuffling 
Because the cells recovered from 128 mM arsenate did not contain plasmid 
DNA, the shuffling for round 4 was performed on the PCR product which was 
obtained from the chromosomal DNA of the cells selected in round 3. This 
PCR product was combined with a 10-fold lower molar amount of the plasmid 
DNA obtained from round 2 cells, and the mixture was fragmented, shuffled 
and selected as for earlier rounds. 
Cloning and Characterization of the Integrated Operon 
The conventional PCR product which was obtained from the chromosomal DNA of 
cells grown at 400 mM arsenate was cloned into the polylinker site of 
pUC19. This construct was similar to pGJ103 except that it lacked the 200 
bp inverted homologous DNA flanking the arsenate operon. Cells containing 
this plasmid were resistant only up to about 10 mM arsenate. The reason 
for this loss of arsenate resistance level is not known. The DNA sequence 
of this cloned chromosomal operon showed thirteen base changes relative to 
the original sequence. The arsR gene contained two silent mutations (T389C 
and T429C. The arsB gene contained ten base changes, and one base change 
occurred in the non-coding area past the end of the arsC gene (G2469C). of 
the ten base changes in arsB, three resulted in amino acid alterations: 
base T1281C change resulted in amino acid change L232S, base T1317C change 
resulted in amino acid change F244S, and base T1853C change resulted in 
amino acid change Y423H, all three involving a change toward a more 
hydrophilic residue via a T to C transition. The seven silent mutations 
were T961G, A976G, T1267C, A1402G, T1730C, T1819C and T1844C. 
Arsenate Reductase Activity 
The activity of arsenate reductase by whole mutant cells after the third 
cycle was increased about 50-fold (FIG. 13) to the wild type initial 
strain with plasmid pGJ103. This increase in whole cell reductase rate 
appeared to be more dependent on an increase in rate reduction and not on 
an enhanced affinity of arsenate for the cells (data not shown). This is 
consistent with the finding that the mutations occurred in the efflux 
transport protein and not in the arsenate reductase itself. 
Although the present invention has been described in some detail by way of 
illustration for purposes of clarity of understanding, it will be apparent 
that certain changes and modifications may be practiced within the scope 
of the claims. 
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# SEQUENCE LISTING 
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(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: 
# 40 GGCA CTTTTCGGGG AAATGTGCGC 
- (2) INFORMATION FOR SEQ ID NO:32: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: 
# 40 TATT TTTGGCCACC ATGGCCTAAA 
- (2) INFORMATION FOR SEQ ID NO:33: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: 
# 40 TCCG CTCATGAGAC AATAACCCTG 
- (2) INFORMATION FOR SEQ ID NO:34: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: 
# 40 TATT GAAAAAGGAA GAGTATGAGT 
- (2) INFORMATION FOR SEQ ID NO:35: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: 
# 40 TCGC CCTTATTCCC TTTTTTGCGG 
- (2) INFORMATION FOR SEQ ID NO:36: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: 
# 40 TTTT GCTCACCCAG AAACGCTGGT 
- (2) INFORMATION FOR SEQ ID NO:37: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: 
# 40 GAAG ATCAGTTGGG TGCACGAGTG 
- (2) INFORMATION FOR SEQ ID NO:38: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: 
# 40 ATCT CAACAGCGGT AAGATCCTTG 
- (2) INFORMATION FOR SEQ ID NO:39: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: 
# 40 AGAA CGTTTTCCAA TGATGAGCAC 
- (2) INFORMATION FOR SEQ ID NO:40: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40: 
# 40 TGTG GCGCGGTATT ATCCCGTATT 
- (2) INFORMATION FOR SEQ ID NO:41: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: 
# 40 AACT CGGTCGCCGC ATACACTATT 
- (2) INFORMATION FOR SEQ ID NO:42: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: 
# 40 TGAG TACTCACCAG TCACAGAAAA 
- (2) INFORMATION FOR SEQ ID NO:43: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: 
# 40 ATGA CAGTAAGAGA ATTATGCAGT 
- (2) INFORMATION FOR SEQ ID NO:44: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: 
# 40 GTGA TAACACTGCA GCCAACTTAC 
- (2) INFORMATION FOR SEQ ID NO:45: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45: 
# 40 AGGA CCGAAGGAGC TAACCGCTTT 
- (2) INFORMATION FOR SEQ ID NO:46: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: 
# 40 GATC ATGTAACTCG CCTTGATCGT 
- (2) INFORMATION FOR SEQ ID NO:47: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: 
# 40 ATGA AGCCATTCCA AACGACGAGC 
- (2) INFORMATION FOR SEQ ID NO:48: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: 
# 40 TGTA GCCATGGCAA CAACGTTGCG 
- (2) INFORMATION FOR SEQ ID NO:49: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: 
# 40 GAAC TACTTACTCT AGCTTCCCGG 
- (2) INFORMATION FOR SEQ ID NO:50: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50: 
# 40 GGAT GGAGGCGGAT AAAGTTGCAG 
- (2) INFORMATION FOR SEQ ID NO:51: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: 
# 40 GGCC CTTCCAGCTG GCTGGTTTAT 
- (2) INFORMATION FOR SEQ ID NO:52: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: 
# 40 GCCG GTGAGCGTGG GTCTCGCGGT 
- (2) INFORMATION FOR SEQ ID NO:53: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53: 
# 40 GGCC AGATGGTAAG CCCTCCCGTA 
- (2) INFORMATION FOR SEQ ID NO:54: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54: 
# 40 GACG GGGAGTCAGG CAACCATGGA 
- (2) INFORMATION FOR SEQ ID NO:55: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55: 
# 40 ATCG CTGAGATAGG TGCCTCACTG 
- (2) INFORMATION FOR SEQ ID NO:56: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56: 
# 40 TGTC AGACCGGCCT GTCAGGCCTC 
- (2) INFORMATION FOR SEQ ID NO:57: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57: 
# 40 GATT TAAAACTTCA TTTTTAATTT 
- (2) INFORMATION FOR SEQ ID NO:58: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58: 
# 40 AGAT CCTTTTTGAT AATCTCATGA 
- (2) INFORMATION FOR SEQ ID NO:59: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59: 
# 40 ACCC AACTTAATCG CCTTGCAGCA 
- (2) INFORMATION FOR SEQ ID NO:60: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60: 
# 40 GCTG GCGTAATAGC GAAGAGGCCC 
- (2) INFORMATION FOR SEQ ID NO:61: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61: 
# 40 CCAA CAGTTGCGTA GCCTGAATGG 
- (2) INFORMATION FOR SEQ ID NO:62: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62: 
# 40 CGGT ATTTTCTCCT TACGCATCTG 
- (2) INFORMATION FOR SEQ ID NO:63: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63: 
# 40 GCAT ATGGTGCACT CTCAGTACAA 
- (2) INFORMATION FOR SEQ ID NO:64: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64: 
# 40 ATAG TTAAGCCAGC CCCGACACCC 
- (2) INFORMATION FOR SEQ ID NO:65: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65: 
# 40 GCGC CCTGACGGGC TTGTCTGCTC 
- (2) INFORMATION FOR SEQ ID NO:66: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66: 
# 40 GACA AGCTGTGACC GTCTCCGGGA 
- (2) INFORMATION FOR SEQ ID NO:67: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67: 
# 40 GTTT TCACCGTCAT CACCGAAACG 
- (2) INFORMATION FOR SEQ ID NO:68: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68: 
# 40 CGCG CGTTTCGGTG ATGACGGTGA 
- (2) INFORMATION FOR SEQ ID NO:69: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69: 
# 40 CAGC TCCCGGAGAC GGTCACAGCT 
- (2) INFORMATION FOR SEQ ID NO:70: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70: 
# 40 CCGG GAGCAGACAA GCCCGTCAGG 
- (2) INFORMATION FOR SEQ ID NO:71: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71: 
# 40 TGGC GGGTGTCGGG GCTGGCTTAA 
- (2) INFORMATION FOR SEQ ID NO:72: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72: 
# 40 CAGA TTGTACTGAG AGTGCACCAT 
- (2) INFORMATION FOR SEQ ID NO:73: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73: 
# 40 CGCA CAGATGCGTA AGGAGAAAAT 
- (2) INFORMATION FOR SEQ ID NO:74: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74: 
# 40 TTCG CCATTCAGGC TACGCAACTG 
- (2) INFORMATION FOR SEQ ID NO:75: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:75: 
# 40 GTGC GGGCCTCTTC GCTATTACGC 
- (2) INFORMATION FOR SEQ ID NO:76: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76: 
# 40 GATG TGCTGCAAGG CGATTAAGTT 
- (2) INFORMATION FOR SEQ ID NO:77: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77: 
# 40 TTCC CAGTCACGAC GTTGTAAAAC 
- (2) INFORMATION FOR SEQ ID NO:78: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78: 
# 40 CTTG CATGCCTGCA GGTCGACTCT 
- (2) INFORMATION FOR SEQ ID NO:79: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79: 
# 40 CCGA GCTCGAATTC GTAATCATGG 
- (2) INFORMATION FOR SEQ ID NO:80: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:80: 
# 40 TGTG AAATTGTTAT CCGCTCACAA 
- (2) INFORMATION FOR SEQ ID NO:81: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81: 
# 40 AGCC GGAAGCATAA AGTGTAAAGC 
- (2) INFORMATION FOR SEQ ID NO:82: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82: 
# 40 GTGA GCTAACTCAC ATTAATTGCG 
- (2) INFORMATION FOR SEQ ID NO:83: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:83: 
# 40 CTTT CCAGTCGGGA AACCTGTCGT 
- (2) INFORMATION FOR SEQ ID NO:84: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:84: 
# 40 AATC GGCCAACGCG CGGGGAGAGG 
- (2) INFORMATION FOR SEQ ID NO:85: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:85: 
# 40 CGCT CTTCCGCTTC CTCGCTCACT 
- (2) INFORMATION FOR SEQ ID NO:86: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:86: 
# 40 TCGT TCGGCTGCGG CGAGCGGTAT 
- (2) INFORMATION FOR SEQ ID NO:87: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:87: 
# 40 GGTA ATACGGTTAT CCACAGAATC 
- (2) INFORMATION FOR SEQ ID NO:88: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:88: 
# 40 AAGA ACATGTGAGC AAAAGGCCAG 
- (2) INFORMATION FOR SEQ ID NO:89: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:89: 
# 40 GTAA AAAGGCCGCG TTGCTGGCGT 
- (2) INFORMATION FOR SEQ ID NO:90: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:90: 
# 40 CCCC CCTGACGAGC ATCACAAAAA 
- (2) INFORMATION FOR SEQ ID NO:91: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:91: 
# 40 AGGT GGCGAAACCC GACAGGACTA 
- (2) INFORMATION FOR SEQ ID NO:92: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:92: 
# 40 TTCC CCCTGGAAGC TCCCTCGTGC 
- (2) INFORMATION FOR SEQ ID NO:93: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:93: 
# 40 CCTG CCGCTTACCG GATACCTGTC 
- (2) INFORMATION FOR SEQ ID NO:94: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:94: 
# 40 GGAA GCGTGGCGCT TTCTCAATGC 
- (2) INFORMATION FOR SEQ ID NO:95: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:95: 
# 40 TCAG TTCGGTGTAG GTCGTTCGCT 
- (2) INFORMATION FOR SEQ ID NO:96: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:96: 
# 40 GCAC GAACCCCCCG TTCAGCCCGA 
- (2) INFORMATION FOR SEQ ID NO:97: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:97: 
# 40 GGTA ACTATCGTCT TGAGTCCAAC 
- (2) INFORMATION FOR SEQ ID NO:98: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:98: 
# 40 TATC GCCACTGGCA GCAGCCACTG 
- (2) INFORMATION FOR SEQ ID NO:99: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:99: 
# 40 AGCG AGGTATGTAG GCGGTGCTAC 
- (2) INFORMATION FOR SEQ ID NO:100: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:100: 
# 40 TGGC CTAACTACGG CTACACTAGA 
- (2) INFORMATION FOR SEQ ID NO:101: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:101: 
# 40 TCTG CGCTCTGCTG AAGCCAGTTA 
- (2) INFORMATION FOR SEQ ID NO:102: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:102: 
# 40 TGGT AGCTCTTGAT CCGGCAAACA 
- (2) INFORMATION FOR SEQ ID NO:103: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:103: 
# 40 GGTG GTTTTTTTGT TTGCAAGCAG 
- (2) INFORMATION FOR SEQ ID NO:104: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:104: 
# 40 AAAA AGGATCTCAA GAAGATCCTT 
- (2) INFORMATION FOR SEQ ID NO:105: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:105: 
# 40 GTCT GACGCTCAGT GGAACGAAAA 
- (2) INFORMATION FOR SEQ ID NO:106: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 56 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:106: 
- CGCGAGACGA AAGGGCCTCG TGATACGCCT ATTTTTATAG GTTAATGTCA TG - #ATAA 
56 
- (2) INFORMATION FOR SEQ ID NO:107: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 47 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:107: 
# 47AGAA ACCATTATTA TCATGACATT AACCTAT 
- (2) INFORMATION FOR SEQ ID NO:108: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:108: 
# 40 TGAG TTTTCGTTCC ACTGAGCGTC 
- (2) INFORMATION FOR SEQ ID NO:109: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:109: 
# 40 ATCA AAGGATCTTC TTGAGATCCT 
- (2) INFORMATION FOR SEQ ID NO:110: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:110: 
# 40 TCTG CTGCTTGCAA ACAAAAAAAC 
- (2) INFORMATION FOR SEQ ID NO:111: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:111: 
# 40 GGTT TGTTTGCCGG ATCAAGAGCT 
- (2) INFORMATION FOR SEQ ID NO:112: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:112: 
# 40 AAGG TAACTGGCTT CAGCAGAGCG 
- (2) INFORMATION FOR SEQ ID NO:113: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:113: 
# 40 TCCT TCTAGTGTAG CCGTAGTTAG 
- (2) INFORMATION FOR SEQ ID NO:114: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:114: 
# 40 CTCT GTAGCACCGC CTACATACCT 
- (2) INFORMATION FOR SEQ ID NO:115: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:115: 
# 40 TTAC CAGTGGCTGC TGCCAGTGGC 
- (2) INFORMATION FOR SEQ ID NO:116: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:116: 
# 40 CCGG GTTGGACTCA AGACGATAGT 
- (2) INFORMATION FOR SEQ ID NO:117: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:117: 
# 40 GCGG TCGGGCTGAA CGGGGGGTTC 
- (2) INFORMATION FOR SEQ ID NO:118: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:118: 
# 40 TTGG AGCGAACGAC CTACACCGAA 
- (2) INFORMATION FOR SEQ ID NO:119: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:119: 
# 40 GTGA GCATTGAGAA AGCGCCACGC 
- (2) INFORMATION FOR SEQ ID NO:120: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:120: 
# 40 GGCG GACAGGTATC CGGTAAGCGG 
- (2) INFORMATION FOR SEQ ID NO:121: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:121: 
# 40 GAGC GCACGAGGGA GCTTCCAGGG 
- (2) INFORMATION FOR SEQ ID NO:122: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:122: 
# 40 TTTA TAGTCCTGTC GGGTTTCGCC 
- (2) INFORMATION FOR SEQ ID NO:123: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:123: 
# 40 TCGA TTTTTGTGAT GCTCGTCAGG 
- (2) INFORMATION FOR SEQ ID NO:124: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:124: 
# 40 AAAA ACGCCAGCAA CGCGGCCTTT 
- (2) INFORMATION FOR SEQ ID NO:125: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:125: 
# 40 TTTG CTGGCCTTTT GCTCACATGT 
- (2) INFORMATION FOR SEQ ID NO:126: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:126: 
# 40 CCCT GATTCTGTGG ATAACCGTAT 
- (2) INFORMATION FOR SEQ ID NO:127: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:127: 
# 40 GCTG ATACCGCTCG CCGCAGCCGA 
- (2) INFORMATION FOR SEQ ID NO:128: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:128: 
# 40 AGTC AGTGAGCGAG GAAGCGGAAG 
- (2) INFORMATION FOR SEQ ID NO:129: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:129: 
# 40 ACCG CCTCTCCCCG CGCGTTGGCC 
- (2) INFORMATION FOR SEQ ID NO:130: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:130: 
# 40 TGGC ACGACAGGTT TCCCGACTGG 
- (2) INFORMATION FOR SEQ ID NO:131: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:131: 
# 40 GCAA CGCAATTAAT GTGAGTTAGC 
- (2) INFORMATION FOR SEQ ID NO:132: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:132: 
# 40 CCAG GCTTTACACT TTATGCTTCC 
- (2) INFORMATION FOR SEQ ID NO:133: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:133: 
# 40 GGAA TTGTGAGCGG ATAACAATTT 
- (2) INFORMATION FOR SEQ ID NO:134: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:134: 
# 40 ATGA CCATGATTAC GAATTCGAGC 
- (2) INFORMATION FOR SEQ ID NO:135: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:135: 
# 40 CTCT AGAGTCGACC TGCAGGCATG 
- (2) INFORMATION FOR SEQ ID NO:136: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:136: 
# 40 CGTC GTTTTACAAC GTCGTGACTG 
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