Ligase chain reaction with endonuclease IV correction and contamination control

The present invention involves methods of improving the Ligase Chain Reaction (LCR.TM.) amplification schemes by modifying at least one probe end so that the probability of the probe contributing to spurious ligation and signal development is greatly reduced. Only after specific hybridization of the modified probe with true target are the modified ends "corrected" by endonuclease IV in a target dependent fashion to allow participation of the probe in the enzymatic ligation reaction. Specific modifications include 3' phosphate blocking groups and nucleic acid overhangs containing an abasic site at the point of ligation. Further embodiments include probes modified to contain ribonucleotide moieties which, after amplification, can be cleaved by RNase to destroy the amplification products and reduce the risk of contamination.

BACKGROUND 
This invention relates to methods of amplifying target nucleic acids and, 
particularly, to methods of performing ligase chain reaction 
amplifications wherein at least one of the probes is reversibly modified 
at the ligation site so that it is not a substrate for the enzyme 
catalyzed ligation. Exemplary modifications include chemical blockage of 
reactant groups, or an abasic site and the addition of one or more nucleic 
acid bases to form an "overhang". The modified end prevents or reduces 
target independent spurious signal development and is later corrected in a 
target dependent manner to enable amplification. 
Oftentimes, the feasibility of a nucleic acid based diagnostic assay is 
dependent on the ability to amplify the signal generated by only a few 
molecules of target. Although signal amplification is one potential 
solution, target amplification is often the preferred solution in nucleic 
acid based assays. Target amplification involves the repeated copying or 
duplication of sections of the nucleic acid designated as the target 
sequence. 
One mechanism for target amplification is known as ligase chain reaction 
(LCRT.TM.). In LCR.TM., two primary probes (first and second, both of same 
sense) and two secondary probes (third and fourth, both of opposite sense 
with respect to primary probes) are employed in excess. The first probe 
hybridizes to a first segment of the target strand and the second probe 
hybridizes to a second segment of the target strand, the first and second 
segments being contiguous so that the 3' hydroxyl end of an "upstream" 
probe abuts the 5' phosphate end of a "downstream" probe, and so that a 
ligase can covalently ligate the two probes into a fused ligation product. 
In like manner, LCR.TM. employs upstream and downstream secondary probes. A 
third probe (downstream secondary) can hybridize to the first probe 
(upstream primary) and a fourth probe (upstream secondary) can hybridize 
to the second probe (downstream primary) in a similar abutting fashion. Of 
course, if the target is initially double stranded, the secondary probes 
can also hybridize to the target complement in the first instance. Once 
the fused strand of primary probes is separated from the target strand, it 
will hybridize with the third and fourth (secondary) probes which can be 
ligated to form a complementary, secondary fused product. In order to 
understand LCR.TM. and the improvements described herein, it is important 
to realize that the fused products are functionally equivalent to either 
the target or its complement. By repeated cycles of hybridization and 
ligation, amplification of the target sequence is achieved. This technique 
is described more completely in EP-A-320 308, the entire disclosure of 
which is incorporated herein by reference. 
One of the great strengths of amplification reactions is their ability to 
detect exceedingly small numbers of target molecules. However, it is 
important that the amplification process be highly specific since the 
amplification of non-target sequences along with signal could potentially 
impair the reliability of the amplification process. One potential problem 
associated with ligase chain reaction is background signal caused by 
target independent ligation of the probes. Since the third probe 
hybridizes to the first probe and the fourth probe hybridizes to the 
second probe, the probes, which are added in excess, can easily form 
duplexes among themselves. These duplexes can become ligated independently 
of the presence of target to form a fused product which is then 
indistinguishable from the desired amplified target, yet which is still 
capable of supporting further amplification. Although target independent 
ligation of these duplexes is a relatively rare event, it is sufficiently 
common to cause undesirably high background signals in highly amplified 
diagnostic assays. 
EP-A-439 182 (corresponding to parent application Ser. No. 07/634,77 1 ) 
describes several mechanisms by which this background or spurious signal 
in LCR.sup.m can be reduced. One such mechanism involves 3' blocking 
groups or abasic sites that are "corrected" in the presence of target to 
yield ends that are ligation competent, i.e. ends that possess the 3' 
hydroxyl substrate necessary for ligation. The present invention expands 
and develops these mechanisms, particularly with regard to the use of 
endonuclease IV as the correction enzyme. 
Levin, et al, "Metalloenzymes in DNA Repair", J. Biol. Chem. 
266(34):22893-22898 (1991) have demonstrated that Endonuclease IV in 
native form contains zinc, and that inactive enzyme (purified in a metal 
free buffer) can be reactivated by the addition of certain divalent 
cations. In particular, Co.sup.2+ and Mn.sup.2 + at 200 .mu.M were 
effective to reactivate the enzymes, depending on the method (EDTA or 
1,10-phenanthroline) of inactivation. Johnson and Demple, J. Biol. Chem. 
263(34):18009-18016 (1988) have shown that the activity of a related 
enzyme, yeast 3' phosphoglycoaldehyde diesterase, is enhanced by 
concentrations of Co.sup.2+ from 3 .mu.M to about 3 mM, above which the 
cation was inhibitory. 
A second potential problem associated with nucleic acid amplification 
systems is the potential for airborne and carryover contamination. Due to 
the exponential increase in target sequences, there is an increased 
potential for some of these molecules to contaminate an untested sample, 
and to render it falsely positive. Several methods have been described for 
reducing such contamination. They generally involve destroying 
substantially all the amplified products either immediately after 
amplification or immediately prior to the next amplification cycle. 
One such contamination control method is taught in co-owned, co-pending 
application Ser. No. 07/863,622, filed Apr. 3, 1992. 
Another method is taught by Walder, et al EP-A-496 483. This document 
describes the incorporation of ribonucleotides into PCR primers followed 
by destruction of the amplification products with RNase or alkaline 
hydrolysis. While the authors allege that their method is useful in 
transcription based amplification and in the ligase chain reaction, they 
have provided no conditions or demonstration of utility except in PCR. 
It is well known in the art that DNA ligases will not ligate DNA probes 
hybridized to a fibonucleotide target. But WO91/17270 describes an LCR 
variation using fibonucleotide residues at the point of ligation. These 
residues can later be cleaved by alkali or enzymes to destroy the 
amplification product and prevent contamination. 
However, there is no teaching of using fibo-modified probes in combination 
with 3' blocking groups as in the present invention. The present invention 
provides a mechanism for reducing or eliminating contamination in LCR.TM. 
using endonuclease IV correction methods. It has been discovered that DNA 
probes having a single ribonucleotide bearing a 3' blocking phosphate 
group can be used in LCR.TM.. When so used, the probes alleviate the 
background caused by target independent ligation and, at the same time, 
provide a mechanism to control contamination. 
SUMMARY OF THE INVENTION 
In a first aspect (the "basic modified" method), the invention relates to a 
method for amplifying a target nucleic acid sequence using LCR, said 
method including: (a) providing an excess of at least two sets of two 
probes, the 3' end of an upstream probe being ligated to the 5' end of a 
downstream probe in the presence of target to form a primary ligation 
product and the second set of probes hybridizing to the primary ligation 
product and being ligated to each other to form a secondary ligation 
product; (b) repeatedly denaturing the hybridized strands, reannealing 
additional probes and ligating them; and (c) detecting to what extent 
ligation products have formed, wherein the improvement comprises: 
(a) providing in at least one of the upstream probes a 3' end modification 
such that the probe is incapable of ligation to its downstream partner, 
said 3' end modification being correctable substantially only when the 
modified probe is hybridized to the target sequence; 
(b) hybridizing the modified probe to the target, if present, to form a 
modified probe-template complex; 
(c) correcting the modification in a target dependent manner using 
endonuclease IV activity to create a 3' hydroxyl end, thus allowing the 
corrected probe to be ligated to its downstream partner; 
(d) ligating the corrected probe to its downstream partner to form an 
amplification product; and 
(e) dissociating the amplification product from the target and repeating 
the hybridization, correction and ligating steps to amplify the desired 
target sequence. 
Preferably, the 3' modification comprises a blocking moiety (the "blocking" 
method) such as a blocking moiety of the form: 
##STR1## 
wherein Z is selected from the group consisting of --H; --(CH.sub.2).sub.n 
CHO, where n is from 1 to about 3; -deoxyribose; and-dideoxyribose. A 
simple phosphate (Z.dbd.H) will do. 
The correcting solution preferably includes available divalent cobalt or 
manganese ion at a concentration of at least about 0.05 mM. The upper 
range may be 10mM or more, but more preferred ranges include from 0.05 mM 
to about 2.0 mM, and from about 0.5 mM to about 1.0 mM. 
In a variation of the blocking method,the above modified LCR method 
includes two sets of two probes which are oligodeoxyfibonucleotide probes, 
except that at least one of said probes includes at least one 
ribonucleotide residue, preferably the terminal residue containing the 3' 
modification. Similar blocking modifications may be used, including 
phosphate at the 3' position of the terminal ribonucleotide residue. 
Consequently, the blocking method may further comprise, after the 
detection step, a step of cleaving ligation products using RNase or 
alkali. In addition or in the alternative, this aspect of the method may 
further comprise, prior to amplification, a step of cleaving ligation 
products using RNase. 
In yet another aspect (the "abasic" method), the blocking moiety may be a 
nucleic acid overhang containing an abasic residue immediately 3' to the 
point of intended ligation. In this case, correction of the modification 
comprises cleavage of said modified probe on the 5' side of said abasic 
site, substantially only when said modified probe is hybridized to target 
or to ligation product. 
The correcting solution again preferably includes available divalent cobalt 
or manganese ion at a concentration of at least about 0.05 mM. The upper 
range may be 10 mM or more, but more preferred ranges include from 0.05 mM 
to about 2.0 mM, and from about 0.5 mM to about 1.0 mM. 
In a variation of the abasic method two sets of two probes are 
oligodeoxyribonucleotide probes, except that at least one of said probes 
includes at least one ribonucleotide residue, preferably immediately 5' to 
the abasic site. Consequently, the abasic method may further comprise, 
after the detection step, a step of cleaving ligation products using RNase 
or alkali. In addition or in the alternative, this aspect of the method 
may further comprise, prior to amplification, a step of cleaving ligation 
products using RNase. 
In any of the above described methods, detection may be by means of a first 
hapten (or other specific binding member) attached to the primary upstream 
and secondary downstream probes; and by a reporter or second hapten (or 
other specific binding member) attached to the primary downstream and 
secondary upstream probes. In both cases, the haptens and reporters should 
be attached to the probes by means which do not significantly affect the 
hybridization and/or ligation of the probes. This may be done, for 
example, by attachment at the "outside ends" of the probes. Alternatively, 
detection may be by means of a blocking moiety which comprises a 
detectable label and said detecting is by means of monitoring the release 
of detectable label from the modified probe. 
Another aspect of the invention involves a diagnostic kit comprising in 
combination the following reagents in one or more suitable containers: 
(a) two pairs of probes hybridizable with target wherein at least one of 
the probes is modified such that, when hybridized, a ligase is 
substantially incapable of acting on the modified probe as its substrate; 
the two probes capable of hybridizing to target in positions such that, 
upon correction of said modified probe, the two probes can be ligated to 
one another 
(b) a first enzyme reagent having ligase activity for assembling an 
amplification product; and 
(c) a second enzyme reagent having endonuclease IV activity capable of 
correcting the modified probe in a target dependent manner to allow the 
probe-template complex to be acted upon by the ligase reagent. 
The kit may also include any or all of: a) a buffer or means for preparing 
a buffer containing from 0.05 mM to about 2.0 mM of divalent cobalt ion; 
and b) an RNase reagent or an alkaline reagent. 
A final aspect of the invention is a nucleic acid probe substantially free 
of naturally occurring nucleic acid fragments, said probe having at least 
three deoxyribonucleotides covalently linked by phosphodiester linkages to 
define a 5' and a 3' end of the probe, the probe further comprising a 
ribonucleotide at the 3' end, the 3' position of the ribonucleotide having 
attached thereto a group of the formula: 
##STR2## 
wherein Z is selected from the group consisting of --H; --(CH.sub.2).sub.n 
CHO, where n is from 1 to about 3; -deoxyribose; and -dideoxyribose. 
Preferably, Z is hydrogen and the oligonucleotide comprises from about 12 
to about 50 deoxyribonucleotides.

DETAILED DESCRIPTION 
For purposes of this invention, the target sequence is described to be 
single stranded. However, this should be understood to include the case 
where the target is actually double stranded but is simply separated from 
its complement prior to hybridization with the probes/primers. In the case 
of double stranded target, secondary, third and fourth probes will also 
participate in the initial step by hybridizing to the target complement. 
In the case of single stranded target, the secondary probes will not 
participate in the initial hybridization step, but will participate in 
subsequent hybridization steps. Target sequences may comprise 
deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). 
It is to be understood that the term "bases" shall refer to Guanine (G), 
Cytosine (C), Adenine (A) and Thymine (T) when the context is that of DNA; 
and Guanine (G), Cytosine (C), Adenine (A) and Uracil (U) in the context 
of RNA. The term also includes analogs and derivatives of the bases named 
above, provided they can undergo hydrogen bonding of base pairs 
characteristic of the natural bases. Exemplary base "analogs" can be found 
in 1114 Official Gazette, at 43. Although the degenerate base Inosine (I) 
may be employed with probes used in this invention, it is not preferred to 
use I within modified portions of the probes according to the invention. 
Individual nucleotides or bases are said to be "complementary" if they 
result in canonical base pairing; e.g. C with G, and A with T or U. 
Throughout this application, the "prime" (') designation is used to 
indicate a complementary base or sequence. One oligonucleotide is 
"complementary" to another if it hybridizes to the other and has 
substantially complementary base pairings in the hybridized region. Thus, 
probe A can be complementary to A' even though it may have ends not 
coterminal with A'. The same is true of B and B'. As a result of this 
definition, "complementary" oligonucleotide sequences encompass sequences 
that have mismatched base pairs in the hybridizable region, provided they 
can be made to hybridize under assay conditions. 
It is an important feature of the present invention that, instead of using 
complementary pairs of probes capable of forming ligatable, blunt-ended 
duplexes, at least one probe of one of the probe pairs initially includes 
a "modified" end which renders the resultant primary or secondary probe 
not a suitable substrate for the ligase catalyzed fusion. A "modified end" 
is defined with respect to the point of ligation rather than with respect 
to its complementary probe. A "modified end" has either (1) a blocking 
moiety (or additional base residues) on a group (e.g. the 5' phosphate or 
the 3' hydroxyl) which, under ordinary LCR.TM. conditions, obligatorily 
participates in the ligase catalyzed fusion (See e.g. probe A of FIG. 2); 
or (2) omitted bases to create a "gap" between one probe terminus and the 
next probe terminus. 
By convention in this application, a modified end of the first type is 
referred to as an "overhang", the overhang being an additional blocking 
moiety or additional base residues which, when hybridized to the target 
sequence extends beyond the point of ligation. The term "overhang" is not 
to be confused with an "extension" of one probe with respect to its 
complementary probe, resulting from the fact that they need not be 
coterminal. A modified end of the second type is referred to herein as a 
"recess", the recess being the gap between two primary or secondary probes 
after hybridizing to the target. The presence of these modified ends 
reduces the falsely positive signal created by target independent ligation 
of complementary probe duplexes to one another in the absence of target. 
"Correction" of the modification is subsequently carded out to render the 
probes ligatable. As used herein "correction" refers to the process of 
rendering, in a target dependent manner, the two primary probes or the two 
secondary probes, or both, ligatable to their same sense partners. Thus, 
only those probes hybridized to target, target complement or 
polynucleotide sequences generated therefrom are capable of being 
"corrected". "Correction" can be accomplished by several procedures, 
depending on the type of modified end used, although endonuclease IV 
corrections are examined herein. 
As used herein, "point of ligation" or "intended point of ligation" refers 
to a specific location between two probe partners that are to be ligated 
in a template-dependent manner. It is the site at which the "corrected" 
upstream probe lies adjacent its downstream partner in 5' phosphate- 3' 
hydroxyl relationship. For each set of four LCR.TM. probes there are two 
"points of ligation", a point for the primary probe partners and a point 
for the secondary probe partners. In conventional LCR.TM., typically the 
two points of ligation are opposite one another, thus forming blunt ended 
duplexes when the probe pairs hybridize to one another. In the present 
invention, the points of ligation may be opposite one another or displaced 
from one another (preferably with 3' extensions) by one or more bases. The 
exact point(s) of ligation varies depending on the embodiment and, thus, 
this term is further defined in the context of each embodiment. 
Each of the probes may comprise deoxyribonucleic acid (DNA) or ribonucleic 
acid (RNA). It is a routine matter to synthesize the desired probes using 
conventional nucleotide phosphoramidite chemistry and the instruments 
available from Applied Biosystems, Inc, (Foster City, Calif.); DuPont, 
(Wilmington, Del.); or Milligen, (Bedford, Mass.). Phosphorylation of the 
5' ends of the probes, a necessity for ligation by ligase, may be 
accomplished enzymatically by a kinase, as is known in the art, or by any 
chemical synthetic method known to phosphorylate 5' ends. Commercial 
reagents are available for this purpose for use with automated synthesis. 
As will be seen, similar methods and reagents are used to place a blocking 
phosphate on a 3' probe end. 
In general, the methods of the invention comprise repeated steps of (a) 
hybridizing the modified probes to the target (and, if double stranded so 
that target complement is present, to the target complement); (b) 
correcting the modification in a target dependent manner to render the 
probes ligatable; (c) ligating the corrected probe to its partner to form 
a fused or ligated product; and (d) dissociating the fused product from 
the target and repeating the hybridization, correction and ligation steps 
to amplify the desired target sequence. Steps (a), (c) and (d) are 
essentially the same for all of the embodiments and can be discussed 
together. They are generally the same steps that one would employ in 
conventional LCR.TM.. Step (b) varies depending on the type of 
modification employed and. correction by endonuclease IV is discussed 
herein. 
Hybridization of modified probes to target (and optionally to target 
complement) is adequately explained in the prior art; e.g EP-320 308 and 
EP 439 182. Probe length, probe concentration, GC content and stringency 
of conditions all affect the degree and rate at which hybridization will 
occur. Preferably, the probes are sufficiently long to provide the desired 
specificity; i.e, to avoid being hybridizable to random sequences in the 
sample. Typically, probes on the order of 15 to 100 bases serve this 
purpose. Presently preferred are probes having a length of from about 15 
to about 40 bases. 
The probes are generally added in approximately equimolar concentration 
since they are expected to react stoichiometrically. Each probe is present 
in a concentration ranging from about 5 nanomolar (nM) to about 90 nM; 
preferably from about 10 nM to about 85 nM. The optimum quantity of probe 
used for each reaction also varies depending on the number of cycles which 
must be performed. Optimum concentrations can readily be determined by one 
of ordinary skill in this art 
The stringency of conditions is generally known to those in the art to be 
dependent on temperature, solvent and other parameters. Perhaps the most 
easily controlled of these parameters is temperature and thus it is 
generally the stringency parameter varied in the performance of LCR.TM.. 
Since the stringency conditions required for practicing this invention are 
not unlike those of ordinary LCR.TM., further detail is deemed 
unnecessary, the routine practitioner being guided by the examples which 
follow. 
The next step in the general method follows the specific correction step 
and comprises the ligation of one probe to its adjacent partner. Thus, 
primary upstream probes are ligated to their associated primary downstream 
probes and secondary upstream probes are ligated to their associated 
secondary downstream probes. An "adjacent" probe is either one of two 
probes hybridizable with the target in a contiguous orientation, one of 
which lies with its phosphorylated 5' end in abutment with the 3' hydroxyl 
end of the partner probe. "Adjacent" probes are created upon correction of 
the modified end(s) in a target dependent manner. Since enzymatic ligation 
is the preferred method of covalently attaching two adjacent probes, the 
term "ligation" will be used throughout the application. However, 
"ligation" is a general term and is to be understood to include any method 
of covalently attaching two probes. 
The conditions and reagents which make possible the preferred enzymatic 
ligation step are generally known to those of ordinary skill in the art 
and are disclosed in the references mentioned in background. Ligating 
reagents useful in the present invention include prokaryotic ligases such 
as E coli ligase, T4 ligase and Thermus thermophilus ligase (e.g., ATCC 
27634) as taught in EP-320 308 and EP-A-373 962. This latter ligase is 
presently preferred for its ability to maintain activity during the 
thermal cycling of LCR.TM.. Suitable thermally stable ligases are 
commercially available from New England Biolabs, Inc. (Beverly, Mass.), 
Epicentre Technologies, Inc. (Madison, Wisc.) and Molecular Biology 
Resources (Milwaukee, Wisc.). Absent a thermally stable ligase, the ligase 
must be added again each time the cycle is repeated. Also of potential 
utility are eukaryotic ligases, e.g. DNA ligase of Drosophilia, reported 
by Rabin, et al., J. Biol. Chem. 261: 10637-10647 (1986). 
Once ligated, the fused probe is dissociated (e.g. melted) from the target 
and, as with conventional LCR.TM., the process is repeated for several 
cycles. The number of repeat cycles may vary from 1 to about 100, although 
from about 15 to about 70 are preferred presently. 
It is desirable to design probes so that when hybridized to their 
complementary (secondary) probes, the ends away from the point of intended 
ligation (i.e. "outside" ends) are not free themselves to participate in 
other unwanted ligation reactions. Thus, ligatable sticky or blunt outside 
ends should be avoided. Free 5' terminal phosphates should be avoided or 
eliminated, especially if such sticky or blunt ends must be used. This can 
be accomplished either through synthesizing oligonucleotide probes (which 
normally carry no 5' terminal phosphate groups), or through the use of 
phosphatase enzymes to remove terminal phosphates (e.g. from 
oligonucleotides generated through restriction digests of DNA). 
Alternatively, ligation of the outside ends of the probes can be prevented 
by blocking the end of at least one of the probes with a "hook" or other 
reporter molecule or marker moiety as will be described in detail below. 
It is also desirable to design the probes so that substantially all of the 
amplification products made can be selectively inactivated or destroyed to 
reduce the risk of contamination. To be effective, such inactivation must 
destroy substantially all the amplification products and it may occur at 
various times during the process. Generally inactivation may occur 
immediately after amplification, after detection or immediately prior to 
the next LCR.TM. reaction. For convenience these will be referred to 
herein as "postamplification", "post-detection" and "pre-amplification", 
respectively. An inactivation method that is used pre-amplification must 
selectively destroy amplification product without destroying the reactant 
probes and reagents. An inactivation method that is used post-detection 
does not share this constraint. 
For example, a ribonucleotide residue can be incorporated into an otherwise 
deoxyribo-oligonucleotide and the resultant product can be cleaved by 
RNase or alkaline hydrolysis conditions. Particularly useful among the 
RNases reported in the literature are RNaseH, which cleave ribonucleotides 
in RNA:DNA duplexes and in single stranded RNA. Although E coli. Rnase 
generally prefers a string of about 4 ribonucleotides for efficient 
cutting, Walder, et al. have reported a human RNase H activity in K562 
erythroleukemia cells, designated RNaseHI (see EP-A-496 483), which is 
said to cleave mixed R/DNA:DNA duplexes when only a single ribo residue is 
present. The procedure by which this is accomplished is described in 
greater detail in EP-A-496 483 and in WO91/17270. 
It is also possible to cleave the ribo-modified products using alkaline 
conditions. In the context of this application, "alkaline conditions" 
refers to conditions above a pH of 7.0 which are sufficient to effect a 
hydrolysis of the phosphodiester bond adjacent a ribonucleotide moiety. 
Usually a pH above about 10 for 0.5 to 2 hours with heat will produce 
hydrolysis. Typical conditions are known in the art and include by way of 
example, not limitation, treatment with 0.6N NaOH for 30 min to 1 h at 
90.degree. C. or with 30-40 mM KOH or NaOH for 1-1.5 hours. A 36 mM 
solution of KOH produces a pH of about 11. Other probe modifications that 
permit destruction after ligation are also within this invention. 
MODIFIED ENDS CORRECTABLE BY ENDONUCLEASE IV 
As mentioned, a first embodiment involves a modified end wherein a blocking 
moiety or additional bases are added to the 3' end of at least one 
upstream probe, beyond the point of intended ligation. The blocking moiety 
or the additional bases comprise the "overhang" and are the reason 
blunt-end ligation is not possible. 
Modified Probe Reagents. In a first variation depicted schematically in 
FIG. 2 and exemplified in Examples 5-7, the overhang comprises a chemical 
blocking agent, R. It is well known that the standard DNA ligase reaction 
requires that the substrate strands present a 3' hydroxyl and a 5' 
phosphate at the point of ligation. Several modifications, particularly at 
the 3' hydroxyl group, are known to introduce an R group which will render 
the modified end incapable of participating in a ligase reaction, but 
which can be removed when the modified strand is part of a double stranded 
structure. Such modifications include the following illustrative R groups 
attached to the 3' oxygen in place of the hydrogen atom: 
##STR3## 
wherein Z is selected from the group consisting of --H; --(CH.sub.2).sub.n 
CHO, where n is from 1 to about 3, preferably 1 or 2;.-deoxyribose; and 
-dideoxyribose. 
The synthesis of probes having ends suitably modified with an R group is 
well known in the art. For example, chemical synthesis of oligonucleotides 
containing a 3' phosphate group has been described by Markiewicz and 
Wyrzykiewicz, Nucl. Acids Res. 17:7149-7158 (1989). Larger blocking 
groups, which may have the advantage of hiding the 3' phosphate from 
non-specific phosphatases that might be present in some samples, are 
conveniently prepared by creating the oligonucleotide probe with terminal 
transferase and dUTP or ddUTP, followed by treatment with uracil 
glycosylase. Purification of uracil glycosylase is taught by Lindahl, et 
al, J.B.C. 252:3286-3924 (1977). In the case of dUTP addition, treatment 
with strong alkali following the uracil glycosylase treatment can be used 
to prepare the glycoaldehyde derivative. It is to be understood that the 
examples of R groups given above are illustrative only, and that one of 
ordinary skill could synthesize many variants which would work equally 
well. In addition, example 16 describes a convenient automated synthesis 
of a probe having a 3' terminal phosphate. 
In another variation, the probes are modified to contain both a 3' 
ribonucleotide moiety and a 3' phosphate blocking moiety. Synthesis of 
such probes is described in Examples 14-17. These probes have dual 
advantages. They avoid the problem of target independent ligation by 
virtue of the 3' blocking phosphate. In addition, they avoid the problems 
of contamination because the amplification products can be cleaved and 
destroyed at the ribonucleotide residue by various RNases or alkaline 
conditions. 
Any contamination control method that cleaves the amplification product may 
be used post-detection. Either RNase or alkaline conditions can be used to 
cleave the ligation product at the ribonucleotide when the reaction is 
completed. However, for preamplification contamination control, RNase is 
preferred. While alkaline conditions will cleave ligation products to 
leave a 5' hydroxyl, it also demonstrates a tendency to act on unligated 
ribo-modified probe reagents to convert 3' phosphates to 2' phosphates. It 
is not known whether or how efficiently the 2' phosphated probes can be 
ligated. In contrast, preferred RNases cut the ligation products leaving a 
5' hydroxyl and a 3' phosphate. In other words, RNase cleaves a 
phosphodiester bond on the 3' or downstream side of the ribo residue, a 
bond that does not even exist in the unligated modified reagents. Thus, 
RNase can be used as a pre-amplification contamination control without 
damaging the amplification reagents which still need to perform. 
The RNase may be, and preferrably is, thermolabile since it should not 
retain activity after LCR.TM. begins. 
In yet another variation of overhanging ends, the overhang consists of 
additional nucleic acid bases which can be cleaved off once the probes are 
hybridized to target. This variation is depicted schematically in FIG. 3 
and exemplified in Examples 3-4 and 8-9. The overhang prevents ligation at 
the intended point of ligation by virtue of its bulk, and stereochemically 
blocks or masks the group(s) which obligatorily participate in the ligase 
reaction (as described above for blocked ends). What distinguishes this 
from the simple chemical blockage described above is the nature and size 
of the "blocking" group (i.e., the overhang). It is by nature composed of 
nucleic acid residues colinear with the probe molecule. However, the size 
of the group is too large to permit the modified end of the molecule to 
remain in the vicinity of the ligation point when hybridized to a target. 
Although several classes of overhangs are possible, (three are described in 
the parent applications) the phosphate-blocked and abasic site overhangs 
are further considered herein. In general, the overhang should be 
complementary to the target so that its removal can be template dependent 
as is described below. The overhang may be from 1-10 bases, preferably 
from 1-5 bases in length. The synthesis of oligonucleotides with abasic 
sites has been described in the literature. See, for example, Takeshita, 
et al, J. Biol. Chem. 262:10171-10179 (1987); and Eritja, et al. 
Nucleosides & Nucleotides 6(4):803-814 (1987). Modified oligonucleotide 
probes can be synthesized so as to position an abasic site immediately 3' 
to the point of ligation on the probe intended to donate its 3' end. 
For both blocked and abasic modified probes, the probes are preferably 
designed to minimize correction that might take place while the probe is 
hybridized to its complementary probe (as opposed to correction while 
hybridized to true target). With reference to FIG. 2 for example, when the 
5' terminus of A' is coterminal with the 3' phosphate blocked probe A 
there may be some tendency for endonuclease IV to recognize this as its 
double stranded substrate and to cleave a 3' phosphate from A even in the 
absence of target. Similarly, in FIG. 3, when the 5' terminal base of A' 
is opposite the abasic site, x, of probe A, and even when opposite the 
residue immediately 5' of the abasic residue in probe A, there may be some 
tendency for endonuclease IV to recognize this as its double stranded 
substrate and to cleave the overhang from A at the abasic site even in the 
absence of target. Both of these adverse situations can be minimized by 
staggering or offsetting the ends of modified probes A (and/or B') so that 
they extend as single strands beyond the ends of their complementary 
probes. Furthermore, in the case of the abasic modification, the abasic 
site itself preferably lies beyond the 5' end of the complementary probe 
by at least one or more bases. In other words, the 5' terminal residue of 
the complementary probe A' lies opposite a nucleotide residue in the 
upstream probe A which is at least one residue 5' to the site of the 
modification. 
In the phosphate blocked situation, this is illustrated by example 7, using 
probe AA123-1P(20) complementary to AA123-3(18), and probe AA123-4P(22) 
complementary to AA123-2 (a 20-mer). Similarly, in the abasic site 
situation, this is demonstrated by example 8 using probes AA 123-1E 1 
complementary to AA 123-3(18), and probe AA123-4E1 complementary to 
AA123-2 (a 20-mer). In each of these cases, the 3' modification extends 
beyond the 5' end of the complementary probe, virtually assuring that 
endonuclease IV will not mistake this duplex for its true substrate, 
modified probe on complete target strands. 
Enzymology. The enzyme endonuclease IV (Siwek, et al, Nucl. Acids Res. 
16:5031-5038 (1988) sometimes referred to herein as "Endo IV") and a 
variety of other naturally occurring enzymes are capable of removing 
various blocking groups to expose a 3' hydroxyl group if and substantially 
only if the strand containing the blocking group is hybridized to a 
complementary strand. For example, Doetsch and Cunningham, Mutation 
Research, 236:173-201 (1990) describe in detail the enzymology of 
endonucleases and the chemistry of several different reactive abasic 
sites. The same enzyme has also been shown to cleave a polynucleotide at 
an abasic site if the polynucleotide is hybridized to a complementary 
strand. Endonuclease IV is a class II AP endonuclease, which effects 
cutting on the 5' side of the abasic site, leaving a 3' hydroxyl end on 
the polynucleotide. By both its position and its chemical nature, the 
polynucleotide is now capable of being joined by ligase to an adjacent 
probe. 
While use of the endonuclease IV referred to herein is clearly within the 
scope of the invention, it will be recognized that many other equivalent 
correcting reagents may be employed. For example, other enzymes may be 
discovered that have a similar ability to correct the modifications 
substantially only when the modified probe is hybridized to target. Also, 
it may be found that the entire enzyme is not essential, but that some 
fragment or digest of the enzyme will have the desired activity. Finally, 
it may be that desired activity may be obtained by recombinantly produced 
polypeptides having only a fraction of the length of the native protein. 
All such variations are deemed equivalents of Endonuclease IV for purposes 
of this invention. 
If the endonuclease IV enzyme is not thermostable, it should be re-added at 
each cycle of LCR.TM.. However, it is preferred to use endonuclease 
isolated or recombinantly engineered from a thermos table species, such as 
the T. thermophilus Endo IV described in copending and co-owned U.S. 
applications Ser. Nos. 07/860,861 and 07/869,306, filed on Mar. 31, 1992 
and Apr. 16, 1992, respectively, the entire disclosures of which have been 
incorporated herein by reference. E. coli strain CS1 carries plasmid pTT7 
containing an insert coding for this enzyme was deposited with the 
American Type Culture Collection (ATCC) at 12301 Parklawn Drive, 
Rockville, Md. on Apr. 9, 1992 and has been given Accession No.68950. In 
the context of the present application, "thermostable" means that the 
enzyme reagent retains a substantial portion of its activity at 
temperatures in excess of about 70.degree. C.; preferably in excess of 
about 80.degree. C. 
It has been shown by Levin, et at, "Metalloenzymes in DNA Repair", J. Biol. 
Chem. 266(34):22893-22898 (1991) that Endonuclease IV in native form 
contains Zinc. These researchers have also shown that endonuclease IV 
which has been purified in a metal free buffer is inactive, but it can be 
reactivated by the addition of certain divalent cations. In particular, 
Co.sup.2+ and Mn.sup.2+ at 200 .mu.M were effective to reactivate the 
enzymes, depending on the method (EDTA or 1,10-phenanthroline) of 
inactivation. Others have shown that the activity of a related enzyme, 
yeast 3' phosphoglycoaldehyde diesterase, is enhanced by concentrations of 
Co.sup.2+ from 3 gM to about 3 mM, above which the cation was inhibitory. 
However, it has surprisingly been found that Co.sup.30 is inhibitory to 
the performance of ligase and LCR.TM. generally. For example, see Examples 
12 and 13, infra, wherein concentrations above about 2 mM prevented all 
amplification from 10.sup.6 targets. Thus, it was necessary to find the 
window of concentration of Co.sup.2+ which enabled the endonuclease IV 
without inactivating the LCR.TM. (apparently the ligase itself). Since 
Co.sup.2+ is very tightly bound by chelators such as EDTA, and it is 
difficult to completely remove EDTA often used in the preparation of 
samples, it is useful to refer to "available" cobalt ion, which is the 
amount in excess of that bound by any chelator present. If careful 
measures are taken to avoid introducing or to completely remove chelator, 
then the "available" cobalt ion concentration approximates the actual 
concentration. It is believed that the available concentration of 
Co.sup.2+ should be about 0.05 to about 2.0 mM, usually about 0.1 to about 
1.5 mM, preferably about 0.5 to about 1 mM. Cobalt ion is suitably 
supplied as any common salt, such as the dichloride salt. 
Applicants have also discovered surprisingly that the divalent cation 
Mn.sup.2+ will substitute for Co.sup.2+ but not for Mg.sup.2+ in the 
LCR.TM. involving endonuclease IV. This is surprising because literature 
reports establish that Mn.sup.2+, as the sole divalent cation, will 
support ligase activity and endonuclease IV activity. However, Mn.sup.2+ 
alone is not sufficient to support the LCR or endo IV modified LCR. The 
presence of Mg.sup.2+ is still a requirement for the amplification 
reactions. Preferably the Mg.sup.2+ concentration is at least about 0.5 
mM, and ideally is from about 5 to about 20 mM. 
Detection. Following amplification, the amplified sequences can be detected 
by monitoring the formation of ligated product using a number of 
conventional technologies known in the art. In one preferred way, 
formation of the ligated product is monitored using the fact that a new 
covalent bond is formed between the first and second probes of the same 
sense. Thus, amplification product is longer than the individual probes 
and can be separated form unligated probes on this basis. Separation may 
easily be achieved by gels or by affinity members or "hooks". A "hook" is 
any moiety having a specific ligand-receptor affinity. It may be, for 
example, a hapten or a segment of polynucleotide. A hook may be attached 
to one probe and a label may be attached to the other probe of same sense. 
Ligation joins the label to the affinity moiety and separated label can be 
measured on a solid phase following separation. 
Alternatively, hooks may be provided at the available outside ends of at 
least two probes (opposite ends of fused product), and preferably to the 
outside ends of all four probes. Typically, the hook(s) at one end of the 
fused product (e.g. the 5' end of A and the 3' end of A') comprises a 
first hapten capable of being immobilized by a reagent (such as antibody 
or avidin) coated onto a solid phase. The hook(s) at the other end (e.g. 
the 3' end of B and the 5' end of B') contains a different antigen or 
hapten capable of being recognized by a label or a label system such as an 
antibody-enzyme conjugate. In the case of an enzyme conjugate, a substrate 
is then added which is convened by the enzyme to a detectable product. 
Exemplary hapten hooks include many drugs (e.g. digoxin, theophylline, 
phencyclidine (PCP), salicylate, etc.), T3, biotin, fluorescein (FITC), 
dansyl, 2,4-dinitrophenol (DNP); and modified nucleotides such as 
bromouracil and bases modified by incorporation of a 
N-acetyl-7-iodo-2-fluorenylamino (AIF) group; as well as many others. 
Certain haptens are disclosed in co-pending, co-owned patent applications 
U.S. Pat. No. 7/808,508 (adamantaneacetic acids), U.S. Pat. No. 07/808,839 
(carbazoles and dibenzofurans), both filed Dec. 17, 1991, U.S. Pat. No. 
07/858,929 (acridines), and U.S. Pat. No. 07/858,820 (quinolines), both 
filed Mar. 27, 1992 (collectively referred to herein as the "hapten 
applications"). The entire disclosure of each of the above hapten 
applications is incorporated herein by reference. 
Virtually any hapten can be used with the present invention. The invention 
requires only that a specific binding partner is known or can be prepared 
(a definitional property of "hapten") and that the hapten can be coupled 
to the probe such that it does not interfere with hybridization or 
ligation. Many methods of adding haptens to probes are known in the 
literature. Enzo Biochemical (New York) and Clontech (Palo Alto) both have 
described and commercialized probe labelling techniques. For example, a 
primary amine can be attached to a 3' oligo end using 3'-Amine-ON 
CPG.sup.m (Clontech, Palo Alto, CA). Similarly, a primary amine can be 
attached to a 5' oligo end using Aminomodifier II.RTM. (Clontech). The 
amines can be reacted to various haptens using conventional activation and 
linking chemistries. 
Publications WO92/10505, published 25 June 1992 and WO 92/11388 published 9 
July 1992 teach methods for labelling probes at their 5' and 3' ends 
respectively. According to one known method for labeling an 
oligonucleotide, a label-phosphoramidite reagent is prepared and used to 
add the label to the oligonucleotide during its synthesis. For example, 
see Thuong, N. T. et al., Tet. Letters, 29(46):5905-5908 (1988); or Cohen, 
J. S. et al., U.S. patent application 07/246,688 (NTIS ORDER No. 
PAT-APPL-7-246,688) (1989). 
In another embodiment of this invention, detection is achieved not by 
directly measuring formation of the ligated product, but by measuring 
release of the blocking moiety or the overhang. This can easily be done if 
the blocking moiety or overhang contains a detectable label. In this case, 
a reduction in signal associated with the solid phase indicates the 
presence of target. In a preferred variation the label is a fluorophore 
having a first characteristic spin property when attached to the probe, 
and a second, distinguishable spin property when released from the probe. 
Such labels are well known in the an of fluorescence polarization assays. 
See, for example, EP-A-382 433 (ICI). Coupling these labels to blocking 
moiety or to a nucleic acid overhang is a matter of routine chemistry. 
EXAMPLES 
The invention will now be described further by way of examples. The 
examples are illustrative of the invention and are not intended to limit 
it in any way. 
Examples 1-2 below, illustrate enhanced LCR.TM. using probes having 
modified ends which are corrected by endonuclease IV. Both blocking 
phosphate groups and abasic site overhangs are demonstrated. The probe 
sequences are given in Tables I-IV below. The probes are specific for a 
target DNA sequence at position 123 or 250 in Actinobacillus 
actinomycetemcomitans, hence the designation "AA123" or "AA250". The next 
numeral (after the dash) designates the position of the probe in a set of 
four: Probes numbered -1 and -2 have the same 5'-3' orientation while 
probes numbered -3 and -4 have the opposite sense. Probes numbered -1 and 
-3 hybridize, as do probes numbered -2 and -4. 
"P" and "p" indicate a phosphate group. This is normal and required on a 5' 
terminus, but serves as a ligation blocking modification on a 3' end. 3' 
phosphorylated probes were synthesized by initiating automated synthesis 
with 2-[[2-[(4,4'-dimethoxytrityl)oxy]ethyl]sulfonyl]ethyl 2-cyano-ethyl 
N,N-diisopropylphosphoramidire (Horn, T and Urdea, M Tet. Lett. 27 
4705+(1986)) as reported (Ashely, GW and Kushland, DM Biochemistry 
30:2927-2933 (1991)), followed by the sequential addition of 
ribonucleofide and deoxyribonucleotide cyanoethyl phosphoramidites using 
an automated DNA synthesizer. 
"E" and "x" designate an abasic site (described further below). A number 
(1, 3 or 5) following the "E" designates the length of complementary bases 
(overhang) beyond the abasic site. Numbers in parentheses represent probe 
lengths. Abasic probes were synthesized on automated instruments using 
modified phosphoramidite reagents according to the method of Eritja, et 
al. Nucleosides & Nucleotides 6(4):803-814 (1987). All probes are 
oligodeoxyribonucleotides except as specified. 
The target DNA used in examples 1-12 and 14-17 was a plasmid containing an 
898 base pair insert isolated from Actinobacillus actinomycetemcomitans. 
(ATCC Acc. No. 53219). The plasmid was digested with EcoRI and PstI to 
liberate an approximately 1000 base pair fragment. Plasmid concentrations 
were determined spectrophotometrically assuming an O.D. equal to 1.0 
corresponds to a DNA concentration equal to 50 .mu.g/mL. Target DNA 
solutions were made by serially diluting the digested plasmid in 5 mM Tris 
pH 7.8, 0.1 mM EDTA and 300 .mu.g/mL human placental DNA. 
All reactions, unless otherwise stated, were performed in LCR Buffer (45 mM 
EPPS pH 7.8, 80 mM KCl, 10 mM MgCl.sub.2, 10 mM NH.sub.4 Cl and 0.5 mM 
NAD.sup.+) supplemented with acetylated bovine serum albumin (BSA) and 
temperature cycling was achieved with a COY Model 50 temperature cycler. 
Reactions were terminated by transferring aliquots into Stop Buffer (80% 
formamide, 20 mM EDTA, 0.05% (w:v) xylene cyanol and 0.05% bromophenol 
blue). The ligated and unligated products were resolved on a 
16.times.20.times.0.04 cm 15% polyacrylamide gel containing 8.3 M urea in 
80 mM Tris, 80 mM boric acid pH 8.0, 1.0 mM EDTA. The gel was 
autoradiographed, the autoradiograph used as a template to excise the 
ligated and unligated probes and the amount of radioactivity in each band 
was measured by liquid scintillation counting. The percentage of 
radioactivity in the ligated product was calculated as a function of the 
total counts in each lane. 
Unless otherwise stated, the following abbreviations have the meaning 
indicated. 
______________________________________ 
BSA bovine serum albumin 
EDTA a metal chelator, ethylenediamine tetraacetic 
acid 
EPPS a buffer comprising N-(2-hydroxyethyl)- 
piperazine-N'-(3-propanesulfonic acid) 
HPLC high performance liquid chromatography 
NAD or NAD.sup.+ 
nicotine adenine dinucleotide 
Tris a buffer comprising tris(hydroxymethyl)amino- 
methane 
TTh Thermus thermophilus 
______________________________________ 
TABLE I 
__________________________________________________________________________ 
AA PROBE SETS TO Position 123 of 
ACTINOBACILLUS ACTINOMYCETEMCOMITANS 
Designation 
SEQUENCE SEQ ID No. 
__________________________________________________________________________ 
AA123-1 (20) 
5'-TTGTCGAGCACCTTGAATAA 
3' 1 
AA123-1P (20) 
5'-TTGTCGAGCACCTTGAATAAp 
3' 2 
AA123-1-E1 
5'-TTGTCGAGCACCTTGAATAAxT 
3' 3 
AA123-1-E3 
5'-TTGTCGAGCACCTTGAATAAxTAA 
3' 4 
AA123-1-E5 
5'-TTGTCGAGCACCTTGAATAAxTAATG 
3' 5 
AA123-2 5'- 
pTTAATGGCTTCGATTGGGCT-3' 6 
AA123-3 (20) 
3'-AACAGCTCGTGGAACTTATTp 
5' 7 
AA123-3 (18) 
3'-AACAGCTCGTGGAACTTAp 
5' 8 
AA123-4P (22) 
3'- 
pTTAATTACCGAAGCTAACCCGA-5' 9 
AA123-4P (20) 
3'- 
pAATTACCGAAGCTAACCCGA-5' 10 
AA123-4 (20) 
3'- 
AATTACCGAAGCTAACCCGA-5' 11 
AA123-4-E1 
3'- 
TxTTAATTACCGAAGCTAACCCGA-5' 12 
AA123-4-E3 
3'- 
CTTxTTAATTACCGAAGCTAACCCGA-5' 
13 
AA123-4-E5 
AACTTxTTAATTACCGAAGCTAACCCGA-5' 
14 
__________________________________________________________________________ 
where x = abasic site; p = 3' phosphate blocking group; and p = 5' 
phosphate group (normal ligation substrate) 
TABLE II 
__________________________________________________________________________ 
AA PROBE SETS TO Position 250 of 
ACTINOBACILLUS ACTINOMYCETEMCOMITANS 
Designation 
SEQUENCE SEQ ID No. 
__________________________________________________________________________ 
AA250-1 
5'-CCGATTGCAATGTAATATCGACGTC 3' 
15 
AA250-1E5 
5'-CCGATTGCAATGTAATATCGACGTCxTCGGC 
3' 
16 
AA250-2 
5' pGTCGGGCAAATAATTCGCCAC-3' 17 
AA250-3 (24) 
3'-GCTAACGTTACATTATAGCTGCAGp 
5' 18 
AA250-3 (22) 
3'-GCTAACGTTACATTATAGCTGCp 
5' 19 
AA250-4 (19) 
3'- 
CAGCCCGTTTATTAAGCGG-5' 20 
AA250-4 (21) 
3'- 
AGCAGCCCGTTTATTAAGCGG-5' 21 
__________________________________________________________________________ 
where x = abasic site; p = 3' phosphate blocking group; and p = 5' 
phosphate group. (normal ligation substrate) 
TABLE III 
__________________________________________________________________________ 
Synthetic Target Molecules 
__________________________________________________________________________ 
For AA123 Probe Sets 
AA123TAR-3/4 
##STR4## 22 
AA123TAR-1/2 
##STR5## 23 
Substrate For Abasic Nuclease Detection 
E4SUB1 
##STR6## 24 
__________________________________________________________________________ 
where x = abasic site. 
EXAMPLE 1: Blunt-End LCR.TM. 
LCR.TM. was performed using the blunt-ended probe set (see Table I) 
consisting of AA123-1(20), AA123-2, AA123-3(20) and AA123-4(20) in a 20 gL 
reaction volume containing LCR Buffer supplemented with 10 .mu.g/mL BSA 
and 300 ng of human placental DNA. Each probe was present at 83 nM 
(approximately 5% of probe 2 was 3' -end labeled with [.alpha.-.sup.32 
P]-cordycepin triphosphate to enable detection) and the final 
concentration of Thermus thermophilus (Tth) DNA ligase equalled 0.15 
.mu.g/mL. Duplicate reactions containing either zero or 10.sup.6 molecules 
of target DNA were performed. The samples were overlaid with 15 gL of 
mineral oil and the temperature cycle consisted of a 90.degree. C. 
incubation for 30 seconds followed by a 50.degree. C. incubation for 30 
seconds. At specified cycles (see Table E-1 ), 1.4 gL aliquots were 
removed, mixed with 2.0 .mu.L of stop buffer, heated to 90.degree. C. for 
2 min and applied to a denaturing polyacrylamide gel. Table E-1 shows the 
average percent ligated and the ratio of (+)/(-) target for the duplicate 
reactions. The data show that 10.sup.6 targets are distinguishable from 
zero targets. However, other data show that detection of fewer than 
10.sup.5 were not adequately reproducible using unmodified probes. 
TABLE E-1 
______________________________________ 
Blunt-End LCR 
Percent Ligated 
Cycle No. 
0 Targets 10.sup.6 Targets 
Ratio (+/-) Target 
______________________________________ 
25 1.65 32.17 19.5 
28 5.76 41.88 7.3 
31 14.55 52.27 3.6 
34 23.28 59.97 2.6 
37 34.11 65.20 1.9 
______________________________________ 
EXAMPLE 2: Demonstration of Blocking Effect of Abasic Modification on 
Ligation 
3'[.alpha.-.sup.32 P]-cordycepin labeled AA123-2 was incubated at 
50.degree. C. for 40 min with either AA123-1E1, AA123-1E3, or AA123-1E5 
(see Table I), the complementary sequence AA123TAR(1/2)(Table III), and 
Tth DNA ligase in the presence (+) or absence (-) of Tth endonuclease IV. 
An identical set of reactions employing the AA123-4E1, AA123-4E3 and 
AA123-4E5 (Table I) with AA123TAR(3/4) (Table III) and 3'[.alpha.-.sup.32 
P]-cordycepin labeled AA123-3(18) was also performed. The ligated and 
unligated products were resolved on a 20.times.40.times.0.04 cm 12.5% 
polyacrylamide gel containing 8.3 M urea in 80 mM Tris, 80 mM boric acid 
pH 8.0, 1.0 mM EDTA. FIG. 4 is an autoradiograph showing that the abasic 
probes with abasic extensions are not suitable substrates for DNA ligase; 
and that the blocking extensions are removed by endonuclease IV to render 
probes -1 and -4 ligatable to probes -2 and -3, respectively. Other data 
(not shown) confirm the same results are achieved with 3'-PO.sub.4 
blocking groups and furthermore, the correction and/or ligation of the 
probes with endonuclease IV and/or DNA ligase occurs only in the presence 
of the correct complementary sequence. 
Examples 3-4, and 8-9 relate to LCR reactions using probes modified to 
contain abasic sites followed by extensions. Examples 5-7 relate to LCR 
reactions using probes modified to contain 3' blocking phosphate groups. 
EXAMPLE 3: LCR Using One Abasic Site Probe 
LCR was performed using probes AA123-1(20), AA123-2 and AA123-3(18) and 
AA123-4E5 from Table I in a Coy model 50 thermocycler set for 95.degree. C 
for 30 sec, followed by 55.degree. C. for 110 sec. Reactions were run in 
20 .mu.L volume containing 45 mM EPPS pH 7.8, 80 mM KCl, 10 mM MgCl.sub.2, 
0.5 mM NAD.sup.+, 1 mM CoCl.sub.2, and 300 ng human placental DNA. Unless 
otherwise indicated, probes were present at 83 nM (about 5% of probe 
AA123-2 was 3' end labeled with radioactive [.alpha.-.sup.32 P]-cordycepin 
triphosphate to enable detection) and Tth ligase (varying amounts, see 
data table E-3). Target was zero, 10.sup.2, 10.sup.3 or 10.sup.4 molecules 
of EcoR1 and PstI digested AA DNA as shown. 
The data is presented in Table E-3 below as a signal-to-background ratio. A 
signal which is consistently at least 3-4 times that of background is 
generally sufficient to distinguish target from background. 
TABLE E-3 
______________________________________ 
Signal-to-Background Ratio 
Endo IV Cycle 10.sup.3 
10.sup.4 
10.sup.5 
Fourth Probe 
Dilution No. Targets 
Targets 
Targets 
______________________________________ 
AA123-4E5 
1:5000 30 12.0 
AA123-4E5 
1:2000 30 9.7.sup.1 
AA123-4E5 
1:10000 30 22.4.sup.2 
AA123-4E5 
1:50000 30 1 3.5 11.6 
40 3.1 6.5 9.8 
______________________________________ 
.sup.1 Under identical conditions, unmodified probe AA1234 produced a S/B 
ratio of only 1.6. 
.sup.2 Under identical conditions, probe AA1234E3 also produced a S/B 
ratio of 20 at 30 cycles. 
EXAMPLE 4: Comparison of Different Abasic Site Probes 
Example 3 is repeated, except probe concentration was reduced to 17 nM in a 
reaction volume of 50 .mu.L, and the fourth probe is either AA123-4E5 or 
AA123-4E3. The data is shown below, as a signal-to-background ratio. 
TABLE E-4 
______________________________________ 
Signal-to-Background Ratio 
Endo IV 
Fourth Probe 
Dilution Cycle No. 10.sup.3 Targets 
10.sup.5 Targets 
______________________________________ 
AA123-4E3 
1:5000 40 1 6.7 
AA123-4E5 40 1 5.0 
AA123-4E3 
1:10000 40 1 7.7 
45 1 6.2 
AA123-4E5 40 1 11.5 
45 1 5.0 
______________________________________ 
EXAMPLE 5: LCR Using One 3' Phosphate Blocked Probe 
LCR was performed using probes AA123-1(20), AA123-2, AA123-3(18) and AA 
123-4P(22) from Table I under conditions as in Example 3, above. The data 
is presented in Table E-5 below as a signal-to-background ratio. 
TABLE E-5 
______________________________________ 
Signal-to-Background Ratio 
Endo IV 
Dilution Cycle No. 10.sup.3 Targets 
10.sup.4 Targets 
______________________________________ 
1:10000 28 1 3.3 
31 2.4 5.8 
34 2.3 3.8 
1:5000 25 1 1.8 
28 2.0 3.8 
31 2.0 3.2 
34 1.6 2.0 
______________________________________ 
EXAMPLE 6: LCR Using Blunt-End 3' Phosphate Blocked Probes 
LCR was performed using the blunt-ended, 3'-phosphorylated probe set (see 
Table I) consisting of AA123-1P(20), AA123-2, AA123-3(20) and AA123-4P(20) 
in a gL reaction volume containing LCR Buffer supplemented with 50 
.mu.g/mL BSA, 0.5 mM CoCl.sub.2 and 300 ng of human placental DNA. Each 
Probe was present at 83 nM (approximately 5% of probe 2 was 3'-end labeled 
with [.alpha.-.sup.32 P]-cordycepin triphosphate to enable detection) and 
the enzymes Tth DNA ligase and Tth endonuclease IV were present at 0.15 
.mu.g/mL and 4.1 .mu.g/mL respectively. Duplicate reactions for zero, 
10.sup.2, and 10.sup.3 molecules of target DNA were performed. The samples 
were overlaid with 15 .mu.L of mineral oil and the temperature cycle 
consisted of a 95.degree. C. incubation for 30 seconds followed by a 
55.degree. C. incubation for 110 seconds. At specified cycles (see Table 
E-6), 1.4 .mu.L aliquots were removed, mixed with 2.0 .mu.L of stop 
buffer, heated to 90.degree. C. for 2 min and applied to a denaturing 
polyacrylamide gel. Table E-6 is the average percent ligated and the ratio 
of (+)/(-) target for the duplicate reactions. It is observed that 103 
targets are distinguishable from zero targets. 
TABLE E-6 
______________________________________ 
Blunt-End 3'-PO.sub.4 Probes 
Percent Ligated (+Target/-Target) 
Cycle No. 0 Targets 10.sup.2 Targets 
10.sup.3 Targets 
______________________________________ 
31 0.91 1.90 (2.1) 
2.07 (2.3) 
34 1.50 2.04 (1.4) 
4.24 (2.8) 
37 2.34 4.40 (1.9) 
9.84 (4.2) 
______________________________________ 
EXAMPLE 7: LCR Using Non-blunt 3' Phosphate Blocked Probes 
LCR was performed using the non-blunt probe set (see Table I) consisting of 
AA123-1P(20), AA123-2, AA123-3(18) and AA123-4P(22) in a 20 .mu.L reaction 
containing LCR Buffer supplemented with 10 .mu.g/mL BSA, 0.5 mM CoCl.sub.2 
and 300 ng of human placental DNA. Each Probe was present at 83 nM 
(approximately 5% of probe 2 was 3'-end labeled with [.alpha.-.sup.32 
P]-cordycepin triphosphate to enable detection) and the enzymes Tth DNA 
ligase and Tth endonuclease IV were present at 0.15 .mu.g/mL and 4.1 
.mu.g/mL respectively. Duplicate reactions for zero, 10.sup.2, and 
10.sup.3 molecules of target DNA were performed. The samples were overlaid 
with 15 .mu.L of mineral oil and the temperature cycle consisted of a 
95.degree. C. incubation for 30 seconds followed by a 55.degree. C. 
incubation for 110 seconds. At specified cycles (see Table E-5), 1.4 .mu.L 
aliquots were removed, mixed with 2.0 .mu.L of stop buffer, heated to 
90.degree. C. for 2 min and applied to a denaturing polyacrylamide gel. 
Table E-7 is the average percent ligated and the ratio of (+)/(-) target 
for the duplicate reactions. It is observed that 10.sup.3 targets are 
distinguishable from zero targets. 
TABLE E-7 
______________________________________ 
Overhang 3'-PO.sub.4 Probes 
Percent Ligated (+Target/-Target) 
Cycle No. 0 Targets 10.sup.2 Targets 
10.sup.3 Targets 
______________________________________ 
28 0.75 0.88 (1.2) 
2.18 (2.9) 
31 2.15 2.48 (1.2) 
5.66 (2.6) 
34 4.67 6.02 (1.3) 
11.48 (2.5) 
37 8.21 9.45 (1.2) 
16.69 (2.1) 
______________________________________ 
EXAMPLE 8: LCR Using Probes Modified with an Abasic Site and an Extension 
of One Base. 
LCR was performed using the non-blunt probe set (see Table I) consisting of 
AA123-1E1, AA123-2, AA123-3(18) and AA123-4E1 in a 20 .mu.L reaction 
volume containing LCR Buffer supplemented with 50 .mu.g/mL BSA, 0.5 mM 
CoCl.sub.2 and 300 ng of human placental DNA. Each probe was present at 83 
nM (approximately 5% of probe 2 was 3'-end labeled with 
[.alpha.-32P]-cordycepin triphosphate to enable detection) and the enzymes 
Tth DNA ligase and Tth endonuclease IV were present at 0.15 .mu.g/mL and 
41 .mu.g/mL respectively. Duplicate reactions for zero, 10.sup.3, and 
10.sup.4 molecules of target DNA were performed. The samples were overlaid 
with 15 .mu.L of mineral oil and the temperature cycle consisted of a 
95.degree. C. incubation for 30 seconds followed by a 55.degree. C. 
incubation for 240 seconds. At specified cycles (see Table E-8), 1.4 
.mu.L aliquots were removed, mixed with 2.0 .mu.L of stop buffer, heated 
to 90.degree. C. for 2 min and applied to a denaturing polyacrylamide gel. 
Table E-8 is the average percent ligated and the ratio of (+)/(-) target 
for the duplicate reactions. It is observed that 10.sup.3 targets are 
distinguishable from zero targets. 
TABLE E-8 
______________________________________ 
Abasic Probes with a One Base Extension 
Percent Ligated 
Cycle 0 Targets 10.sup.3 Targets 
10.sup.4 Targets 
______________________________________ 
35 0 0.98 6.99 
40 0 4.07 13.96 
45 0 6.27 19.23 
50 0 8.74 23.50 
______________________________________ 
EXAMPLE 9: LCR Using Endonuclease IV Activity Isolated From Sulfolobus 
solfataricus 
T A: Enzyme Isolation: Sulfolobus solfataricus (ATCC 35091) was grown as 
suggested by the ATCC. 20 grams of cells frozen in media were thawed and 
mixed with 10 mL of 50 mM Tris pH 7.4, 5% (w:v) glycerol, 0.5 mM 
dithiothreitol to yield a volume of 70 mL. 3.0 mL of 1.0 M Tris pH 7.4 was 
added and the cells were crushed by two passages through a French press at 
14,000 psi. The mixture was centrifuged for 30 min. at 40,000 g. 
Approximately 66 mL of supernatant was collected, diluted with 0.5 volumes 
of glycerol and stored at -20.degree. C. 20 mL of the lysate was diluted 
with 50 mL of 20 mM potassium phosphate pH 7.0, 1.0 mM dithiothreitol, 5% 
glycerol (v:v) ("Buffer A") and loaded onto a 1.6x9.5 cm column of Blue 
Sepharose (Pharmacia) equilibrated with Buffer A. The column was washed 
with 15 mL Buffer A and developed with a linear gradient from 0.1M to 
0.74M NaCl in Buffer A. 3.2 mL fractions were collected and 1.25 mL of 
glycerol were added to each of 45 fractions collected. The fractions were 
tested for endonuclease IV activity employing the oligonucleotide E4SUB 1 
(see Table III) labelled with [.alpha.-.sup.32 P] cordycepin by terminal 
deoxynucleotidyl transferase and resolving the cut and uncut oligos on a 
denaturing polyacrylamide gel. Fractions 27-44 were pooled, made 0.15 mM 
in CoCl.sub.2, and heated to 80.degree. C. for 2.5 min. yielding a final 
volume of 59 mL. To concentrate the sample it was necessary to remove 
debris by centrifuging at 28,000 g for 15 min., passing the supernatant 
through a 0.2 .mu.M filter and finally concentrating 3-fold in an Areicon 
Centfiprep 10 filter. 3.0 mL of the concentrated protein solution was 
diluted with 30.0 mL of 25 mM Tris pH 7.4, 1.0 mM MgSO.sub.4, 50 .mu.M 
CoCl.sub.2 and 5% (v:v)glycerol ("Buffer B") and loaded onto a 4.0 mL 
heparin-agarose column equilibrated with Buffer B. The column was 
developed using a gradient of 0.25 M to 0.5 M KCl in Buffer B collecting 
1.0 mL fractions. Fractions were assayed for abasic nuclease activity and 
fractions 9-11 were pooled. The 3.0 mL were transferred to 25 mM EPPS pH 
7.7, 0.1 M KCl, 1 mM MgCl.sub.2, 50 .mu.M CoCl.sub.2 and 5% (v:v) glycerol 
("Buffer C") by passing the sample over a BioRad 10DG column equilibrated 
with Buffer C. 4.0 mL was collected and concentrated to ca. 100 .mu.L in 
an Amicon Centriprep 10. This sample was heated to 90.degree. C. for 5 min 
and was found to be free of both single- and double-stranded nuclease 
activity using oligodeoxyribonucleotides AA250-1 and AA250- 3 as 
substrates. 
T B: LCR Reactions: LCR was performed comparing the blunt-ended probe 
set consisting of AA250-1, AA250-2, AA250-3(24) and AA250-4(19) with the 
non-blunt probe set with one abasic probe consisting of AA250-1E5, 
AA250-2, AA250-3(22) and AA250-4(21 ) in a 20 .mu.L reaction volume 
containing LCR Buffer supplemented with 10 .mu.g/mL BSA, 2.0 mM CoCl.sub.2 
and 300 ng of human placental DNA. Each probe was present at 83 nM 
(approximately 5% of probe 2 was 3'-end labeled with [.alpha.-.sup.32 
P]-cordycepin triphosphate to enable detection) and the Tth DNA ligase was 
present at 0.15 .mu.g/mL. The purified SulfoIobus solfataricus 
endonuclease IV activity was used at a final dilution of 1:10. Duplicate 
reactions for zero and 10.sup.6 molecules of target DNA were performed for 
both blunt and non-blunt probe sets. The samples were overlaid with 15 
.mu.L of mineral oil and the temperature cycle consisted of a 95.degree. 
C. incubation for 30 seconds followed by a 55.degree. C. incubation for 60 
seconds. At specified cycles (see Table E-9), 1.4 .mu.L aliquots were 
removed, mixed with 2.0 .mu.L of stop buffer, heated to 90.degree. C. for 
2 min and applied to a denaturing polyacrylamide gel. Table E-9 gives the 
average percent ligated and the ratio of 10.sup.6 targets/zero targets for 
the duplicate reactions in each case. It is observed that the ratio of 
10.sup.6 targets/zero targets is better with the abasic extension modified 
non-blunt probe set. This improvement is due to a decrease in the signal 
arising in the reactions containing zero target molecules whereas the 
percent ligated in the reactions containing 10.sup.6 target molecules is 
the same for both probe sets. 
TABLE E-9 
______________________________________ 
LCR with Endo IV from Sulfolobus solfataricus 
Ratio (+/-) 
Cycle No. 
4th Probe 0 Targets 10.sup.6 Targets 
Target 
______________________________________ 
30 AA123-1 3.9 24.1 6.2 
30 AA123-1E5 0.5 26.1 54.0 
35 AA123-1 6.8 38.0 5.6 
35 AA123-1E5 3.1 35.8 11.7 
40 AA123-1 18.7 51.1 2.7 
40 AA1E5 15.3 52.8 3.5 
______________________________________ 
Examples 10-13 relate to the effect of divalent cations on LCR and 
Endonuclease IV modified LCR. 
EXAMPLE 10: Effect of MnCl.sub.2 on LCR and the Improvement with 
Endonuclease IV 
LCR was performed using the non-blunt probe set (see Table I) consisting of 
AA123-1P(20), AA123-2, AA123-3(18) and AA123-4P(22) in a 20 .mu.L reaction 
volume containing LCR Buffer supplemented with 10 .mu.g/mL BSA, 0.1 mM 
MnCl.sub.2 and 300 ng of human placental DNA. In this experiment probe 
AA123-1P(20) was haptenated with biotin at its 5' terminus and AA123-2(20) 
was haptenated at its 3' terminus with digoxigenin for a purpose not 
related to this experiment. Each probe was present at 83 nM (approximately 
5% of probe 3 was 3'-end labeled with [.alpha.-.sup.32 P]-cordycepin 
triphosphate to enable detection) and the enzymes Tth DNA ligase and Tth 
endonuclease IV were present at 0.15 .mu.g/mL and 4.1 .mu.g/mL 
respectively. Duplicate reactions for zero and 10.sup.3 molecules of 
target DNA were performed. The samples were overlaid with 15 .mu.L of 
mineral oil and the temperature cycle consisted of a 95.degree. C. 
incubation for 30 seconds followed by a 55.degree. C. incubation for 110 
seconds. At specified cycles (see Table E-10), 1.4 .mu.L aliquots were 
removed, mixed with 2.0 .mu.L of stop buffer, heated to 90.degree. C. for 
2 min and applied to a denaturing polyacrylamide gel. Table E-10 gives the 
average percent ligated and the ratio of (+)/(-) target for the duplicate 
reactions. It is observed that 10.sup.3 targets are not distinguishable 
from zero targets. 
TABLE E-10 
______________________________________ 
Overhang 3'-PO.sub.4 Probes with MnCl.sub.2 
Percent Ligated 
Cycle No. 
0 Targets 10.sup.3 Targets 
Ratio (+/-) Target 
______________________________________ 
25 3.29 2.47 0.8 
35 23.65 23.30 1.0 
______________________________________ 
EXAMPLE 11: Effect of CoCl.sub.2 Concentrations on LCR with Unmodified, 
Non-blunt Probes 
LCR was performed using the unmodified, non-blunt probe set (see Table I) 
consisting of AA123-1(20), AA123-2, AA123-3(18) and AA123-4(22) in a 20 
.mu.L reaction volume containing LCR Buffer supplemented with various 
amounts of CoCl.sub.2 as indicated in Table E-11, 10 .mu.g/mL BSA and 300 
ng of human placental DNA. In this experiment probe AA123-1P(20) was 
haptenated with biotin at its 5' terminus and AA123-2(20) was haptenated 
at its 3' terminus with digoxigenin. Each probe was present at 83 nM 
(approximately 5% of probe 3 was 3'-end labeled with [.alpha.-.sup.32 
P]-cordycepin triphosphate to enable detection) and the final 
concentration of Tth DNA ligase equalled 0.15 .mu.g/mL. Duplicate 
reactions containing either zero or 10.sup.6 molecules of target DNA were 
performed. The samples were overlaid with 10 .mu.L of mineral oil and the 
temperature cycle consisted of a 95.degree. C. incubation for 30 s 
followed by a 55.degree. C. incubation for 110 s. At 20 and 30 cycles 1.4 
.mu.L aliquots were removed, mixed with 2.0 .mu.L of stop buffer, heated 
to 90.degree. C. for 2 min and applied to a denaturing polyacrylamide gel. 
Table E-11 is the average percent ligated and the ratio of (+)/(-) target 
for the duplicate reactions. It is observed that CoCl.sub.2 has an 
inhibitory effect on the extent of amplification in the reactions both 
with and without target DNA. This implies that CoCl.sub.2 has an 
inhibitory effect on Tth DNA ligase. 
TABLE E-11 
______________________________________ 
Effects of CoCl.sub.2 Concentration on LCR 
Percent Ligated 
Cycle CoCl.sub.2 
No. (mM) 0 Targets 
10.sup.6 Targets 
Ratio (+/-) Target 
______________________________________ 
20 0.0 0.19 4.60 24.2 
0.5 0.32 3.13 9.8 
1.0 0.37 0.90 2.4 
2.0 0.0 0.0 N.A. 
30 0.0 12.28 22.42 1.8 
0.5 2.68 12.96 4.8 
1.0 0.75 5.88 7.8 
2.0 0.0 0.0 N.A. 
______________________________________ 
EXAMPLE 12: LCR with Modified and Unmodified Probes in the Absence of 
MgCl.sub.2 and CoCl.sub.2 
LCR was performed in a buffer containing 47 mM EPPS pH 7.8, 80 mM KCl, 10 
mM NH.sub.4 Cl.sub.2, 5 mM MnCl.sub.2, 10 .mu.g/mL BSA and 15 .mu.g/mL 
human placental DNA and no MgCl.sub.2 or CoCl.sub.2. Four sets of probes 
were used in this experiment, representing 3'-phosphorylated ends, abasic 
sites with extensions of 3 or 5 bases, and no modifications, as follows 
(see Table I): 
______________________________________ 
unmodified, non-blunt set 
AA123-1(20), AA123-2, AA123-3(18), 
and AA123-4(22) 
3'-phosphorylated set 
AA123-1P(20), AA123-2, 
AA123-3(18), and AA123-4P(22) 
abasic extension set 
AA123-1E3, AA123-2, AA123-3(18), 
and AA123-4E3 
abasic extension set 
AA123-1E5, AA123-2, AA123-3(18), 
and AA123-4E5 
______________________________________ 
Duplicate reactions containing either zero or 10.sup.4 molecules of target 
DNA were performed. The samples were overlaid with 15 .mu.L of mineral oil 
and the temperature cycle consisted of a 95.degree. C. incubation for 30 s 
followed by a 55.degree. C. incubation for 110 s. After 60 cycles 3.0 
.mu.L aliquots were removed, mixed with 3.0 .mu.L of stop buffer, heated 
to 90.degree. C. for 2 min and applied to a denaturing polyacrylamide gel. 
No amplification occurred with any of the probe sets indicating that 
MnCl.sub.2 at 5 mM cannot substitute for 10 mM MgCl.sub.2 supplemented 
with low concentrations of either CoCl.sub.2 or MnCl.sub.2. 
Analogous LCR assays incorporating the same buffer as above but with 10 mM 
MnCl.sub.2 and an unmodified probe set consisting of AA123-1(20), AA123-2, 
AA123-3(18) and AA123-4(22) also showed no amplification out to 44 cycles. 
EXAMPLE 13: LCR and Endo IV LCR with varying Cobalt Concentrations. 
Endo IV-LCR assays were performed in a reaction mix consisting of 50 mM 
EPPS pH 7.8, 5mM MgCl.sub.2, 20.mu.g/ml BSA, 1.times.10.sup.12 of the 
oligos given in Table IV below, 215 units Thermus thermophilus DNA ligase, 
a 1.5.times.10.sup.-4 dilution of T. thermophilus endonuclease IV, and 
various concentrations of CoCl.sub.2 ranging from 500.mu.M to 2 mM in a 
final reaction volume of 20 microliters. The oligos are specific for 
positions 6693-6739 of the Chlamydia trachomaas cryptic plasmid as given 
by Hatt, C. et al. Nuc. Acids Res. 16:4053-4067 (1988). Oligos #1 and #4 
include 3' phosphate blocking groups as shown. 
TABLE IV 
__________________________________________________________________________ 
DNA PROBE SETS TO Position 6693-6739 of 
Chlamydia trachomatis cryptic plasmid 
Designation 
SEQUENCE SEQ ID No. 
__________________________________________________________________________ 
#1 5' F1-GATACTTCGCATCATGTGTTCCp 
3' 
35 
#3 5' pGGAGTTTCTTTGTCCTCCTATAACG-Bio 3' 
36 
#2 3' F1-CTATGAAGCGTAGTACACAAGGp 
5' 
37 
#4 3' pCCTCAAAGAAACAGGAGGATATTGC-Bio 5' 
38 
__________________________________________________________________________ 
where: p = 3' - phosphate blocking group; and p = a normal 5' phosphate 
group; Fl = a fluorescein moiety; and Bio = a biotin moiety 
Endo IV-LCR cycling conditions were 95.degree. C. for 30 seconds then 
55.degree. C. for 110 seconds repeated thirty times in a Coy thermocycler. 
Negative reactions were set up with 300 nanograms of human placental DNA 
in dH.sub.2 O. Positive reactions contained either 10.sup.4 or 10.sup.6 
molecules of a synthetic DNA oligonucleotide corresponding to map 
positions 6693-6739 of the C. trachomatis plasmid sequence in a background 
of 300 ng of placental DNA. Following amplification, reactions were 
diluted 1:1 with IMx diluent buffer, and the LCR amplification products 
were detected via a sandwich immunoassay performed using the Abbott 
IMx.RTM. automated immunoassay system. Results are shown in Table E-13 
below. It can be seen that at cobalt concentrations of 1.0 mM or less, 
10.sup.6 targets are distinguishable from no target; while at greater 
cobalt concentrations, target was not distinguishable. 
TABLE E-13 
______________________________________ 
IMx rate count 
Amount of CoCl.sub.2 concentration 
Target 0.5 mM 1 mM 2 mM 
______________________________________ 
0 27.8 8.8 23.5 
10.sup.4 47.3 8.9 19.8 
10.sup.6 390.0 71.5 16.3 
______________________________________ 
Examples 14-17 relate to the use of LCR reactions using endonuclease IV 
correctable modified probes which also contain ribonucleotide residues. 
The ribonucleotide residues permit selective destruction of the 
amplification products using RNase or alkali as a means for controlling 
possible contamination, as is taught in WO91/17270 for conventional LCR 
reactants. The probes used in these examples are shown in Table V below. 
TABLE V 
__________________________________________________________________________ 
Mixed DNA/RNA PROBE SETS TO Position 123 of 
ACTINOBACILLUS ACTINOMYCETEMCOMITANS 
Designation 
SEQUENCE SEQ ID No. 
__________________________________________________________________________ 
AA123-1R (20) 
5'TTGTCGAGCACCTTGAATAA 
3' 25 
AA123-1RP (20) 
5'TTGTCGAGCACCTTGAATAAp 
3' 26 
AA123-2 5' pTTAATGGCTTCGATTGGGCT-3' 
6 
AA123-3 (20) 
3'AACAGCTCGTGGAACTTATTp 
5' 7 
AA123-4R (20) 
3' AATTACCGAAGCTAACCCGA-5' 
27 
AA123-4RP (20) 
3' pAATTACCGAAGCTAACCCGA-5' 
28 
__________________________________________________________________________ 
where: A is adenosine ribonucleotide; p = 3' - phosphate blocking group; 
and p = a normal 5' phosphate group. 
EXAMPLE 14: Preparation of Mixed Ribo- and Deoxyribo-Oligonucleotides 
Having 3' Phosphate Blocking Groups 
The mixed ribo- and deoxyribo-oligonucleotides AA 123-1R and AA123-4R (see 
Table V) were prepared by initiating synthesis on a solid phase support 
beating an adenosine ribonucleotide. Synthesis was continued with the 
sequential addition of deoxyribonucleotide phosphoramidites using an 
automated DNA synthesizer. The resulting mixed oligonucleotide 
"ribo-modified" probe) was cleaved from the support and deprotected with 
37% NH.sub.4 OH at 55.degree. C. for 12 hours and purified by 
reverse-phase HPLC on a C18 column. 
Radioactive phosphate moieties were added in two steps to make 
oligodeoxyribonucleotides having at the 3' terminal residue a 2'-OH and 
3'-.sup.32 PO.sub.4 group. First, the ribo-modified probes AA123-1R or 
AA123-4R were incubated for 1.5 h at 37.degree. C. with 20 pmol of 
[.alpha.-.sup.32 P]-cordycepin 5'-triphosphate (5000 Ci/mmol) and 10 units 
of deoxynucleotidyl terminal transferase in a total reaction volume of 15 
.mu.L buffered with 140 mM sodium cacodylate pH 7.2, 1 mM CoCl.sub.2, 0.1 
mM dithiothreitol. The unreacted cordycepin was separated from the 
oligonucleotide on a 1.0 mL Sephadex Go50 column equilibrated with 5 mM 
Tris pH 8.0, 0.1 mM EDTA. Two drop fractions (ca. 75 .mu.L) were collected 
and the elution profile was monitored by counting 1.0 .mu.L of each 
fraction in 4.0 mL of liquid scintillation cocktail. The fractions 
containing the oligonucleotide were pooled. The resulting oligonucleotide 
contains a single .sup.32 P-labeled 3'-phosphodiester bond adjacent to the 
unique 2'-OH group at the 3' terminus. 
Second, the phosphodiester bond between the oligonucleotide and the 
cordycepin was cleaved with T2 RNase, which cuts 3' to the adenosine 
residue, liberating the cordycepin deoxyribonucleoside and the desired 
fibo-modified probes. Approximately 0.2 pmol of .sup.32 P labeled AA 
123-1R or AA 123-4R were incubated with 2.5 units of T2 RNase for 1 h at 
37.degree. C. in a total reaction volume of 4.0 .mu.L buffered with 50 mM 
potassium acetate, pH 5.2. The reaction was terminated by adding 5.0 .mu.L 
of stop buffer and 1.0 .mu.L of 5' LCR buffer and 5.0 .mu.L was loaded on 
a 20.times.40.times.0.04 cm denaturing 12.5% polyacrylamide gel and 
electrophoresed for 1.75 h at 30W. As can be observed in FIG. 5 (lanes 3 
and 11), the products AA 123-1RP(20) and AA123-4RP(20), resulting from 
RNase digestion of the [.alpha.-.sup.32 P]-cordycepin labeled AA 123-1R 
and AA 123-4R migrate faster than the undigested oligonucleotides (lanes 2 
and 10). Although the products of digestion with T2 RNase are expected to 
be decreased in length by only one base, they are observed to migrate with 
an Rf value similar to that of the faint failure sequence that is two 
bases shorter than the undigested material. This is expected since the 
correlation between length and Rf values of DNA fragments is based on all 
the DNA fragments having the same mass to charge ratio but, the additional 
negative charge associated with the 3'-phosphorylated oligonucleotide will 
increase the charge to mass ratio, resulting in an increased Rf value. 
EXAMPLE 15: Activity of Endonuclease IV on Ribo-Modified Probes 
The 3'-phosphorylated oligonucleotides AA123-1RP(20) and AA123-4RP(20) were 
further analyzed to determine if the 3'-PO.sub.4 group attached to a 
ribonucleoside could be removed by Tth endonuclease IV, and if the 
enzymatic removal required that AA 123 1RP(20) and AA 123-4RP(20) be 
hybridized to the complementary synthetic target oligonucleotides AA 
123TAR(1/2) or AA 123TAR(3/4), respectively, (see Table III) so that the 
3'-PO.sub.4 is located in a double-stranded region. This was achieved by 
digesting aliquots of the [.alpha.-.sup.32 P]-cordycepin labeled AA123-1R 
and AA123-4R with T2 RNase, as in example 14, followed by a second 
digestion with Tth endonuclease IV in the presence or absence of 
AA123TAR(1/2) or AA123TAR(3/4) in LCR buffer. As required, 1.7 pmol of 
AA123TAR(1/2) or AA123TAR(3/4) and Tth endonuclease IV, to a final 
concentration equal 4.1 .mu.g/mL, were added and the volume was adjusted 
to 10 .mu.L with water. The samples were incubated for 1 h at 55.degree. 
C., 5.0 .mu.L aliquots were removed into 5.0 .mu.L of stop buffer, and 
then analyzed by denaturing polyacrylamide gel electrophoresis as detailed 
above. As can be seen in FIG. 5 (lanes 4 and 12), the removal of the 
3'-.sup.32 PO.sub.4 from the oligonucleotide occurs only when T2 RNase, 
Tth endonuclease IV and the strand complementary to the labeled 
oligonucleotide are all present. The absence of any one of these three 
materials prevents the removal of the .sup.32 p label from AA123-1RP(20) 
and AA 123-4RP(20). 
It has also been observed (data not shown) that the cleavage of 
AA123-1RP(20) by T2 RNase in the presence of target AA123TAR(1/2) but the 
absence of Tth endonuclease IV results in the same size product as 
observed with T2 RNase in the absence of the complementary target. These 
results demonstrate the reaction product from T2 RNase treatment of the 
[.alpha.-.sup.32 P]-cordycepin labeled oligonucleotide, which should be a 
3'-PO.sub.4 group, is indeed a suitable substrate for endonuclease IV and 
obeys the same double-stranded substrate specificity described for 
endonuclease IV with DNA substrates. 
EXAMPLE 16: Demonstration of Ligation of Endonuclease IV Product 
Part A. 3'phosphorylated probes were synthesized by initiating automated 
synthesis with 2-[[2-(4,4'-dimethoxytrityl)oxy]ethyl]sulfonyl]ethyl 
2-cyano-ethyl N,N-diisopropylphosphoramidite (Horn, T and Urdea, M Tet. 
Lett. 27 4705+(1986)) as reported (Ashely, GW and Kushland, DM 
Biochemistry 30:2927-2933 (1991)), followed by the sequential addition of 
ribonucleotide and deoxyribonucleotide cyanoethyl phosphoramidites using 
an automated DNA synthesizer. The oligonucleotide was cleaved from the 
support and deprotected with 37% NH.sub.4 OH at 55.degree. C. for 12 h and 
purified by reverse-phase HPLC. on a C18 column. 
Part B. It was then demonstrated that when the 3'-phosphate is removed by 
Tth endonuclease IV (as in example 15) the resulting ribo-modified 
oligonucleotide is a suitable substrate for ligation by Tth DNA ligase. 
Duplicate reactions containing 83 nM AA123-1RP(20) from example 14, 16 nM 
AA 123-2 (approximately 25% 3'-labeled with .alpha.-.sup.32 P]-cordycepin 
triphosphate), and 66 nM AA123TAR(1/2) in LCR Buffer supplemented with 2.0 
mM CoCl.sub.2 and 10 .mu.g/mL BSA were incubated in the presence or 
absence of 0.15 .mu.g/mL Tth DNA ligase and/or 4.1 .mu.g/mL Tth 
endonuclease IV at 55 .degree. C. for 1 h. Analogous assays containing 300 
ng of human placental DNA and no AA 123TAR(1/2) were also performed. As 
can be observed in FIG. 6 (lanes 3 and 4), almost all of the AA 123-2 is 
convened into a ligated product only when Tth endonuclease IV, Tth DNA 
ligase and AA123TAR(1/2) are all present. 
It was also observed (FIG. 6, lanes 1 and 2) that a small amount of ligated 
product was formed in the presence of AA123TAR(1/2) and ligase but the 
absence of Tth endonuclease IV. The formation of a ligated product in the 
absence of Tth endonuclease IV implies that the 3' position of AA 123-1RP 
is not completely blocked with a 3'-PO4 group. A 3'-OH group may arise 
from incomplete 3'-phosphorylation during synthesis or the removal of 
and/or exchange of the 3'-PO.sub.4 group with the 2'-OH group during the 
treatment with strong alkali that follows synthesis. If the exchange 
reaction is responsible for generating an unblocked 3'-OH group then it 
must be possible for Tth DNA ligase to use the 2'-PO.sub.4, 3'-OH 
ribonucleoside as a substrate. 
EXAMPLE 17: LCR Using Modified Probes Containing a 3'Ribonucleotide Bearing 
a 3' Phosphate 
LCR was performed using the blunt-end probe set (see Table V) consisting of 
AA123-1RP(20), AA123-2, AA123-3(20) and AA123-4RP(20) in a 20 .mu.L 
reaction volume containing LCR Buffer supplemented with 10 pg/mL BSA, 0.5 
mM CoCl.sub.2 and 300 ng of human placental DNA. Each probe was present at 
83 nM (approximately 5% of probe 2 was 5'-end labeled with 
[.gamma.-.sup.32 P]-adenosine triphosphate to enable detection) and the 
enzymes Tth DNA ligase and Tth endonuclease IV were present at 0.15 
.mu.g/mL and 4.1 .mu.g/mL, respectively. Duplicate reactions for zero, 
10.sup.3, and 10.sup.4 molecules of target DNA were performed. The samples 
were overlaid with 15 .mu.L of mineral oil and the temperature cycle 
consisted of a 95.degree. C. incubation for 30 seconds followed by a 
55.degree. C. incubation for 110 seconds. At specified cycles (see Table 
E-17), 1.7 .mu.L aliquots were removed, mixed with 2.5 .mu.L of stop 
buffer, heated to 90.degree. C. for 2 min and applied to a denaturing 
polyacrylamide gel. Table E-17 gives the average percent ligated and, in 
parentheses, the ratio of (+)/(-) target for the duplicate reactions. It 
is observed that 10.sup.3 targets are distinguishable from zero targets. 
TABLE E-17 
______________________________________ 
LCR with 3'-Ribo; 3'-PO.sub.4 Probes 
Percent Ligated (+Target/-Target) 
Cycle No. 0 targets 10.sup.3 targets 
10.sup.4 targets 
______________________________________ 
28 0.45 1.65 (3.7) 5.87 (13.0) 
31 1.83 5.50 (3.0) 12.08 
(6.6) 
34 6.70 12.87 (1.9) 21.60 
(3.2) 
37 13.35 23.39 (1.8) 28.49 
(2.1) 
______________________________________ 
For Examples 18 and 19 plasmid pUC19 is used as target, hence the 
designation "pUC" in Table VI, below. The next numeral (after the dash) 
designates the position of the probe in a set of four: Probes numbered -1 
and -2 have the same 5'-3' orientation while probes numbered -3 and -4 
have the opposite sense. Probes numbered -1 and -3 hybridize, as do probes 
numbered -2 and -4. "P" and "p" indicate a phosphate group. This is normal 
and required on a 5' terminus, but serves as a ligation blocking 
modification on a 3' end. "E" and "x" designate an abasic site (described 
further below). A number "1" following the "E" designates the length of 
complementary bases (overhang) beyond the abasic site. 
TABLE VI 
__________________________________________________________________________ 
pUC19 Modified Probes 
Designation 
SEQUENCE & ORIENTATION SEQ ID No. 
__________________________________________________________________________ 
pUC-1P 
5'-AATTCGAGCTCGGTACCCp 
3' 29 
pUC-1E1 
5'-AATTCGAGCTCGGTACCCxG 
3' 30 
pUC-2 5'- pGGGGATCCTCTAGAGTCGACCTGCA-3' 
31 
pUC-3 3'-GCTCGAGCCATGGGp 
5' 32 
pUC-4P 
3'- pCCCCTAGGAGATCTCAGCTG-5' 33 
pUC-4E1 
3'- GxCCCCTAGGAGATCTCAGCTG-5' 34 
__________________________________________________________________________ 
EXAMPLE 18: LCR Using a pUC19 Target with 3' Phosphate Blocked Probes 
A probe set is designed to detect the pUC19 target sequence by LCR, with 
reduced background levels. The probe set (see Table VI) features two 
normal probes (pUC-2 and pUC-3) two probes (pUC-1P and pUC-4P) containing 
terminal 3' phosphate blocking groups. 
LCR reactions are performed (substantially as described herein) using 
various amounts of target (pUC19). After the hybridization step of each 
cycle, endonuclease IV purified from E. coli is added to the reaction. 
This can be done under standard LCR conditions, since E. coli endonuclease 
IV is somewhat thermostable. Alternatively, endonuclease IV from a 
thermostable species such as Thermus thermophilus could be used. As a 
control, the LCR is run using the same number of target molecules without 
the addition of endonuclease IV. In these controls a probe set similar to 
the one shown above is used, only the 3' terminal nucleotides (containing 
the 3' phosphates) are not included on probes -1 and -4. 
In both the experimental and control reactions, the rate of appearance of 
ligated product is correlated with the initial number of target molecules 
added. What distinguishes the two protocols is that in the second case, a 
"blank" tube containing no target molecules will give rise to signal at 
about the same rate as a tube containing 1000 target molecules, whereas in 
the case where modified probes and endonuclease IV are used, a "blank" 
tube containing no target molecules will give rise to signal significantly 
more slowly than does a tube containing 1000 target molecules. This 
suppression of background provides an advantage in increasing the usable 
range of sensitivity of the assay. 
Those skilled in the art will immediately appreciate the desirability of 
employing a highly thermostable endonuclease IV, for the same reasons that 
highly thermostable ligases and polymerases are useful and desirable in 
LCR and PCR, respectively. Those skilled in the art will also appreciate 
that other enzymes, either known or not yet known, which can remove 
modifications at the 5' or 3' ends of a DNA strand in a template dependent 
manner leaving the previously blocked 5' phosphate or Y hydroxyl intact, 
can be employed in a manner completely analogous to endonuclease IV as 
described above. 
EXAMPLE 19: LCR Using a pUC19 Target with Abasic Probes 
A probe set is designed to detect the pUC19 target sequence by LCR, with 
reduced background levels. The probe set (see Table VI) features two 
normal probes (pUC-2 and pUC-3) and two modified probes (pUC-1E1 and 
pUC-4E1 ) containing a Y end abasic site followed by one additional normal 
residue complementary to the target. 
LCR reactions are performed as in example 18. Results and interpretation 
will be similar to those of example 18. 
The following text and renumbered examples are incorporated herein from 
U.S. Ser. No. 07/860,861 or 07/869,306, which are now published as 
WO93/20191. 
Reagents & Enzymes 
Media for the growth of bacteria were purchased from Difco (Detroit, Mich., 
USA) or Gibco (Madison, Wisc. USA). Restriction enzymes, T4 DNA ligase, T4 
DNA polymerase, large fragment of the enzyme E. coli DNA polymerase I and 
Polynucleotide kinase were purchased from Bethesda Research Laboratories 
(BRL, Gaithersburg, Md. USA); New England Biolabs (Beverley, Mass. USA); 
Boehringer Mannhelm (Indianapolis, Ind. USA) or PL Laboratories 
(Milwaukee, Wisc. USA). Agarose was from International Biotechnology, Inc. 
Kanamycin was purchased from Sigma. X-gal (5-bromo-4-chloro-3-indoyl- 
D-galactoside) was purchased from BRL. E. coli K12 strains HB101, DH5a, E. 
coli K12/pUC9, E. coli K12/pUC19, and E. coli K12/pBR322 are obtainable 
from BRL or PL Laboratories. 
Buffers are defined as follows: 
TAE (Tris-acetate) defined as 40 mM Tris, 20 mM acetic acid, 2 mM EDTA; LB 
broth defined per liter as 10g BactoTryptone, 5 g yeast extract, 5 g NaCl; 
TB top agar defined as LB broth, defined supra, containing 0.75 % w/v 
agar, 5 mM calcium chloride, 0.2% glucose, 10 mM magnesium sulfate; TE 
defined as 10 mM Tris.cndot.Cl and 1 mM EDTA; 5 X LCR buffer defined as 50 
mM EPPS/K.sup.+ pH 7.6, 10 mM NH.sub.4 Cl, 10 mM MgCl.sub.2, 80 mM KC1, 
100 .mu.g/ml gelatin, and 0.5 mM CoCl.sub.2 ; and SOC. media defined as 20 
g bactotryptone, 5 g yeast extract, 0.5 g NaCl, 2.5 mM KCl, 10 mM 
MgCl.sub.2, 20 mM glucose, pH 7.0 
Example 20: Synthetic Abasic Substrates Which are Suitable in an Assay for 
Class II AP Endonuclease Detection. 
Synthetic substrates comprising hairpin structures containing a synthetic 
abasic site in the double stranded region of the hairpin were synthesized 
by known methods in phosphoramidite chemistry on a DNA synthesizer. The 
substrates (a) are small in size so that they can easily be synthesized in 
high yield and efficiency; (b) have a single abasic site incorporated 
using standard DNA synthesis chemistry; (c) have the abasic site modified 
by a (reduced) furan ring which precludes cleavage by a general 
base-catalyzed .beta.-elimination mechanism (Eritja, et al., Nucleosides 
and Nucleotides 6 (4): 803-814 (1987); (d) result in an easily resolvable 
cleavage product; (e) have a free 3' hydroxyl group for labelling using 
commercially available [.sup.32 P]- labelled nucleotides and terminal 
transferase; and (f) contain palindromic sequences which cause the 
formation of a hairpin. 
The hairpin serves two functions. Firstly, it obviates the need for a 
hybridization step before the oligonucleotide can be used. Secondly, the 
hairpin also has a high melting temperature, which is generally much 
higher than two oligonucleotides of equal double stranded region. This is 
important for use at high temperatures with thermophilic enzymes. Previous 
synthetic substrates were composed of two obligatorily separate 
oligonucleotides. The two strands needed to be kept separate by the need 
to depurinate a single purine residue in a polypyrimidine strand prior 
forming a double-stranded DNA molecule, with a complementary 
purine-containing strand. The latter (purine-containing) strand would need 
to be kept separate from the strand undergoing treatment to depurinate. 
Two hairpin substrates of the present invention are exemplified as follows: 
Hairpin 1 is a 39-residue oligonucleotide (wherein X is an abasic residue) 
having the following structure: 
##STR7## 
Upon cleavage by class II AP endonuclease, the substrate yields a 
7-residue nucleotide product (one nucleotide is actually the abasic 
residue). 
Hairpin 2 is a 45-residue oligonucleotide (wherein X is an abasic residue) 
having the following structure: 
##STR8## 
Upon cleavage by class II AP endonuclease, the substrate yields a 
10-residue nucleotide product. 
The melting temperatures of the hairpin substrates of the present invention 
at 3 .mu.M in 25 mM EPPS pH 7.6, 0.1 M NaCl, 10 mM EDTA were determined to 
be approximately 81.degree. C. (hairpin 1) and approximately 74.degree. C. 
(hairpin 2). 
Both hairpins have design features to minimize 3' exonuclease activity of 
the type exhibited by E. coli exonuclease III. Endonuclease IV activity in 
E. coli is heat stable at 65.degree. C., whereas exonuclease III activity 
is not, and this property is exploited to assay the former in the presence 
of the latter. Hairpin 1 has 5 phosphorothioate linkages between the six 
3' terminal thymidines. These linkages have been shown to resist the 
action of exonuclease III. Hairpin 2 has a 4 nucleotide extension at the 
3' end. It has been shown that exonuclease III, which requires double 
stranded DNA, will not act on a substrate with a 4 nucleotide overhang at 
the 3' end. Cleavage of these substrates was not observed on treatment 
with 0.2 M NaOH for 15 min at 37.degree. C. indicating that the synthetic 
abasic site was stable as predicted. 
The hairpin oligonucleotides are used as substrates in the assay described 
herein for the thermophilic class II AP endonuclease of the present 
invention. An aliquot of the EPPS-containing buffer, along with the 
hairpin substrate, is warmed at 50.degree. C. for &gt;1 min prior to starting 
the assay by addition of enzyme. The enzyme is incubated for a 
pre-determined time ranging from about 5-30 min for crude and partially 
purified extracts and up to 19 h for screening of clone banks, and the 
assay stopped by addition of 20 .mu.l of formamide-dye mix. After heating 
for about 2 min at 95.degree.-100.degree. C., aliquots of about 10-16 ml 
are loaded on a 20% acrylamide/50% w/v urea/TBE gel (15 cm H.times.17 cm 
W.times.0.7 mm thick) and substrates and products localized by 
electrophoresis at 55 V/cm (800 V for 15 cm gel) until the Bromophenol 
blue dye is approximately at the bottom of the gel (about 30-45 min). An 
aluminum plate is fastened to the exposed glass plate to uniformly spread 
and dissipate heat generated during electrophoresis. 
When the gel is finished, it is removed from the plates onto a used piece 
of film, and covered with plastic film, e.g. Saran wrap. The enzyme is 
then localized by autoradiography. For example, the gel is placed on Kodak 
X-OMAT AR film, matching up the film with one of the corners of the gel to 
allow subsequent superimposition for excising bands for counting. The film 
is exposed for 20-100 min at 22.degree. C. (no screen) or 30 min -4 h with 
intensifying screen at -80.degree. C., with the actual time depending on 
the amount of the radioactive label. 
For quantitation, the radioactive bands is excised and radioactivity 
determined by scintillation counting. The exposed film is taped to a 
light-box and the gel placed over the film and taped in place. The 
position of dyes aids alignment. The substrate and product bands are 
excised and placed in 4 ml of scintillation fluid (e.g. Ecolume, ICN 
Biomedicals, Radiochemical Division. Irvine. Calif., U.S.) in a mini-vial 
and counted for 2 minutes or until 40,000 counts have been recorded in the 
.sup.32 p channel of a scintillation counter. 
The assay provides a sensitive, reliable, and rapid manner of detecting 
class II AP endonuclease activity. 
Example 21 
Transduction of nfo::kan mutation from E. coli BW528 into E. coli MM294 
E. coli BW528 cells (Cunningham, et. al. J. Bacteriol.168:1120-1127 (1986) 
were grown overnight (about 16 hours) in LB broth at 37.degree.. Fifty 
microliters (50 .mu.l) was subcultured into 5 ml LB containing 0.2% w/v 
glucose and 5 mM calcium chloride. The cultures were incubated for 30 min 
at 37.degree. with aeration, and 0.1 ml bacteriophage P1 vir was added to 
give ca. 5.times.10.sup.8 phage/ml. The cells were then shaken 3 h for 
phage development and cell lysis. Chloroform (0.1 ml) was added and the 
culture vortexed. The cell debris was pelleted by centrifugation at 4500 X 
g for 10 min, and the supernatants were transferred to fresh sterile tubes 
containing 0.1 ml chloroform. After mixing, the phage preparations were 
stored at 4.degree.. 
Titre (number of viable phage/ml) of the phage preparations was determined 
as follows: the phage preparations were serially diluted 10.sup.3, 
10.sup.5, and 10.sup.6 fold in 10 mM magnesium sulfate and 5 mM calcium 
chloride and 5 btl of each dilution spread as a patch of approximately 1 
cm diameter on a lawn of E. coli MM294 (Meselson, et al., Nature 
271:1110-1114 (1968)cells in TB top agar on a LA plate (LB solidified with 
1.5 % agar). The lawn was made by adding 0.1 ml suspension of E. coli 
MM294 cells in 10 mM magnesium sulfate, 5 mM calcium chloride 
(approximately. 10.sup.9 /ml) to 3 ml molten TB top agar cooled to 
45.degree.. The mixture was then poured onto the surface of a LB plate. 
The plates were incubated at 37.degree. overnight and the number of 
plaques counted at a suitable dilution to calculate the titre. The 
resulting preparation of P1 vir had a titre of 4.times.10.sup.8 plaque 
forming units/mi. 
A 5 ml LB culture of E. coli MM294 was grown overnight, and the cells 
collected by centrifugation at 1500 .times.g for 10 min. The cells were 
resuspended in 2.5 ml 10 mM magnesium sulfate and 5 mM calcium chloride. 
Aliquots (0.1 ml) of the cells were placed in test tubes with 0.01, 0.05, 
0.1 ml P1 vir lysate described above, and incubated 30 min at 30.degree. 
for infection. Sodium citrate (0.1 ml of I M solution) and 1 ml LB were 
added. Cells were grown 1 h at 37.degree. to express the drug resistance 
before 2.5 ml molten (45.degree.) LB top agar (LB with 0.7% agar) was 
added, and the mixture plated on LA plates containing 25 mg/1 kanamycin 
sulfate (LA Km.sub.25). The plates were incubated at 37.degree. for 2 
days. 
Two kanamycin resistant colonies were obtained from the tube with 0.01 ml 
phage. These colonies were streaked out on LA Km.sub.25 plates and a 
single colony tested for loss of class II AP endonuclease activity by 
assay with an abasic substrate. No cutting of the abasic substrate was 
observed, confirming the loss of class II AP endonuclease. This strain was 
named E. coli MM294 nfo::kan. 
Example 22 
Subcloning of the Gene from Thermus thermophilus 
Step A: Isolation of plasmid pCS10 from T. thermophilus 
The clone bank of T. thermophilus of Lauer, et al., J. Bacteriol. 173: 
5047-5053 was used. It had been constructed in E. coli strain HB101 by 
cloning 7- to 30-kb DNA fragments generated by partial Sau3AI digestion of 
T. thermophilus chromosomal DNA into pTR264 digested with BclI and treated 
with calf intestinal phosphatase. 
Plasmid DNA of the Thermus thermophilus clone bank consisting of 4 pools of 
.about.400 clones per pool with an average insert size of 10 kb of 
chromosomal DNA, (DNA concentration ca. 0.5 mg/ml) was diluted 10 fold 
with T.sub.5 E.sub.0.5, and added to the competent E. coli MM294 nfo::kan 
cells prepared as described in Example 1. After 30 min on ice, the tubes 
were heat shocked for 90 sec at 42.degree. C. followed by cooling on ice 
for about 1 min. One ml of SOC medium was added, and the cells shaken for 
1 h at 37.degree. for expression of the tetracycline resistance gene on 
the plasmid. A 50 .mu.l aliquot was plated onto LA tetracycline (5 
.mu.g/ml) and the remainder spun down and plated on a single plate. The 
plates were incubated at 37.degree. overnight. 
The dilute plates contained roughly 200 colonies, and the concentrated 
plate contained over 1000 colonies in the case of all four pools. The 
colonies on the concentrated plate were scraped up in 3 ml LB, and another 
1 ml LB used to wash the surface of the plate. The final recovered volume 
was approx 3 ml. One ml of the resuspended cells was used to inoculate 250 
ml LB Tc5 and cultures grown at 37.degree. for about 3 h. Cells were 
collected by centrifugation, resuspended in crushing buffer (50 mM 
Tris/HCl pH 7.4, 10 mM MgSO.sub.4, 180 .mu.M CoCl.sub.2,5% v/v glycerol), 
and respun. Cells were finally resuspended in 6 ml crushing buffer and 
cells broken at 14,000-16, 000 psi in a French Press. Cell debris was 
removed by centrifugation at 15,000 rpm (26,900 .times.g) for 15 min. A 
200 .mu.l aliquot of the cleared supernatant was made 50 mM in NaCl, and 
heat treated at 90.degree. for 5 min, followed by cooling on ice for 1 
min. Precipitated protein was removed by centrifugation in a microfuge at 
room temp for 5 min. A 10 .mu.l aliquot was then assayed for class II AP 
endonuclease activity against both hairpin substrates #1, and #2. Assays 
were done with and without the addition of herring sperm DNA with 3' 
labelled hairpin and 5.times.LCR buffer. 
A 10 .mu.l sample was removed after 1 h into an equal volume of 98% 
formamide-dye mix and the remainder stopped after 17 h as above. A 15 
.mu.l sample of the stopped assays was run on a 20% 
polyacrylamide/urea/TBE gel (45 min, 800 V), and exposed to film for 100 
min. Class II AP endonuclease activity was obtained from Pool #1 and #3 on 
hairpin 2. Pool #1 was chosen for further work. 
A second transformation of the DNA of the Thermus thermophilus clone bank, 
prepared above, was performed with competent E. coli MM294 nfo::kan cells. 
Eleven plates (labelled A-K) of 50 patches each were made, in duplicate, 
by patching out colonies from the transformation onto LA Tc.sub.5. 
Horizontal rows of each plate (6-10 patches) were scraped into 500 .mu.l 
0.85% saline, 10% glycerol, and resuspended. A 125 .mu.l aliquot of each 
of the pools from a single plate were inoculated into a single 250 ml LB 
Tc.sub.5 culture, such that each culture represented a single plate of 50 
patches. These cultures were grown 16.5 h at 37.degree. with shaking and 
harvested by centrifugation (5 min, 5000 rpm, 6.times.250 ml rotor). The 
cell pellets were washed in 20 ml 0.85% saline, 50 .mu.M CoCl.sub.2, and 
resuspended in 6 ml crushing buffer. Cells were crushed, spun, 
heat-treated and assayed for 1 h as described above. Plates A, B, and I 
showed a product band indicative of class II AP endonuclease activity. 
Plate I was chosen for further investigation. 
Pools of 6-10 patches from plate I of clone bank were screened in the 
following manner. Six 250 ml LB Tc.sub.5 cultures were set up and 
inoculated with pools of 6-10 clones from plate I as for experiment with 
pools of 50. Cultures were grown and extracts made, heat treated and 
assayed as for previous experiment with pools of 50. A 30 min time sample 
(10 .mu.l) was removed from the assay and electrophoresed on 
polyacrylamide as before. Class II AP endonuclease -like activity was 
observed in the culture corresponding to ten patches from rows 1 and 2 of 
plate I. 
Further analysis of these 10 clones indicated that patch #9 was responsible 
for the class II AP endonuclease activity. Patch #9 was streaked out for 
single colonies, and two singles retested for class II AP endonuclease 
activity. Both were positive for cutting of the abasic hairpin #2 in a 30 
min assay. One was chosen for storage. The plasmid was called pCS10, and 
the strain E. coli MM294 nfo::kan (pCS10). 
DNA of pCS10 was purified by alkaline lysis, followed by cesium chloride 
gradient centrifugation from a 250 ml LB Tc.sub.5 culture using standard 
procedures known to those skilled in the art such as is found in Maniatis, 
supra. The DNA preparation had a concentration of 420 .mu.g/ml. 
Step B: Sau3AI digest of pCS10 to prepare pCS11 
Plasmid vector pIC20H was cut with Bam HI and dephosphorylated with 
bacterial alkaline phosphatase (BAP). pIC20H has a polylinker in the a 
fragment of .beta. galactosidase such that inserts into the polylinker 
cause loss of the .beta. galactosidase activity and loss of color 
formation on .beta. galactosidase indicator plates. This allows the 
identification of inserts in the vector. Digestion was done at 37.degree. 
for 90 min, followed by phenol extraction and ethanol precipitation. The 
plasmid DNA was resuspended in 20 .mu.l T.sub.5 E.sub.0.5, and 15 .mu.l 
treated with the phosphatase according to manufacturers instructions. The 
reaction was incubated 1 h at 65.degree.. 30 .mu.l of H.sub.2 O and 1 
.mu.l 0.25 .mu.l EDTA were added and the reaction heated 10 min at 
50.degree.. The digest was phenol-extracted and ethanol-precipitated. The 
plasmid DNA pellet was resuspended in 10 .mu.l T.sub.5 E.sub.0.5. 
Next, plasmid pCS10 was partially digested with Sau3AI 37.degree. for 20 
minutes and stopped by heating 10 minutes at 68.degree.. A .mu.l aliquot 
was run on an agarose gel to determine degree of cutting. Digestions 3, 4, 
and 5 were not significantly digested, so they were redigested by addition 
of 2 .mu.l 1:20 Sau3AI to each tube for 20 min followed by heat 
inactivation at 65.degree. for 10 min. The whole digest was run on a 1% 
agarose gel in TAE. After staining, agarose slices were cut out under long 
wave uv, corresponding to 1-1.5 kb, 1.5-2 kb, 2-3 kb and 3-4 kb. The DNA 
was isolated using the Gene-clean kit (BIO-101, La Jolla, Calif.) and 
resuspended in approximately 15 .mu.l T.sub.5 E.sub.0.5. These fractions 
were then ligated to plasmid pIC20H prepared above. (Marsh, et al, Gene 
32, 481-485, 1984). 
Ligations (10 .mu.l) were set up containing 0.3 .mu.g pIC20H (1 .mu.l) and 
2 .mu.l of one of the size fractions isolated from agarose. After 
overnight ligation at 16.degree., 1 .mu.l of the ligations was transformed 
into 50 .mu.l DH5aF' according to manufacturers instructions, except that 
the cells were grown out for 1 h in 2 ml SOC before spreading onto LA 
Amp.sub.100 X-Gal plates. For the 1-1.5 kb size range, approximately 205 
white and 7 blue colonies appeared in total. A second transformation was 
done with the ligation of 1-1.5 kb fragments for more transformants. 
Plasmid DNA minipreparations were done on 10 transformants. HindlII 
digestions showed that the insertion was in the selected size range in all 
cases. 
Eight plates of 50 colonies each of the 1-1.5 kb size range (total=400 
colonies) were patched out on LA Amp.sub.100 X-Gal in duplicate. Plates 
were incubated overnight at 37.degree.. Groups of 10 patches from one of 
the duplicate plates was scraped up and resuspended in 0.5 ml 0.85% 
saline, 10% glycerol. 0.25 ml of each of the five pools from the same 
plate were inoculated into LB Ampre.sub.100, and grown for 4 h at 
37.degree.. Cells were harvested and extracts made and assayed as for the 
previous screening of the clone bank. Assays were stopped at 40 min and 
samples electrophoresed as before. Plates #1 and #2 both showed activity. 
Subpools of 10 colonies from both these plates were grown and extracts 
tested for class II AP endonuclease activity. Pool D (#Patches 31-40) from 
plate #1 and pool B (#Patches 11-20) from plate #2 were identified as 
giving activity. These colonies were individually tested and #36 (plate 
#1) and patch #20 (plate #2) were identified. These were purified through 
single colonies on LA Amp.sub.100, and DNA preps made by CsCl-gradient 
centrifugation Restriction mapping indicated identical 1.4 kb inserts in 
the two plasmids The plasmid from plate#1, patch #36 was named pCS11. 
The 1.4 kb insert in pCS11 was restriction mapped with Barn HI, BglII, 
PstI, SacI, SmaI, SalI, XbaI and XhoI. The insert contained sites for 
BglII, SacI and XhoI, but no sites for the other enzymes. The sites for 
BglII, SacI and XhoI were roughly equally spaced 0.3-0.4 kb apart and this 
was exploited for subcloning into M13 for sequencing. 
Step C: Subcloning of pCS11 into M13mp18 for Sequencing 
Four micrograms of pCS11 was digested with 10-15 units of the appropriate 
restriction enzyme in a 20 .mu.l reaction using the recommended buffer at 
37.degree. for 2.5 h. Digests were stopped with 2 .mu.l gel loading 
buffer, and 10 .mu.l electrophoresed on a 1.2% agarose gel in TAE. Bands 
corresponding to the desired fragment (see Table below) were excised and 
the DNA isolated using the Prep-a-Gene Kit (BioRad). The DNA was 
resuspended in 15 .mu.l T.sub.5 E.sub.0.5. Ligations were set up with 13 
.mu.l DNA fragment (approx. 0.15 .mu.g) and 0.1 .mu.g M13 cut as shown in 
table below. The M13 (0.5 .mu.g) was cut with the appropriate enzymes at 
37.degree. for 1.5 h, phenol extracted and ethanol precipitated, and the 
pellet resuspended in 10 .mu.l T.sub.5 E.sub.0.5. It was then 5' 
dephosphorylated with bacterial alkaline phosphatase (30 Units) in a 15 
.mu.l reaction for 1 h at 65.degree.. Phosphatase was removed by phenol 
extraction and the DNA recovered by ethanol precipitation as above. The 
DNA was resuspended in 10 .mu.l T.sub.5 E.sub.0.5.) 
______________________________________ 
Fragment of M13 derivatives 
M13 digested 
pCS11 Size used with: 
______________________________________ 
PstI - Asp718 
1.5 kb M13mp18 & PstI/Asp718 
M13mp19 
HindIII - BglII 
0.4 kb M13mp18 & HindIII/Bam HI 
M13mp19 
BglII - SacI 
0.4 kb M13mp18 & Bam HI/SacI 
M13mp19 
SacI - SmaI 
0.65 kb M13mp18 & SacI/SmaI 
M13mp19 
SacI - Xho I** 
0.35 kb M13mp18 & SacI/SalI 
M13mp19 
______________________________________ 
Sequencing of M13 clones was done using the method of Sanger et. al., 1977. 
Nat. Acad. Sci. USA 74:5463-5467, employing M13 templates prepared as 
described by Messing, 1983. Methods in Enzymol. 101:20-78. Sequencing of 
double stranded templates was performed as described by Zhang et al., 
1988. Nucleic Acids Res. 16:1220. 
The DNA sequence of the inset in pCS11 was 1469 base pairs. Translation of 
the sequence in all reading frames was done. A single reading frame of 813 
base pairs (including TAA stop codon) encoding a 270 amino acid 
polypeptide was deduced from the sequence. The ATG start codon was 
preceded by a weak Shine-Dalgarno (ribosome binding) site for E. coli. The 
protein had a predicted mol. wt. of 29,088 and a predicted isoelectric 
point of 6.17. The G-C. content of the coding region was 71.1%. A series 
of 10 synthetic DNA sequencing primers, spaced roughly 200-250 base pairs 
apart, were designed and used to re-sequence the coding region. This 
sequence is shown in FIG. 1A of WO93/20191 which corresponds to U.S. Ser. 
No. 07/860,861 and 07/869,306. 
Example 23 
Insertion of a Ribosome Binding Sequence 5' to the T. thermophilus Class II 
AP endonuclease Coding Sequence for Efficient Expression in E. coli 
The polymerase chain reaction (PCR) was used to insert a ribosome binding 
sequence (i.e., Shine-Dalgarno sequence) particularly effective in E. coli 
at the 5'-end of the class II AP endonuclease gene. The PCR product was 
about 151 base pairs, and contained about 122 nucleotides of the 5'-end of 
the coding sequence. The intact class II AP endonuclease gene was then 
reassembled in vitro prior to overexpression in a suitable vector. 
A 46-mer oligonucleotide PCR primer having the sequence 
GGCTAGCCCGGGATCCAGGAGGTATAAAAATGCCGCGCTACGGGTT 3' (SEQ ID No. 41) 
(ribosome binding sequence and start codon are underlined) contained in 
order from the 5' end: NheI, SmaI and BamHI restriction sites followed by 
a Shine-Dalgarno sequence (AGGAGGT) placed six base pairs from the ATG 
start codon and followed by seventeen nucleotides homologous to the T. 
thermophilus class II AP endonuclease gene. An internal leftward reading 
sequencing primer having the sequence 5'-CAGCTCCGCGGGGCTTTT-3 ' (SEQ ID 
No. 42) was used as the other PCR primer. PCR was done using standard 
methodology (McConlogue, et al., Nucleic Acids Res., 16:9869 (1988)) 
except that a mixture of 7-deaza-dGTP and dGTP (in a 3:1 ratio) was used. 
A gel-purified fragment of plasmid pCSll (8 ng) which had been restricted 
with HaeII and XmaIII was used as the target for PCR. This resulted in a 
fragment of about 150 bases which included the restriction sites and the 
ribosome binding sites (the Shine-Dalgarno sequence). This PCR product was 
reamplified by a second round of PCR and then treated with mung bean 
nuclease (to give DNA blunt ends) for 35 min at 30.degree., extracted with 
phenol, and precipitated with ethanol. The DNA was resuspended in 10 .mu.l 
H.sub.2 O, heated 5 min at 60.degree. to inactivate the nuclease, and 4 
.mu.l used for ligation. 
The product was then ligated into pUC19 (see Yanisch-Perron et al., 1985, 
supra; Roberts,1986, supra) and transformed into competent E. coli DH5aF 
cells according to manufacturers instructions up to the heat shock step. 
Cells were then grown in SOC medium for 1 h at 37.degree. prior to plating 
on LA Amp.sub.100 X-Gal IPTG plates. After overnight incubation, 3 white 
and 1 light blue colony were obtained together with 76 blue 
(non-recombinant) colonies. 
DNA plasmid minipreparations were done and the results showed the expected 
size in two of the clones, designated PCR#2 and PCR #4. DNA sequence 
analysis demonstrated that the clones contained the ribosome binding site 
upstream of the ATG start codon. 
Example 24 
Reassembly of the Intact T. thermophilus gene and Construction of Plasmid 
pTT1 
Plasmid pCS11 (2.5 mg) was restricted with BglII and HindIII and the 
digests electrophoresed on agarose. An approximately 1.1 kb fragment was 
isolated and the DNA recovered using the Prep-a-Gene kit (BioRad) in a 
final volume of 15 .mu.l. A 6 .mu.l aliquot of this was ligated into PCR#2 
and PCR#4 which had been restricted with BglII and HindIII and 
dephosphorylated with phosphatase. The ligations were transformed into 
DH5a cells; plasmid DNA minipreparations were made for ten of the 
transformants from each cloning and the DNA cut with BamHI and HindIII. 
One ligation product into pPCR#4 contained the correct 1.1 kb insertion 
and was designated pPCR#4-8. This construct was then digested with BamHI 
and HindIII. An approximate 1.3 kb band was isolated and ligated into 
plasmid pGL516x (pGL516 of Lauer, et al., supra, modified by addition of 
XhoI linker at Bst X site in the lambda promoter) which had been 
restricted with BamHI and HindIII and dephosphorylated. 
The ligation was electroporated into E. coli CS1 and the transformants 
selected for ampicillin resistance. Resultant clones were tested for the 
insert. Some transformants seemed to have a double insertion. Candidates 
were grown and extracts from the clones tested for class II AP 
endonuclease activity. A clone, pTT1, identified as an overproducer, 
contained an insertion of the intact class II AP endonuclease gene 
followed downstream by a tandem copy of a portion of the gene from the 
BglII site to the 3' end, similar to the tandem insertion in pPCR#4-8. 
Example 25 
Reassembly of the Intact T. thermophiIus gene and Construction of 
Overexpressing Plasmids pTT2, pTT3, pTT4, and pTT5 
Plasmid pCS11 was restricted with Bgl II and a 1.1 kb fragment isolated. 
This fragment was then ligated to pPCR#2 and pPCR#4 which had been cut 
with Bgl II to yield plasmids pPM2010 and pPM2020, respectively which now 
contain the reassembled intact class II AP endonuclease gene. 
The class II AP endonuclease fragments in pPM2010 and pPM2020 were excised 
with BamHI and isolated from an agarose gel. Plasmid DNA minipreparation 
was used for isolation of the approximately 1 kb fragment. 
pGL516 (1.5 .mu.g) and pGL516X (1.4 .mu.g) were cut with BamHI, 
dephosphorylated, and phenol extracted/ethanol precipitated. The DNA was 
resuspended in a final volume of 15 .mu.l, and 1.2 .mu.l used in each 
ligation. 
The 1 kb fragments from pPM2010 and pPM2020 were ligated into both pGL516 
and pGL516X (a total of 4 ligations). A 2 .parallel.l aliquot was 
electroporated into CS1 electrocompetent cells. Cells were made 
electrocompetent according to a procedure published by BioRad and provided 
with their electroporation device. The only modification was that the 
cells were grown at 30.degree. rather than 37.degree.. After 
electroporation and outgrowth in SOC medium, the cells were plated on LA 
Amp.sub.100. 
Transformants were patched onto LA Amp.sub.100 and also grown in 2 ml LA 
Amp.sub.100 for DNA minipreparation. The miniprep DNA was double digested 
with BgllI and EcoRI, and run on an agarose gel. The enzymes cut in the 
class II AP endonuclease gene and in the vector at the promoter-distal end 
of the insert to give a 1050 base pair insert piece joined to an 
approximately 375 base pair vector fragment yielding a diagnostic fragment 
of approx. 1425 bp. Several correct candidates were present for each of 
the four ligations. 
One candidate from each cloning was selected and named as follows: 
pTT2: reassembled class II AP endonuclease from pPM2010 in pGL516 
pTT3: reassembled class II AP endonuclease from pPM2020 in pGL516 
pTT4: reassembled class II AP endonuclease from pPM2010 in pGL516X 
pTT5: reassembled class II AP endonuclease from pPM2020 in pGL516X. 
Example 26 
Construction of Plasmids pTT7, pTT8, pTT9, and pTT10 
The class II AP endonuclease gene in pTT3 and pTT5 was partially digested 
with BamHI and samples removed at 5, 10, 15, and 20 min into loading 
buffer. Samples were electrophoresed in an agarose gel and the approx. 6.7 
kb singly cut product excised. The DNA was isolated with the Prep-a Gene 
(BioRad) kit, and digested with NheI for 10 min at 37.degree.. 
Deoxynucleotide triphosphates were added to about 32 .mu.M and Klenow 
fragment was added to fill in the overhangs. After 10 min at 30.degree., 
the reaction was stopped with loading buffer, and the DNA run on a 0.7% 
agarose gel. The upper band corresponding to DNA cut at the promoter 
proximal BamHI site and NheI, was excised and the DNA isolated from the 
gel slice as above. 
The DNA was then self-ligated to reseal the blunted ends, and 
electroporated into CS1 as for construction of pTT3 above. Eight minipreps 
of each cloning were made, double digested with BarnHi +SalI, and 
electrophoresed on a 0.8% agarose gel. 
Two different plasmid derivatives resulted from this experiment. pTT7 and 
pTT8 (from pTT3 and pTT5, respectively) both contained a filled-in 
promoter-proximal BamHI site as the only modification. pTT9 and pTT10 
(from pTT3 and pTT5, respectively) contained a deletion of approximately 
180 base pairs between the promoter proximal BamHI and NheI sites. 
The relative activity of the various class II AP endonuclease producing 
plasmids was tested in a semiquantitative assay with hairpin #2. The class 
II AP endonuclease activity of extracts of CS1 (pTT1) was more than 300 
times higher than extracts of E. coli nfo:kan (pCS11). The relative 
activities of the pTT series of overproducing plasmids was as follows: 
______________________________________ 
pTT1: pTT3: pTT7: pTT9: pTT10: 
______________________________________ 
1: 1.8: 6.8: 2.4: 1.6 
______________________________________ 
Example 27 
Purification of Recombinant T. thermophilus Class II AP endonuclease from 
E. coli CS1 (pTT7) 
Cell paste (70.6 g) of E. coli CS1 (pTT7) was thawed and resuspended in 
211.8 ml (3 vol.) Buffer A (25 mM Tris/HCI pH 7.4, 1 mM MgSO.sub.4, 50 
.mu.M CoCl.sub.2, 5% glycerol) containing 0.1 M NaCl at room temperature 
(22.degree. C.). Cells were broken at 14,000 psi in a French Press in 45 
ml aliquots and the resulting extract centrifuged at 14,000 rpm (23,420 
.times.g) for 20 min to remove cell debris. The supernatant was incubated 
at 37 .degree. C. for 1 h during which time precipitation of some 
contaminating proteins occurred. After centrifugation as above, the 
extract was cooled 2 hours on ice, recentrifuged as above, and the 
supernatant filtered through a 5 mm filter. This yielded 196 ml of 
extract, which was stored at -80.degree. C. 
The filtered extract (196 ml) was thawed, diluted to 500 ml with buffer A, 
and centrifuged at 14,000 rpm for 30 min to remove precipitated protein. A 
100 ml aliquot was loaded onto a 100 ml Blue Sephrose (Pharmacia) radial 
flow column equilibrated in Buffer A at room temperature at a flow rate of 
about 5 ml/min. (The remainder was frozen as 100 ml aliquots at 
-80.degree. C.). The column was then washed with Buffer A until the OD 
returned to nearly baseline. A 500 ml gradient was run from 0-1.0 KCI at 5 
ml/min. Twenty eight 20 ml fractions were collected. Class II AP 
endonuclease eluted mainly in fractions #8-26 which were pooled and stored 
at -80.degree. C. 
The other four 100 ml aliquots of the diluted crude extract were similarly 
chromatographed on Blue Sepharose, and fractions #8-26 pooled. The pools 
from the 5 columns were then pooled and concentrated by ultrafiltration 
(YM1 membrane, Amincon) to about 120 ml and dialysed overnight against 4L 
Buffer A at 4.degree. C. The pool was then centrifuged at 12,000 rpm 
(17,210 .times.g) for 20 min to remove precipitated protein and column 
material, and filtered through a 1.2 micron filter. 
The pool was then divided into three 40 ml portions and one portion was 
loaded onto a 50 ml Heparin agarose (Sigma) radial flow column prepared in 
Buffer A at approx. 1.5 ml/min. After washing unbound protein from the 
column, 150 ml gradient was run from 0.0-1 M KCI at 1.5 ml/min. Fractions 
(3.75 ml) were collected. A major peak of uv-absorbing material with a 
shoulder was observed. Fractions 15-54 contained class II AP endonuclease 
activity, and these were pooled. The other two aliquots were similarly 
processed except that Heparin Sepharose CL-6B was used with similar 
results. Fractions 18-48 contained class II AP endonuclease activity, and 
these were pooled. 
The pooled fractions (about 266 ml) were concentrated by ultrafiltration 
(YM1 membrane, Amincon) to approx. 50 ml and then heat-treated at 
75.degree. C. in a 50 ml polypropylene tube, by placing in a boiling water 
bath, with constant stirring. The temperature was monitored, and the tube 
removed to ice when the temperature reached 75.degree. C. (6.5 min). After 
4 h on ice, the preparation was centrifuged to remove precipitated protein 
as before, and the supernatant filtered through a 0.8 .mu.m filter. The 
pool was then further concentrated to 4.3 ml by ultrafiltration (YM5 
membrane, Amincon) and filtered through a 0.8 .mu.m filter. 
230 .mu.l samples were subjected to HPLC size exclusion chromatography on a 
TSK-G30008W column (7.5 mm dia. .times.250 mm, BioRad) in 50 mM MES, 200 
mM NaCl pH 6.8 at 1 ml/min. One-ml fractions were collected. Activity was 
found in the major 280 nm-absorbing peak (frac. 13-20). 
A total of 19 such runs were done, and the class II AP endonuclease 
fractions pooled. The pool (about 140 ml) was then concentrated to 14.5 ml 
by ultrafiltration (YM5 membrane, Amincon). The final yield of class II AP 
endonuclease was 14.5 ml at 4.45 mg/ml. The enzyme was analyzed for purity 
by SDS-polyacrylamide gel electrophoresis. After staining with Coomassie 
Blue, a major band was visible at about. 29,000 daltons, corresponding to 
the molecular weight predicted for endonuclease IV from the DNA sequence 
(29,088). The purity was approximately 95%. 
Example 28 
Growth of E. coli CS1(pTT7) for Overproduction of Cloned Thermostable Class 
II AP endonuclease 
The same medium was used throughout, and was composed of a 1:1 mixture of 
"2 .times. seed medium" and LB, and included 100 mg per liter ampicillin. 
The "2 .times. seed medium" contained (per liter): 20 g glucose, 2 mM 
MgSO.sub.4, 0.2 mM CaCl.sub.2, 2 ml micronutrients, 44 mM KH.sub.2 
PO.sub.4, 138 mM K.sub.2 HPO.sub.4, 3.4 mM NaCl, 76 mM (NH.sub.4).sub.2 
SO.sub.4, 0.001% vitamin B1, 20 ml Fe citrate solution. [Micronutrient 
solution contained per liter: 0.15 g Na.sub.2 MO.sub.4.2H.sub.2 O, 2.5 g 
H.sub.3 B0.sub.3, 0.7 g CoCl.sub.2.6H.sub.2 O, 0.25 g CuSO.sub.4.5H.sub.2 
O, 1.6 g MnCl.sub.2, and 0.3 g ZnSO.sub.4.7H.sub.2 O. Fe citrate solution 
contained per liter: 0.2 g FeSO.sub.4.7H.sub.2 O, 100 g Na.sub.3 
Citrate.2H.sub.2 O.] 
A 1 ml frozen stock of CS1 (pTT7) (a stationary phase LB culture that had 
been made 5% in glycerol and frozen at -80.degree. C.) was thawed and 
added to 25 ml fermentation medium. The culture was grown 12h at 
30.degree. C., then a 4 ml aliquot added to 950 ml of the same medium and 
grown 12h at 30.degree. C. This was then added to 10 L of medium in a 
Chemap CF 2000 fermentor and grown for 7h at 30.degree. C. (optical 
density at 600 nm approx. 9.5) During growth in the fermentor, the pH was 
monitored constantly and automatically adjusted to 7 with H.sub.2 SO.sub.4 
or NH.sub.3. The culture was also stirred rapidly and bubbled vigorously 
with air. After 7h at 30.degree. C., the temperature was raised to 
42.degree. C. for 2h to induce production of the class II AP endonuclease, 
during which the optical density did not change appreciably. The culture 
was then placed in a container on ice and the cells collected by 
centrifugation at 8,000 rpm (10,800 .times.g) for 15 min in 500 ml 
bottles. The bottles were filled and spun several times to give an 
accumulated cell pellet of roughly 70 g per bottle. The total yield was 
285 g wet weight. The cell paste was frozen in the centrifuge bottles at 
-80.degree. C. 
Example 29 
Identification of Class II AP endonuclease from T. thermophilus 
T. thermophilus HB8 ATCC. 27634 was grown and an extract tested for class 
II AP endonuclease activity using hairpin #1 (defined, supra) as substrate 
in an assay buffer consisting of 25 mM EPPS, pH 7.6, 50 mM NaCl, 1 mM DTT, 
1 mM EDTA and 50 mg/mL bovine serum albumin. The extract gave several 
bands from hairpin #1. However, none corresponded to that expected of 
class II AP endonuclease activity. It appeared that there was rapid 
destruction of the substrate by nuclease activity to give the observed 
bands, despite the presence of EDTA. Crude extracts showed no definitive 
class II AP endonuclease but results indicated the presence of an 
EDTA-resistant nuclease activity, herein defined as endonuclease X. 
Therefore, in order to separate the class II AP endonuclease from 
endonuclease X, the crude extracts were treated with protamine sulfate to 
precipitate the DNA, followed by chromatography over a size exclusion 
column (Ultrogel Ac54, 13 cm.times.1 cm dia.) at 0.5 ml/min at room 
temperature (about 22.degree. C.). Forty 0.2 ml fractions collected and 
the fractions were assayed with hairpin #1 as substrate. The endonuclease 
X activity was recovered in fractions 12-28. In the assay only a single 
band of about 13 nucleotides was obtained from the abasic hairpin #1 
rather than the seven nucleotide fragment expected from the class II AP 
endonuclease activity. This result differed from the crude extract in that 
the crude extract gave multiple bands, but still no class II AP 
endonuclease activity was evident in any of the fractions. 
Subsequentially, T. thermophilus extract (70 ml which had been treated with 
protamine sulfate to precipitate DNA which tends to interfere with the 
chromatography stationary phase) was chromatographed on a DEAE Sepharose 
Fast Flow column (7 cm.times.4.4 cm dia.) equilibrated with 50 mM Tris/HCl 
pH 7.4, 1 mM DTT, 5% v/v glycerol, 25 mM benzamidine C1 using a 800 ml 
gradient from 0-1 M KCl at 4.degree. C. A total of 32.times.12 ml 
fractions followed by 8.times.28 ml fractions were collected. No class II 
AP endonuclease activity was evident in any of the fractions. Endonuclease 
X activity eluted immediately after the flowthrough fraction. The 
endonuclease X fractions (#11-17, 84 ml) were pooled, concentrated to 3 ml 
and chromatographed on a size exclusion column (Sephacryl S100HR, 55 
cm.times.2.2 cm dia.) in order to test the possibility that there was 
class II AP endonuclease in this strain but that it had co-eluted with the 
endonuclease X. 
Fractions off the column were assayed. High endonuclease X was recovered in 
fractions 23 through 41. In addition, fractions 41-47 showed a band that 
corresponded to one expected from an class II AP endonuclease activity. 
In conclusion, class II AP endonuclease activity was identified in T. 
thermophilus after two purification steps which separated class II AP 
endonuclease activity from the endonuclease X activity. 
Example 30 
Demonstration of Class II AP endonuclease Activity in Thermophiles 
Sulfolobus solfataricus and Thermus aquaticus 
Three Thermus strains, T. flavus (ATCC. 33923), T. sp (ATCC. 31674), T. 
aquaticus (ATCC. 25104) and the archebacterium Sulfolobus solfataricus 
(ATCC. 35091) were obtained from the American Type Culture Collection 
(ATCC) and grown using standard culture media recommended by the ATCC, and 
extracts tested with hairpin #1 as defined in Example 9. 
Class II AP endonuclease was clearly seen in the T. aquaticus and in the S. 
solfataricus crude extracts. However, its activity was masked by the 
presence of endonuclease X in T. fiavus and T. sp. 
Example 31 
Purification of Native Class II AP endonuclease from T. aquaticus 
T. aquaticus ATCC. 25104 frozen cell paste (38 g) was thawed, washed in 100 
ml saline (0.85 %w/v NaCl), centrifuged, and resuspended in 2 volumes 
buffer (80 ml; 50 mM Tris/HCl pH 7.4, 5 % glycerol, 0.5 mM DTT). The cells 
were broken and debris removed by centrifugation. The supernatant (93 ml) 
was made 0.4 % w/v in Polymin P/HCl pH 7.9 (3.9 ml of 10% w/v stock) with 
stirring on ice. Comparison of the activity of the supernatant before and 
after Polymin P treatment indicated that significant activity was lost. 
An aliquot (20 ml) of the extract was diluted with 50 ml buffer (20 mM 
potassium phosphate pH 7.0, 1 mM DTT, 10% v/v glycerol) and loaded onto a 
Blue sepharose column at 4.degree. C., and washed with 15 ml buffer. Over 
90% of the protein did not bind to the column as judged by the optical 
density of the material that did not bind. A 2.times.150 ml gradient of 
0.1-1.0 M NaCl was then used to develop the column. Fractions (80 drops, 
about 4 ml, 74 fractions total) were collected, and 2 .mu.l aliquots 
assayed to locate the endonuclease IV peak. Endonuclease IV eluted broadly 
between fractions #4-48. Fractions. #16-40, (roughly 0.3-0.6 M NaCl) 
contained most of the activity. Significant activity was also present in 
the flowthrough fraction, indicating that all of the class II AP 
endonuclease like activity did not bind. Fractions were made roughly 33% 
in glycerol and frozen at - 20.degree. C. 
Pools of fractions #14-19, 20-25, 26-31, and 32-37 were used to demonstrate 
that this activity yielded ligatable ends with abasic substrate and a 
target. Pools of 26-31, and 32-37 were concentrated roughly 3-fold and 
used for heat stability studies resulting in the discovery of the need for 
a divalent metal for stability. 
Example 32 
Heat Stability of Partially Purified Cloned T. thermophilus Class II AP 
endonuclease 
Cloned T. thermophilus class II AP endonuclease from the chromosomal clone 
[MM294 nfo::kan (pCS10)]was partially purified on a Blue Sepharose column. 
Two class II AP endonuclease -containing fractions were eluted in 1 M NaCl 
and concentrated and used to demonstrate heat stability. Heat cycling (20 
.mu.l) was done in LCR buffer. Fifty cycles were done of heating to 
90.degree. (max. rate), holding for 30 sec; cooling to 50.degree. (max. 
rate); and holding at 50.degree. for 45 sec. An aliquot of the reaction 
was then diluted 10-fold and assayed in a modified assay buffer containing 
50 mM EPPS/KOH pH 7.6, 80 mM KCl, 1 mM MgCl.sub.2, 10 mg/1 gelatin, 0.1 mM 
CoCl.sub.2 for 10 min at 50.degree.. In all cases, the degree of cutting 
of the substrate was essentially equal to or greater than the activity of 
the control kept on ice, thus demonstrating the T. thermophilus 
preparation was substantially stable to heat cycling. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 38 
(2) INFORMATION FOR SEQ ID NO: 1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other nucleic acid (synthetic DNA) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: 
TTGTCGAGCACCTTGAATAA20 
(2) INFORMATION FOR SEQ ID NO: 2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other nucleic acid (synthetic DNA) 
(ix) FEATURE: 
(A) NAME/KEY: 3'end phosphorylated 
(B) LOCATION: 20 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: 
TTGTCGAGCACCTTGAATAA20 
(2) INFORMATION FOR SEQ ID NO: 3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other nucleic acid (synthetic DNA) 
(ix) FEATURE: 
(A) NAME/KEY: N represents an abasic site 
(B) LOCATION: 21 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: 
TTGTCGAGCACCTTGAATAANT 22 
(2) INFORMATION FOR SEQ ID NO: 4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other nucleic acid (synthetic DNA) 
(ix) FEATURE: 
(A) NAME/KEY: N represents an abasic site 
(B) LOCATION: 21 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: 
TTGTCGAGCACCTTGAATAANTAA24 
(2) INFORMATION FOR SEQ ID NO: 5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other nucleic acid (synthetic DNA) 
(ix) FEATURE: 
(A) NAME/KEY: N represents an abasic site 
(B) LOCATION: 21 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: 
TTGTCGAGCACCTTGAATAANTAATG26 
(2) INFORMATION FOR SEQ ID NO: 6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 
( B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other nucleic acid (synthetic DNA) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: 
TTAATGGCTTCGATTGGGCT20 
(2) INFORMATION FOR SEQ ID NO: 7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other nucleic acid (synthetic DNA) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: 
TTATTCAAGGTGCTCGACAA20 
(2) INFORMATION FOR SEQ ID NO: 8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other nucleic acid (synthetic DNA) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: 
ATTCAAGGTGCTCGACAA18 
(2) INFORMATION FOR SEQ ID NO: 9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other nucleic acid (synthetic DNA) 
(ix) FEATURE: 
(A) NAME/KEY: 3'end phosphorylated 
(B) LOCATION: 22 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: 
AGCCCAATCGAAGCCATTAATT 22 
(2) INFORMATION FOR SEQ ID NO: 10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other nucleic acid (synthetic DNA) 
(ix) FEATURE: 
(A) NAME/KEY: 3'end phosphorylated 
(B) LOCATION: 20 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: 
AGCCCAATCGAAGCCATTAA20 
(2) INFORMATION FOR SEQ ID NO: 11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other nucleic acid (synthetic DNA) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: 
AGCCCAATCGAAGCCATTAA20 
(2) INFORMATION FOR SEQ ID NO: 12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 
(B) TYPE: nucleic acid 
(C ) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other nucleic acid (synthetic DNA) 
(ix) FEATURE: 
(A) NAME/KEY: N represents an abasic site 
(B) LOCATION: 23 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: 
AGCCCAATCGAAGCCATTAATTNT24 
(2) INFORMATION FOR SEQ ID NO: 13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other nucleic acid (synthetic DNA) 
(ix) FEATURE: 
(A) NAME/KEY: N represents an abasic site 
(B) LOCATION: 23 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: 
AGCCCAATCGAAGCCATT AATTNTTC26 
(2) INFORMATION FOR SEQ ID NO: 14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 28 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other nucleic acid (synthetic DNA) 
(ix) FEATURE: 
(A ) NAME/KEY: N represents an abasic site 
(B) LOCATION: 23 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: 
AGCCCAATCGAAGCCATTAATTNTTCAA28 
(2) INFORMATION FOR SEQ ID NO: 15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 
(B) TYPE: nucleic acid 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: 
CCGATTGCAATGTAATATCGACGTC25 
(2) INFORMATION FOR SEQ ID NO: 16: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 31 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other nucleic acid (synthetic DNA) 
(ix) FEATURE: 
(A) NAME/KEY: N represents an abasic site 
(B) LOCATION: 26 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: 
CCGATTGCAATGTAATATCGACGTCNTCGGC31 
(2) INFORMATION FOR SEQ ID NO: 17: 
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(A) LENGTH: 21 
(B) TYPE: nucleic acid 
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(D) TOPOLOGY: linear 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: 
GTCGGGCAAATAATTCGCCAC 21 
(2) INFORMATION FOR SEQ ID NO: 18: 
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(A) LENGTH: 24 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other nucleic acid (synthetic DNA) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: 
GACGTCGATATTACATTGCAATCG 24 
(2) INFORMATION FOR SEQ ID NO: 19: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other nucleic acid (synthetic DNA) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: 
CGTCGATATTACATTGCAATCG 22 
(2) INFORMATION FOR SEQ ID NO: 20: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other nucleic acid (synthetic DNA) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20: 
GGCGAATTATTTGCCCGAC 19 
(2) INFORMATION FOR SEQ ID NO: 21: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other nucleic acid (synthetic DNA) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: 
GGCGAATTA TTTGCCCGACGA21 
(2) INFORMATION FOR SEQ ID NO: 22: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 40 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
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(ii) MOLECULE TYPE: Other nucleic acid (synthetic DNA) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: 
TTGTCGAGCACCTTGAATAATTAATGGCTTCGATTGGGCT40 
(2) INFORMATION FOR SEQ ID NO: 23: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 40 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other nucleic acid (synthetic DNA) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: 
AGCCCAATCGAAGCCATTAATTATTCAAGGTGCTCGACAA40 
(2) INFORMATION FOR SEQ ID NO: 24: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 45 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii ) MOLECULE TYPE: Other nucleic acid (synthetic DNA) 
(ix) FEATURE: 
(A) NAME/KEY: N represents an abasic site 
(B) LOCATION: 36 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: 
AAAAAAAGCCGGATCCGTACACAACGGATCCGGCTNTTTTTGGGG45 
(2) INFORMATION FOR SEQ ID NO: 25: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other nucleic acid (synthetic DNA) 
(ix) FEATURE: 
(A) NAME/KEY: adenosine ribonucleotide 
(B) LOCATION: 20 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25: 
TTGTCGAGCACCTTGAATAA 20 
(2) INFORMATION FOR SEQ ID NO: 26: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other nucleic acid (synthetic) 
(ix) FEATURE: 
(A) NAME/KEY: adenosine ribonucleotide 
(B) LOCATION: 20 
(ix) FEATURE: 
(A) NAME/KEY: 3'end phosphorylated 
(B) LOCATION: 20 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: 
TTGTCGAGCACCTTGAATAA20 
(2) INFORMATION FOR SEQ ID NO: 27: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other nucleic acid (synthetic DNA) 
(ix) FEATURE: 
(A) NAME/KEY: adenosine ribonucleotide 
(B) LOCATION: 20 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: 
AGCCCAATCGAAGCCATTAA20 
(2 ) INFORMATION FOR SEQ ID NO: 28: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other nucleic acid (synthetic DNA) 
(ix) FEATURE: 
(A) NAME/KEY: adenosine ribonucleotide 
(B) LOCATION: 20 
(ix) FEATURE: 
(A) NAME/KEY: 3'end phosphorylated 
(B) LOCATION: 20 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: 
AGCCCAATCGAAGCCATTAA20 
(2) INFORMATION FOR SEQ ID NO: 29: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other nucleic acid (synthetic DNA) 
(ix) FEATURE: 
(A) NAME/KEY: 3'end phosphorylated 
(B) LOCATION: 18 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: 
AATTCGAGCTCGGTACCC18 
(2) INFORMATION FOR SEQ ID NO: 30: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other nucleic acid (synthetic DNA) 
(ix) FEATURE: 
(A) NAME/KEY: N represents an abasic site 
(B) LOCATION: 19 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30: 
AATTCGAGCTCGGTACCCNG 20 
(2) INFORMATION FOR SEQ ID NO: 31: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other nucleic acid (synthetic DNA) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31: 
GGGGATCCTCTAGAGTCGAC CTGCA25 
(2) INFORMATION FOR SEQ ID NO: 32: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other nucleic acid (synthetic DNA) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: 
GGGTACCG AGCTCG14 
(2) INFORMATION FOR SEQ ID NO: 33: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other nucleic acid (synthetic DNA) 
(ix) FEATURE: 
(A) NAME/KEY: 3'end phosphorylated 
(B) LOCATION: 20 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33: 
GTCGACTCTAGAGGATCCCC20 
(2) INFORMATION FOR SEQ ID NO: 34: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other nucleic acid (synthetic DNA) 
(ix) FEATURE: 
(A) NAME/KEY: N represents an abasic site 
(B) LOCATION: 21 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34: 
GTCGACTCTAGAGGATCCCCNG22 
(2 ) INFORMATION FOR SEQ ID NO: 35: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other nucleic acid (synthetic DNA) 
(ix) FEATURE: 
(A) NAME/KEY: 3'end phosphorylated 
(B) LOCATION: 22 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35: 
GATACTTCGCATC ATGTGTTCC22 
(2) INFORMATION FOR SEQ ID NO: 36: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other nucleic acid (synthetic DNA) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36: 
G GAGTTTCTTTGTCCTCCTATAACG25 
(2) INFORMATION FOR SEQ ID NO: 37: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other nucleic acid (synthetic DNA) 
(x i) SEQUENCE DESCRIPTION: SEQ ID NO: 37: 
GGAACACATGATGCGAAGTATC22 
(2) INFORMATION FOR SEQ ID NO: 38: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other nucleic acid (synthetic DNA) 
(ix) FEATURE: 
(A) NAME/KEY: 3'end phosphorylated 
(B) LOCATION: 25 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38: 
CGTTATAGGAGGACAAAGAAACTCC25