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Description  |
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BACKGROUND OF THE INVENTION
This invention relates to the detection of specific sequences of
nucleotides in a variety of nucleic acid samples, and more particularly to
those which contain a sequence characterized by a difference in a single
base pair from a standard sequence.
In recent years it has been found that many human diseases can be traced
directly to genetic mutations. Some commonly known examples include cystic
fibrosis, muscular dystrophy, Tay-Sachs disease, hemophilias,
phenylketonuria and sickle-cell anemia. Of the over 500 recognized genetic
diseases, many can be traced to single base pair mutations.
Two important techniques have been developed in the art for directly
detecting these single base pair mutations. However, neither of these
approaches can be easily automated. An automated technique is desirable
since it has the potential to decrease labor time, decrease the level of
skill required, and should increase reliability. In the first of these
prior art techniques, the presence or absence of the mutation in a subject
is detected by analysis of a restriction digest of the subject's DNA using
Southern blotting. (E. Southern, "Detection of Specific Sequences Among
DNA Fragments Separated by Gel Electrophoresis," Journal of Molecular
Biology, 98, (1975), 503). For example, sickle-cell disease results from a
mutation that changes a glutamic acid residue, coded by the triplet GAG,
for a valine residue, coded by GTG, at position 6 in the .beta.-globin
chain of hemoglobin. In the mutation of A to T in the base sequence of the
.beta.-globin gene, a restriction site for the enzyme MstII (as well as
sites for other restriction enzymes) is eliminated. The sickle hemoglobin
mutation can therefore be detected by digesting sickle-cell and normal DNA
with MstII and using Southern blotting to distinguish the restriction
fragments. Normal DNA will generate an MstII fragment 1.1 kilobases long
whereas sickle-cell DNA will generate a fragment 1.3 kilobases long.
The specifics of the Southern blot technique are as follows. First, the
sample DNA is cut with a restriction enzyme (in this case MstII), and the
resultant fragments are separated, based on their size, typically by
agarose gel electrophoresis. The gel is then laid onto a piece of
nitrocellulose, and a flow of an appropriate buffer is set up through the
gel, perpendicular to the direction of electrophoresis, toward the
nitrocellulose filter. The flow causes the DNA fragments to be carried out
of the gel onto the filter, where they bind, so that the distribution of
the DNA fragments in the gel is replicated on the nitrocellulose. The DNA
is then denatured and fixed onto the filter. A radioactively labeled
probe, complementary to the sequence under study, is then hybridized to
the filter, the probe hybridizing to the specific fragment containing the
sequence under study. Autoradiography of the nitrocellulose filter then
identifies which fragment or fragments contain the sequence under study,
each fragment being identified according to its molecular weight. A
variation on this technique is to hybridize and do autoradiography
directly in the gel, rather than on a nitrocellulose filter. Also, other
restriction enzymes may be used provided one of the resulting fragments
contains the mutation site of interest.
This direct Southern blot approach used for sickle-cell disease cannot be
used, however, for genetic diseases where the mutation does not alter a
restriction site, for example, as in .alpha..sub.1 -antitrypsin
deficiency, a genetic disease which subjects the individual to greatly
increased risk of developing pulmonary emphysema or infantile liver
cirrhosis. There, the mutant gene has a single base change (G.fwdarw.A)
that leads to an amino acid substitution (GLU.fwdarw.LYS) at residue 342,
thereby producing a non-functional protein. This substitution does not,
however, create or destroy a restriction site for any of the currently
known restriction enzymes as in sickle-cell anemia. Hence, a
straightforward analysis of restriction fragments to search for an altered
restriction site is not possible. A technique has been developed, however,
which can be used in this situation. (See "Detection of Sickle-cell
.beta..sup.s -globin Allele by Hybridization with Synthetic
Oligonucleotides," by Brenda J. Conner, et al., Proc. Natl. Acad. Sci.,
Vol 80, pp. 278-282, (January 1983), and "Prenatal Diagnosis of
.alpha..sub.1 -Antitrypsin Deficiency by Direct Analysis of the Mutation
Site in the Gene," by Vincent J. Kidd, et al., New England Journal of
Medicine, Vol. 310, No. 10, (March 1984).) This second technique is to
synthesize a 19-base long oligonucleotide (hereinafter a 19-mer) that is
complementary to the normal .alpha..sub.1 -antitrypsin sequence around the
mutation site. The 19-mer is labeled and used as a probe to distinguish
normal from mutant genes by raising the stringency of hybridization to a
level at which the 19-mer will hybridize stably to the normal gene, to
which it is perfectly complementary, but not to the mutant gene, with
which it has the single base pair mismatch. (By stringency, it is meant
the combination of conditions to which nucleic acids are subject that
cause the duplex to dissociate, such as temperature, ionic strength, and
concentration of additives, such as formamide. Conditions that are more
likely to cause the duplex to dissociate are called "higher" stringency,
e.g., higher temperature, lower ionic strength, and higher concentration
of formamide.) Similarly, if it is desired to detect the mutant gene,
instead of the normal gene, a 19-mer is used which is complementary to the
mutant .alpha..sub.1 -antitrypsin sequence around the mutant site. Hence,
by using synthetic probes complementary to the sequence of interest in a
Southern blot analysis, and varying the stringency, normal and mutant
genes can be distinguished.
Although this latter technique is straightforward, it is not without
difficulties, especially if an automated procedure is desired. Generally,
both the matched and mismatched probes undergo hybridization to the
fragment excised by the restriction enzyme, the matched probe being bound
at all 19 bases, and the mismatched probe at most 18 bases. Hence, the
relative difference in binding energy between the two probes is quite
small. Thus changes in stringency must be delicate enough to dissociate
the mismatched probe without also dissociating the matched probe. This
would not be a problem with respect to automating the technique were it
not for the considerable retention of 19-mer probes by high-molecular
weight DNA, presumably due to DNA sequences in the human genome that are
somewhat homologous with the synthetic DNA probes used, although this
cannot be stated with certainty. This large excess of somewhat homologous
sequences in comparison with the .alpha..sub.1 -antitrypsin gene obscures
the experimental results and must be treated as background noise in any
automated technique and is presently resolved by subjecting the sample to
gel electrophoresis and Southern blotting. (See FIGS. 1A and 1B showing
the Southern blots for the .alpha..sub.1 -antitrypsin detection scheme
reported by Kidd, et al., above.) In this particular instance, this
background did not interfere with the diagnosis since a previously
developed restriction map indicated that only the band at 2.4 kilobases
was relevant. However, it can be seen that most of the probe actually
bound is not in the 2.4 kilobase band. In this instance, the time and
labor consuming restriction digest and electrophoresis were carried out to
separate the DNA sequence of interest, the 2.4 kilobase fragment, from the
bulk of the DNA, thereby essentially eliminating background problems.
In most situations involving genetic disease, such restriction maps will
already be available, so that the above technique can be quite generally
applicable. However, such a technique is not easily automated, just as the
previous technique used for sickle-cell disease is not easily automated.
What is needed is a technique for detecting single base pair differences
between sequences of nucleotides which does not require the use of
restriction enzymes, gel electrophoresis, or time consuming
autoradiography, and which is readily amenable to automation.
SUMMARY OF THE INVENTION
The invention provides a method for diagnosis of genetic abnormalities or
other genetic conditions which can be readily automated. The method takes
advantage of the low probability that a particular diagnostic sequence
which may be found in a number of irrelevant locations in the genome, will
be extended by the same or similar contiguous sequence at these irrelevant
locations. By requiring that both the diagnostic and contiguous sequence
be present, background noise caused by spurious binding of the diagnostic
sequence is eliminated, and the necessity for separating the relevant
sequence from the background using such techniques as electrophoresis or
chromatography is eliminated.
In one aspect, the invention relates to a method for determining the
presence or absence of a target sequence in a sample of denatured nucleic
acid which entails hybridizing the sample with a probe complementary to a
diagnostic portion of the target sequence (the diagnostic probe), and with
a probe complementary to a nucleotide sequence contiguous with the
diagnostic portion (the contiguous probe), under conditions wherein the
diagnostic probe remains bound substantially only to the sample nucleic
acid containing the target sequence. The diagnostic probe and contiguous
probe are then covalently attached to yield a target probe which is
complementary to the target sequence, and the probes which are not
attached are removed. In the preferred mode, one of the probes is labeled
so that the presence or absence of the target sequence can then be tested
by melting the sample nucleic acid-target probe duplex eluting the
dissociated target probe, and testing for the label.
In another approach, the testing is accomplished without first removing
probes not covalently attached, by attaching a hook to the probe that is
not labeled, so that the labeled target probe may be recovered by catching
the hook.
In both instances, the presence of both the diagnostic probe and the
contiguous probe is required for the label to appear in the assay. This
eliminates the background which had previously been segregated by the size
separations accomplished by the Southern blot technique. Hence, the
predominant detriments of the prior art techniques have been eliminated,
i.e., no treatment with restriction enzymes is required, no gel
electrophoresis is required, and no autoradiography is required.
The above method is directly applicable to detecting genetic diseases,
particularly those resulting from single base pair mutations, and may be
made more precise by comparative results from tests wherein each of the
normal and abnormal sequence is made the target sequence. For example, in
this embodiment, two diagnostic probes are synthesized, one for the normal
gene and one for the mutated gene, and the above method is carried out for
each probe independently. The DNA from individuals homozygous for the
normal gene will show a high count of label for the probe specific to the
normal gene and a low count for the gene specific for the mutated gene.
Similarly, DNA from individuals homozygous for the mutated gene, will show
a high count for the probe specific to the mutated gene and a low count
for the normal probe. Heterozygous individuals will show a count for both
the normal probe and mutated probe which are equal and intermediate
between the high and low counts for homozygous and heterozygous
individuals. Use of only one diagnostic probe, preferably specific to the
mutated sequence of interest is also possible. Using the general method
described earlier, the detection scheme is first calibrated so that the
amount of label expected corresponding to homozygous normal, homozygous
mutant, and heterozygous individuals is known. Then the method is carried
out on the sample DNA, and the amount of label detected is compared with
the calibration.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A shows the results of a test for .alpha..sub.1 -antitrypsin
deficiency. The test used a Southern blot for DNA samples from cloned
.alpha..sub. -antitrypsin gene (pAT9.6), the MM (normal-normal) and ZZ
(deficient-deficient) homozygote cellular DNA controls, the parents, and
the fetus at risk, hybridized to a normal M oligomer probe. The band at
2.4 kb indicates the presence of the normal gene.
FIG. 1B shows the same DNA samples hybridized to a deficient Z oligomer
probe. The band at 2.4 kb indicates the presence of the deficient gene.
FIGS. 2A, 2B, and 2C illustrate various stages of the method of the
invention.
FIG. 3 shows the results of a melting curve analysis of a 15-mer hybridized
to a .lambda.-phage DNA sample and of an 80-mer made up of the same 15-mer
ligated to a 65-mer which was hybridized adjacent to the 15-mer on the
same substrate.
DETAILED DESCRIPTION OF THE INVENTION
For the purposes of the subsequent description, the following definitions
will be used:
"Target sequence" is a nucleic acid sequence, the presence or absence of
which is desired to be detected. In the context of a preferred application
of the method of the invention, it is a sequence which forms part of a
coding region in a gene associated with a genetic disease, such as
sickle-cell anemia. In many such diseases, the presence of the genetic
aberration is characterized by small changes in the coding sequence; most
frequently, normal individuals have sequences which differ by only one
nucleotide from the corresponding sequences present in individuals with
the genetic "deficiency." In the method of the invention, either the
normal or altered sequence can be used as the target sequence.
"Diagnostic portion" refers to that portion of the target sequence which
contains the nucleotide modification, the presence or absence of which is
to be detected. "Contiguous portion" refers to a sequence of DNA which is
a continuation of the nucleotide sequence of that portion of the sequence
chosen as diagnostic. The continuation can be in either direction.
It will be recognized, based on the disclosure below, that the precise
position of the selected diagnostic portion is arbitrary, except that it
must contain the nucleotide(s) which differentiate the presence or absence
of the target sequence. Thus, the contiguous portion continues the
sequence of this arbitrarily chosen diagnostic portion.
"Hybridization" and "binding" in the context of probes and denatured DNA
are used interchangeably. Probes which are hybridized or bound to
denatured DNA are aggregated to complementary sequences in the
polynucleotide. Whether or not a particular probe remains aggregated with
the polynucleotide depends on the degree of complementarity, the length of
the probe, and the stringency of the binding conditions. The higher the
stringency, the higher must be the degree of complementarity, and/or the
longer the probe.
"Covalently attaching" refers to forming a covalent chemical bond between
two substances.
"Ligating" refers to covalently attaching two polynucleotide sequences to
form a single sequence. This is typically performed by treating with a
ligase which catalyzes the formation of a phosphodiester bond between the
5' end of one sequence and the 3' end of the other. However, in the
context of the invention, the term "ligating" is intended to also
encompass other methods of covalently attaching such sequences, e.g., by
chemical means, and the terms "covalently attaching" and "ligating" will
be used interchangeably.
"Probe" refers to an oligonucleotide designed to be sufficiently
complementary to a sequence in a denatured nucleic acid to be probed, in
relation to its length, to be bound under selected stringency conditions.
"Diagnostic probe" refers to a probe which is complementary to the
diagnostic portion.
"Contiguous probe" refers to a probe which is complementary to the
contiguous portion.
"Target probe" refers to a probe which is complementary to the target
sequence and which is made by covalently attaching (ligating) the
diagnostic probe and the contiguous probe.
"Hook" refers to a modification of a probe that enables the experimenter to
rapidly and conveniently isolate probes containing this modification by
"catching" the hook. The interaction between hook and catching mechanism
can be, for example, covalent bonding or ligand receptor binding of
sufficient tightness. Such hooks might include antigens which can be
recovered by antibody, specific DNA sequences which can be recovered by
complementary nucleic acids, and specific reactive chemical groups which
can be recovered by appropriate reactive groups.
"Label" refers to a modification to the probe nucleic acid that enables the
experimenter to identify the labeled nucleic acid in the presence of
unlabeled nucleic acid. Most commonly, this is the replacement of one or
more atoms with radioactive isotopes. However, other labels include
covalently attached chromophores, fluorescent moieties, enzymes, antigens,
groups with specific reactivity, chemiluminescent moieties, and
electrochemically detectable moieties, etc.
In a preferred embodiment of the method, certain preliminary procedures are
necessary to prepare the sample nucleic acid and the probes before the
assay may be performed.
Sample Preparation
The sample nucleic acid is denatured and usually immobilized, typically by
being bound to a solid support such as nitrocellulose filter paper.
Techniques for denaturing and binding are well known in the art. (See for
example, P. 331, Molecular Cloning, by Maniatis, Fritsch, and Sambrook,
Cold Spring Harbor Laboratory, 1982, reproduced as Appendix A herein.)
The non-specific binding sites available in the system are then blocked. In
the typical case using nitrocellulose filter paper as the solid support,
the nucleic acid-paper is treated so that additional nucleic acid will not
become irreversibly bound to the paper. This is accomplished, for example,
by incubating the nucleic acid and filter for two hours at 80.degree. C in
40x Denhardt's solution (40x Denhardt's solution is 8 g/l bovine serum
albumin, 8 g/l polyvinyl pyrolidone and 8 g/l Ficoll). Then the 40x
Denhardt's is removed.
Probe Preparation
In those cases where the diagnostic probe and the contiguous probe are not
already available, they must be synthesized. Apparatus for such synthesis
is presently available commercially, such as the Applied Biosystems 380A
DNA synthesizer and techniques for synthesis of various nucleic acids are
available in the literature.
In one embodiment, the probes are prepared for ligation, e.g., if ligase is
to be used, the probe which will have its 5' end adjacent the 3' end of
the other probe when hybridized to the sample nucleic acid is
phosphorylated in order to later be able to form a phosphodiester bond
between the two probes. One of the probes is then labeled. This labeling
can be done as part of the phosphorylation process above using radioactive
phosphorus, or can be accomplished as a separate operation by covalently
attaching chromophores, fluorescent moieties, enzymes, antigens,
chemiluminescent moieties, groups with specific binding activity, or
electrochemically detectable moieties, etc. (Appendix B provides a
detailed description for 5' end labeling with 32.sub.P using T.sub.4
polynucleotide kinase.)
As another aspect of the invention, the diagnostic and contiguous probes
useful for the invention may be packaged as a test kit. A diagnostic probe
and a contiguous probe for detecting a particular target sequence
associated with a genetic disease are synthesized and one labelled. One of
the probes is also terminated in a manner that permits ligation to the
other probe. The two probes can then be packaged with appropriate
instructions so that the method may be practiced.
METHOD OF THE INVENTION
Step 1
Hybridize the probe which will remain bound under the more stringent
conditions. (Generally, either probe may be longer, and thus remain bound
under more stringent conditions. However, for some sequences of
nucleotides, the shorter probe may be the one more strongly bound.) The
sample nucleic acid is incubated with this probe under conditions that
promote specific binding of the probe to only the complementary sequences
in the sample nucleic acid.
Step 2
Hybridize the probe which will remain bound under the less stringent
conditions. Again, the sample nucleic acid is incubated, this time with
the other probe under conditions which will promote specific binding to
only complementary sequences in the sample nucleic acid. Since probe from
Step 1 will remain bound under the less stringent conditions required for
this probe, hybridization of this probe to the sample nucleic acid will
not materially affect the earlier hybridization.
Substep 1a or 2a
Remove a substantial portion of the diagnostic probe which is not perfectly
bound. (If the probe bound in Step 1 is the diagnostic probe, this is Step
1a and should be performed before Step 2, whereas, if the probe bound in
Step 2 is the diagnostic probe, this is Step 2a and should follow Step 2.)
This is accomplished by washing the sample nucleic acid at an appropriate
stringency to dissociate from the nucleic acid any diagnostic probe which
is not perfectly bound (i.e., not bound at all sites in the diagnostic
portion) while leaving intact that which is perfectly bound. This
procedure relies on the fact that there is an energy difference between
probes which are perfectly bound and those which are hot. In the situation
under study, this difference may be quite small, since it can be the
result of a single base pair mismatch in an entire diagnostic portion.
Hence, the stringency needs to be carefully controlled during this removal
process.
Step 3
Ligate the two probes. The sample nucleic acid with the two probes bound
thereto is treated with ligase or treated chemically to covalently attach
the two probes, the 5' phosphate of one probe to the 3' hydroxyl of the
other probe, at sites where they are hybridized to the nucleic acid
adjacent to each other.
Step 4
Increase the stringency to remove nearly all of the labeled probe that is
not ligated to the other probe in Step 3 (>99%).
Following Step 4, for practical purposes, all that remains in the system is
labeled target probe and any of several techniques can be used to detect
it. For example, the label can be detected directly by autoradiography or
counting, since the background from the non-specific binding of the
labeled probe has been removed. In many situations, however, it is
preferable and more easily quantitated to further increase the stringency
to remove the target probe and to measure the amount of label coming off
as the labeled target probe dissociates from the target sequence, a
procedure which is easily automated.
In addition, this elution process provides more accurate results than
detecting the bound target probe in situ, since it eliminates further
background which can be contributed by irreversible binding of the labeled
probe directly to the filter paper in those instances where the sample
preparation has not been as effective as desired in blocking non-specific
binding sites.
FIGS. 2A through 2C illustrate the principles of the above method at
several stages, and particularly with regard to the detection of the
diagnostic probe. It is assumed, for purposes of discussion, that the
contiguous probe is the one which is more tightly bound, and that
diagnostic probe is labeled.
FIG. 2A corresponds to the stage immediately after Steps 1 and 2, but
before Step 2a, i.e., immediately following hybridization of a diagnostic
probe 13 and a contiguous probe 15. In the Figure, the sample nucleic
acid, represented by several denatured strands 11, has diagnostic portions
D and a contiguous portion C. Where contiguous portion C and diagnostic
portion D are adjacent designates the target sequence. Also included are
portions X which correspond to any portion of the sample that is
sufficiently similar in sequence to the diagnostic portions that the
diagnostic probe will hybridize to that section. Areas N indicate any site
in the system that will bind probes non-specifically. For purposes of
illustration, it is assumed in FIG. 2A that 80% of the diagnostic
sequences present will bind diagnostic probe and that 20% of the X sites
bind probe and that all N sites bind probe. If the amount of diagnostic
probe were measured at this stage to determine the number of target
sequences present, there would be excessive background noise as occurred
in the prior art due to the binding from all N sites, from D sites outside
of the target sequence, and from a percentage of the X sites.
FIG. 2B illustrates the results of the method immediately after Step 4,
where the diagnostic and contiguous probes have been ligated to form
target probes 17 and the stringency has been increased to the point where
all unligated diagnostic probes are no longer hybridized to the sample
nucleic acid. If there are only few N sites present at this stage, the
amount of the diagnostic probe can be measured in situ to determine the
number of target sequences present.
FIG. 2C shows the stage after elution, i.e., where all the probes are
removed, including target probes 17. Here, the amount of diagnostic probe
which was originally hybridized to the target sequence can be determined
without the background noise contributed by the binding to N sites, to X
sites, and to D sites not in the target sequence.
The following example is provided as a specific application of the above
technique and should not be construed to limit the scope of the invention.
Bacteriophage .lambda.-DNA was chosen as the sample nucleic acid in this
example for two reasons. First, the expression of the .lambda. genome is
generally regarded as a model for genetically determined differentiation
processes occurring in cells of higher organisms. Because of its model
nature, .lambda. phage was felt to provide an adequate example for a
demonstration of the method of the invention. Second, .lambda. phage has
been well studied and is readily available.
EXAMPLE 1
Sample bacteriophage .lambda.-DNA was obtained from Bethesda Research
Laboratories in Gaithersburg, Md., Catalogue Number 5250. The target
sequence chosen corresponds to the sequence of nucleotides starting at
base 145 and ending at base 224. (See "Nucleotide Sequence of cro, c II,
and part of the O gene in Phage .lambda.-DNA," E. Schwarz, et al., Nature,
272, Mar. 30, 1978.)
The diagnostic portion chosen was a 15-mer corresponding to the sequence of
nucleotides starting at base 145 and ending at 159 in the .lambda.-DNA.
The diagnostic probe, a 15-mer complementary to the diagnostic sequence,
was synthesized on an Applied Biosystems 380A DNA synthesizer, and had the
sequence 5'-ATCAGCGTTTATAGT-3'. The contiguous portion chosen was a
sequence 65 bases long contiguous with the diagnostic sequence, i.e.,
beginning at base 160 and ending at base 224. The contiguous probe, a
65-mer complementary to the contiguous portion, was also synthesized on an
Applied Biosystems 380A DNA synthesizer, and had the sequence
5'-GTTATTTATGCTGTTGTTTTTTTGTTACTCGGGAAGGGCTTTACCTCTTCCGCATAAACGCTTCC-3'.
The 15-mer was phosphorylated with .sup.32 P using T.sub.4 polynucleotide
kinase, thereby also radioactively labeling the 15-mer. (See Appendix B
for the specifics of this kinasing process.)
The sample DNA was denatured and immobilized (as described in Appendix A,
except that Schleicher & Schuell BA85 nitrocellulose filter was used cut
in 9mm diameter circles), and non-specific binding sites were blocked, as
described earlier under Sample Preparation. About 0.3 pmol phage
.lambda.-DNA was fixed onto the nitrocellulose filter paper during this
sample preparation process.
Step 1
The 6-mer was hybridized to the .lambda.-DNA by incubating the DNA-paper
with 150 .mu.l 1 2xSSC containing 4 pmol 65-mer for 3 hours at 57.degree.
C, and the 65-mer solution was removed. (2xSSC is 0.3M NaCl, 0.03M Na
citrate pH 7.0.)
Step 2
The 15-mer was hybridized to the .lambda.-DNA by incubating the DNA paper
with 150 .mu.l 2xSSC containing 4 pmol of the labeled 15-mer for 16 hours
at 36.degree. C.
Step 2a
A substantial portion of the 15-mer not perfectly bound (i.e., not bound at
all 15 sites) in Step 2 was removed by washing the DNA-paper in 320ml
2xSSC for 10 minutes at 23.degree. C. and then washing the filter again in
2 .mu.l of the buffer 66 mM Tris HCl at pH 7.5, 6.6 mM MgCl.sub.2, and 10M
dithiothreitol.
Step 3
The two probes were ligated by incubating the DNA-paper in 150 .mu.l of the
buffer used in Step 2a, plus 0.4 mM ATP and 2 .mu.l T4 DNA ligase for 2
hours at 23.degree. C.
Step 4
The stringency was increased to remove nearly all of the labeled probe that
was not ligated in Step 4. This was accomplished by washing the DNA-paper,
by passing 200 .mu.l/min of 2xSSC through the filter at 28.degree. C. for
1 hour. Although it was designed for another purpose, an Applied
Biosystems 470A Protein Sequencer was used for this operation, since it
has a cartridge assembly which can hold a filter and can be programmed to
deliver liquids through the filter at a programmable temperature.
To detect the ligated probes (the target probe), the stringency was further
increased by increasing the temperature 5.degree. C./hr and passing
0.1xSSC with 0.5% sodium dodecyl sulfate through the filter. Fractions of
200 .mu.l each were collected every 45 minutes. The radioactivity of each
fraction was then determined using a liquid scintillation counter and the
number of cumulative counts at the end of each interval was plotted versus
temperature.
FIG. 3 shows the results of this procedure. For comparison purposes, this
was also done for the case where no ligase was used. As can be seen, the
use of the ligase dramatically changed the melting curve relative to the
unligated case. Essentially all of the unligated labeled probe was
dissociated by the time the temperature reached 35.degree. C. Whereas, in
the ligated case, a substantial portion of the labeled 80-mer remained at
35.degree. C. Hence, by restricting the measurement of radioactivity to
material removed from the filter above 35.degree. C., essentially only
ligated probe will be counte | | |
