U.S. patent application number 09/782386 was filed with the patent office on 2001-08-09 for double-stranded conformational polymorphism analysis.
This patent application is currently assigned to Naxcor. Invention is credited to Albagli, David, Atta, Reuel Van, Wood, Michael.
Application Number | 20010012616 09/782386 |
Document ID | / |
Family ID | 21725020 |
Filed Date | 2001-08-09 |
United States Patent
Application |
20010012616 |
Kind Code |
A1 |
Wood, Michael ; et
al. |
August 9, 2001 |
Double-stranded conformational polymorphism analysis
Abstract
Double-stranded conformational polymorphism analysis is
performed by combining a probe comprising a cross-linking agent and
optionally a label with a sample having a target sequence, which
may be complementary or have one or a few mismatches with respect
to the probe sequence. After sufficient time for hybridization
under mild or lesser stringency conditions, hybridized pairs are
irradiated to induce cross-link formation by the cross-linking
agent. The sample is then analyzed by denaturing gel
electrophoresis where the rate of migration depends upon the degree
of complementarity between the probe and the target. For
corroboration, in a second experiment, the probe may be combined
with the sample under high stringency conditions, where it is found
that the formation of cross-linked probe/target is substantially
lower for pairs having mismatches than for fully matched pairs.
After cross-linking, the sample may be separated by gel
electrophoresis, and the amount of cross-linked nucleic acid
determined.
Inventors: |
Wood, Michael; (Palo Alto,
CA) ; Atta, Reuel Van; (Mountain View, CA) ;
Albagli, David; (Palo Alto, CA) |
Correspondence
Address: |
Todd A. Lorenz
FLEHR HOHBACH TEST ALBRITTON & HERBERT LLP
Suite 3400
Four Embarcadero Center
San Francisco
CA
94111-4187
US
|
Assignee: |
Naxcor
|
Family ID: |
21725020 |
Appl. No.: |
09/782386 |
Filed: |
February 12, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09782386 |
Feb 12, 2001 |
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08742376 |
Nov 1, 1996 |
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6187532 |
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60007239 |
Nov 3, 1995 |
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Current U.S.
Class: |
435/6.11 ;
435/6.1; 435/6.12; 435/6.18 |
Current CPC
Class: |
C12Q 1/6827 20130101;
Y10S 435/81 20130101; C12Q 1/6827 20130101; C12Q 2523/101 20130101;
C12Q 2565/131 20130101; C12Q 2565/125 20130101; C12Q 2523/101
20130101; C12Q 2523/313 20130101; C12Q 2523/101 20130101; C12Q
2565/125 20130101; C12Q 2565/131 20130101; C12Q 1/6827 20130101;
C12Q 1/6827 20130101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 001/68 |
Claims
What is claimed is:
1. A method of detecting the presence or absence of at least one
mismatch between a nucleic acid probe and a nucleic acid target,
wherein said probe and target have sequences which differ by not
more than five mismatches, said probe comprising a known sequence
and a photoactivatable cross-linking agent, which when said probe
sequence is hybridized to said target sequence, upon
photoactivation forms a covalent bond between said probe sequence
and said target sequence, said method comprising: combining, in a
hybridizing medium, a nucleic acid sample comprising said target
and said probe under mild stringency hybridizing conditions for a
time sufficient for said target and said probe to hybridize;
irradiating said hybridizing medium to form cross-links between
said probe and target sequence to which said probe is hybridized to
from cross-linked double-stranded nucleic acid; separating nucleic
acid in said hybridizing medium by denaturing electrophoresis and
comparing the migratory rate of said cross-linked double-stranded
nucleic acid to a known mismatched or matched cross-linked
double-stranded nucleic acid standard, whereby the presence or
absence of said at least one mismatch is determined.
2. A method according to claim 1, wherein said probe is labeled
with a detectable label.
3. A method according to claim 1, wherein said sample is prepared
using the polymerase chain reaction and said sample nucleic acid is
labeled with a detectable label.
4. A method according to claim 1, wherein said electrophoresis is
polyacrylamide gel electrophoresis.
5. A method of detecting the presence or absence of at least one
mismatch between a nucleic acid probe and a nucleic acid target,
wherein said probe and target have sequences which differ by not
more than five mismatches, said target sequence comprising a
nucleic acid molecule of from about 25 to 300 nt and said probe
comprising a known sequence of from 15 to 50 nt and a
photoactivatable cross-linking agent, which when said probe
sequence is hybridized to said target sequence, upon
photoactivation forms a covalent bond between said probe sequence
and said target sequence, said method comprising: combining, in a
hybridizing medium, a nucleic acid sample comprising said target
and said probe under mild stringency hybridizing conditions for a
time sufficient for said target and said probe to hybridize;
irradiating at a wavelength in the range of about 300-400 nm said
hybridizing medium to form cross-links between said probe and
target sequence to which said probe is hybridized to cross-linked
double-stranded nucleic acid; separating nucleic acid in said
hybridizing medium by denaturing electrophoresis and comparing the
migratory rate of said cross-linked double-stranded nucleic acid to
a known mismatched or matched cross-linked double-stranded nucleic
acid standard, whereby the presence or absence of said at least one
mismatch is determined.
6. A method according to claim 5, wherein said sample is prepared
by restriction enzyme digestion of genomic DNA.
7. A method according to claim 5, wherein said sample is prepared
using the polymerase chain reaction and said sample nucleic acid is
labeled with a detectable label.
8. A method according to claim 5, wherein said probe is labeled
with a detectable label.
9. A method according to claim 5, wherein said electrophoresis is
polyacrylamide gel electrophoresis.
10. A method of detecting the presence or absence of at least one
mismatch between a nucleic acid probe and a nucleic acid target,
wherein said probe and target have sequences which differ by not
more than five mismatches, said target sequence comprising a
nucleic acid molecule of from about 25 to 300 nt and said probe
comprising a known sequence of from 15 to 50 nt and a
photoactivatable cross-linking agent, which when said probe
sequence is hybridized to said target sequence, upon
photoactivation forms a covalent bond between said probe sequence
and said target sequence, said method comprising: combining, in a
hybridizing medium, a nucleic acid sample comprising said target
and said probe under mild stringency hybridizing conditions
equivalent to a temperature in the range of 25-70.degree. C. and
with 0.1-1.5 M sodium for a time sufficient for said target and
said probe to hybridize; irradiating at a wavelength in the range
of about 300-400 nm said hybridizing medium to form cross-links
between said probe and target sequence to which said probe is
hybridized to from cross-linked double-stranded nucleic acid;
separating nucleic acid in said hybridizing medium by denaturing
gel electrophoresis and comparing the migratory rate of said
cross-linked double-stranded nucleic acid to a known mismatched or
matched cross-linked double-stranded nucleic acid standard, whereby
the presence or absence of said at least one mismatch is
determined.
11. A method according to claim 10, wherein said cross-linking
agent comprises a coumarinyl group.
12. A method of detecting the presence or absence of at least one
mismatch between a nucleic acid probe and a nucleic acid target,
wherein said probe and target have sequences which differ by not
more than five mismatches, said target sequence comprising a
nucleic acid molecule of from about 25 to 300 nt and said probe
comprising a known sequence of from 15 to 50 nt and a
photoactivatable cross-linking agent, which when said probe
sequence is hybridized to said target sequence, upon
photoactivation forms a covalent bond between said probe sequence
and said target sequence, said method comprising: combining, in a
hybridizing medium, a nucleic acid sample comprising said target
and said probe under high stringency hybridizing conditions for a
time sufficient for said target and said probe to hybridize, where
a probe complementary to said target results in at least about a
2-fold greater amount of hybridization than a mismatched probe;
irradiating at a wavelength in the range of about 300-400 nm said
hybridizing medium to form cross-links between said probe and
target sequence to which said probe is hybridized to cross-linked
double-stranded nucleic acid; separating nucleic acid in said
hybridizing medium by denaturing electrophoresis and determining
the amount of cross-linked double-stranded nucleic acid, where the
amount of cross-linked double-stranded nucleic acid is related to
the presence or absence of mismatches between said probe and said
target.
13. A method according to claim 12, wherein said high stringency
conditions are at least equivalent to a temperature in the range of
about 40-70.degree. C. and 0.05 to 0.5 M sodium ion.
14. A kit comprising two probes, characterized by consisting of
from 15 to 50 nt, joined to each of said probes is a
photoactivatable cross-linking agent, each of said probes differing
with the other probe by not more than 3 mismatches, and being
naturally occurring sequences.
15. A kit according to claim 14, wherein said photoactivatable
cross-linking agent comprises a coumarinyl group.
16. A kit according to claim 14, wherein said naturally occurring
sequences are related by one being the mutant of the other.
17. A kit according to claim 14, wherein said naturally occurring
sequences are related by one being the allele of the other.
18. A kit according to claim 14, wherein said probes are labeled
with a detectable label.
19. A kit according to claim 14, wherein each of said probes has a
plurality of cross-linking agents.
Description
INTRODUCTION
TECHNICAL FIELD
[0001] The field of this invention is detecting mutations in
DNA.
BACKGROUND
[0002] The amount of genetic information concerning humans and
other species has been expanded enormously, particularly with the
advent of the human genome project. With identification of all of
the genes present, we will be able to identify mutations associated
with particular phenotypes. There is already a substantial library
of genes, which when mutated, are known to be associated with
various diseases. One need only consider cystic fibrosis,
Huntington's disease, .beta.-thalassemia, sickle-cell anemia, and
the like. In some instances, such as sickle-cell anemia, there is a
common point mutation associated with the disease. In other cases,
such as cystic fibrosis, there are numerous point mutations spread
throughout the genes associated with the disease.
[0003] There are many situations where one would wish to know
whether a patient or other species has a point mutation or a
particular polymorphism of interest. Not only are we interested in
diseases, but particularly with other species, there may be an
interest in knowing whether the host has a particular allele.
[0004] Numerous techniques have been developed to identify
differences between a known and target sequence.
[0005] Allele-specific oligonucleotide (ASO) tests are used to
identify single-nucleotide mismatches or small differences between
a short probe and a target DNA. The target DNA is electrophoresed
through a gel and subsequently transferred to a nylon or
nitrocellulose membrane. A labelled probe is incubated with the
membrane under hybridization conditions which distinguish between
the presence and absence of complementarity. The test is dependent
upon the strict observance of the hybridization and wash conditions
necessary to distinguish between mismatches and
complementarity.
[0006] The polymerase chain reaction (PCR) has been employed to
directly detect sequence differences. One technique known as the
amplification refractory mutation system (ARMS) is based on the
observation that oligonucleotides which are complementary to a
given sequence except for a mismatch of the 3' end will not
function as a primer for PCR. Thus, by appropriate selection of
primer sets and PCR conditions, one can detect a mismatch.
Alternatively, primers may be selected that lead to the formation
of normal or mutated amplification products, resulting in a
restriction site in one or the other sequence.
[0007] Single-stranded conformation polymorphism (SSCP) looks to
the detection of single-base differences due to differences in
migration rates through non-denaturing polyacrylamide gels (PAGE).
After denaturing the target DNA, variations in secondary structure
of single-strand DNA can be detected using a non-denaturing
gel.
[0008] Complementary and mismatched DNA-DNA hybrids denature under
different conditions from one another. This has been exploited by
denaturing gradient gel electrophoresis (DGGE). DGGE gels contain
gradually increasing levels of denaturant causing complementary and
mismatched dsDNA molecules to migrate and denature at different
points in the gel.
[0009] In addition to electrophoresis, there are chemical
techniques that may be employed, such as chemical modifying agents
that cleave the DNA at the mismatched site, e.g. osmium tetroxide,
hydroxylamine, etc.; ribonuclease A cleaves DNA:RNA hybrids at
mismatch points; which are then followed by analysis with PAGE.
Other techniques include heteroduplex analysis and nucleotide
sequence analysis. All of these techniques have limitations in the
strictness of the conditions and control which must be employed,
the complexity of the protocols, limitations on the generality of
the methodology, and the like.
RELEVANT LITERATURE
[0010] Articles which describe various techniques for detecting
mismatches include: Dowton and Slaugh, Clin. Chem. 41:785-794
(1995); Newton et al., Nucl. Acids Res. 17:2503-2516 (1989);
Haliassos et al., Nucl. Acids Res. 17:3606 (1989); Orita et al.,
Proc. Natl. Acad. Sci. USA 86:2766-2770 (1989); Sarkar et al.,
Nucl. Acids. Res. 20:871-878 (1992); Fischer and Lerman, Proc.
Natl. Acad. Sci. USA 80:1579-1583 (1983); Cotton et al., Proc.
Natl. Acad. Sci. USA 85:4397-4401 (1988); Myers et al., Science
230:1242-1246 (1985); and White et al., Genomics 12:301-306
(1992).
SUMMARY OF THE INVENTION
[0011] Methods and compositions are provided for detection of
single or multiple mismatches between a target sequence and a known
sequence. The method comprises hybridizing under not greater than
mild stringency conditions a probe and a target sequence of less
than about 300 bases. The probe comprises the known sequence,
optionally a detectable label, and a cross-linking agent. After
sufficient time for hybridization to occur for a detectable amount
of double-stranded nucleic acid, the conditions of the medium are
changed to induce cross-linking of hybridized pairs. The sample is
then separated using PAGE under denaturing conditions and the
migratory rate of the labelled probe cross-linked to target nucleic
acid determined as against a known standard. A probe/target pair
with mismatches will migrate at a different rate from a
complementary probe/target pair. For confirmation, more stringent
hybridization conditions can be selected where the amount of
hybridization between a mismatched pair of sequences and a matched
pair of sequences is substantially different. The resulting sample
is heated the same way as indicated above, where the amount of
probe which becomes cross-linked is related to the degree of
mismatches between the probe and target, there being a
substantially smaller amount of cross-linked probe in the case of a
mismatch. In accordance with the subject invention, substantially
increased flexibility is obtained as to the conditions which may be
employed for determining the presence of a mutation in a target
sequence.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
[0012] In accordance with the subject invention, probes are
provided to be used in methods for detecting the presence or
absence of mismatches to the probe in a target sequence. The
mismatches may be as a result of a mutation, allelic variation,
species variation, alternative splicing, etc. The mismatch may be
an insertion, deletion or mismatched pairing, usually one or more
point mismatches.
[0013] Generally, the method employs combining the probe, which is
characterized by having a known sequence, optionally a detectable
label and a cross-linking agent, with a target sequence, which may
be present as a major component of the DNA from the target or as
one member of a complex mixture. The target sequence is provided in
single-stranded form. The probe and target sequence are allowed to
hybridize under not greater than mild stringency conditions. After
sufficient time for a sufficient amount of double-stranded nucleic
acid to form, the conditions are changed so as to provide for
cross-linking. After cross-linking has occurred, the sample is then
separated by gel electrophoresis, where the migratory rate of a
mismatched double-stranded nucleic acid is different from the
migratory rate of a complementary double-stranded nucleic acid. The
observed migratory rate of the probe-target double-stranded complex
may be compared with a standard to determine the presence or
absence of mismatches.
[0014] The target DNA may come from any source and will be provided
as an average size in the range of about 25 to 300 nt, more usually
50-250 nt, preferably from about 50-200 nt. The source of DNA may
be prokaryotic or eukaryotic, usually eukaryotic. The source may be
the genome of the host, plasmid DNA, viral DNA, where the virus may
be naturally occurring or serving as a vector for DNA from a
different source, a PCR amplification product, or the like. The
target DNA may be a particular allele of a mammalian host, an MHC
allele, a sequence coding for an enzyme isoform, a particular gene
or strain of a unicellular organism, or the like. The target
sequence may be genomic DNA, cDNA, RNA, or the like.
[0015] Nucleic acids of the desired length can be achieved,
particularly with DNA, by restriction, use of PCR and primers, and
the like. Desirably, at least about 80 mol %, usually at least
about 90 mol % of the target sequence, will have the same size. For
restriction, a frequently cutting enzyme may be employed, usually
an enzyme with a four base consensus sequence, or combination of
restriction enzymes may be employed, where the DNA will be subject
to complete digestion. The mismatch will normally be internal to
the target fragment and will normally not be at a site cleaved by a
restriction enzyme used to digest the sample DNA. Typical sequences
of interest include the mutation in sickle-cell anemia, the MHC
associated with IDDM, mutations associated with cystic fibrosis,
Huntington's disease, .beta.-thalassemia, Alzheimer's disease, and
various cancers, such as those caused by activation of oncogenes
(e.g. ras, src, myc, etc.) and/or inactivation of tumor
suppressants (e.g. p53, RB, etc.). In some cases, such as
sickle-cell anemia, there is a single mutation. In other cases,
such as cystic fibrosis, there are multiple mutations to be
determined. By selection of appropriate restriction enzymes, one
can provide that the region suspected of harboring one or more
mutations is present on a fragment of predetermined size, so that
by using a combination of probes, one can readily detect the
presence of one or more of the mutations in the gene.
[0016] Depending upon the source of DNA, the DNA may be subject to
some purification, such as separation of proteins, removal of
restriction enzyme inhibitors, or the like.
[0017] The probe will generally be of about 15 to 50 nt, more
usually of from about 20 to 35 nt. The probe may have from 1 to 5
cross-linking agents, more usually from about 1 to 3 cross-linking
agents. The cross-linking agents will be selected so as not to
interfere with the hybridization and will generally be positioned
across from a thymidine (T), cytidine (C), or uridine (U) to
provide for cross-linking. A large number of functionalities are
photochemically active and can form a covalent bond with almost any
organic moiety. These groups include carbenes, nitrenes, ketenes,
free radicals, etc. One can provide for a scavenging molecule in
the bulk solution, normally excess non-target nucleic acid, so that
probes which are not bound to a target sequence will react with the
scavenging molecules to avoid non-specific cross-linking between
probes and target sequences. Carbenes can be obtained from diazo
compounds, such as diazonium salts, sulfonylhydrazone salts, or
diaziranes. Ketenes are available from diazoketones or quinone
diazides. Nitrenes are available from aryl azides, acyl azides, and
azido compounds. For further information concerning photolytic
generation of an unshared pair of electrons, see A. Schonberg,
Preparative Organic Photochemistry, Springer-Verlag, N.Y. 1968.
[0018] For the most part, the compounds which are employed for
cross-linking will be photoactivatable compounds which form
covalent bonds with a base, particularly a pyrimidine. These
compounds will include functional moieties, such as coumarin, as
present in substituted coumarins, furocoumarin, isocoumarin,
bis-coumarin, psoralen, etc., quinones, pyrones,
.alpha.,.beta.-unsaturated acids, acid derivatives, e.g. esters,
ketones, and nitrites; azido, etc.
[0019] Another class of photoactive reactants are organometallic
compounds based on any of the d- or f-block transition metals.
Photoexcitation induces the loss of a ligand from the metal to
provide a vacant site available for substitutions. Suitable ligands
include nucleotides. For further information regarding the
photosubstitution of organometallic compounds, see "Organometallic
Photochemistry", G. F. Geoffrey and M. S. Wrighton, Academic Press,
San Francisco, Calif., 1979.
[0020] The probe homologous sequence which binds to the target
sequence will usually contain naturally occurring nucleotides.
However, in some instances the phosphate-sugar chain may be
modified by using unnatural sugars, by substituting oxygens of the
phosphate with sulfur, carbon, nitrogen, or the like, or other
modification which can provide for synthetic advantages, stability
under the conditions of the assay, resistance to enzymatic
degradation, etc.
[0021] The probes may be prepared by any convenient method, most
conveniently synthetic procedures, where the cross-linking modified
nucleotide is introduced at the appropriate position stepwise
during the synthesis. Linking of various molecules to nucleotides
is well known in the literature and does not require description
here. See, for example, "Oligonucleotides and Analogues. A
Practical Approach", Eckstein, F. ed., Oxford University Press,
1991.
[0022] Similarly, the label, if present, may be bonded to any
convenient nucleotide in the probe chain, where it does not
interfere with the hybridization between the probe and the target
sequence. Labels will generally be small, usually from about 100 to
1,000 Da. The labels may be any detectable entity, where the label
may be able to be detected directly, or by binding to a receptor,
which in turn is labelled with a molecule which is readily
detectable. Molecules which provide for detection in
electrophoresis include radiolabels, e.g. .sup.32P, 35S, etc.
fluorescers, such as rhodamine, fluorescein, etc. ligand for
receptors, such as biotin for streptavidin, digoxigenin for
anti-digoxigenin, etc., chemiluminescers, and the like.
Alternatively, no label need be used, where the DNA may be stained
either prior to, during or after the separation, using such stains
as ethidium bromide, ethidium dimer, thiazole orange, thiazole
blue, dimers thereof, or the like. When using PCR, one can provide
for the primer to be labelled, rather than the probe, so that the
primer may provide for the detection. Where a ligand is employed,
the receptor may be labelled with any of the directly detectable
labels.
[0023] The method is performed by combining the probe with the
target DNA. Usually, the target DNA will be estimated to be present
in the range of about 10.sup.-20 to 10.sup.-8 moles, more usually
in the range of about 10.sup.-17 to 10.sup.-10 moles. The probe may
be present in equivalent amount or large excess, generally in
excess not more than 10.sup.9- fold, more usually in excess not
more than 10.sup.7- fold, based on the estimated amount of target
nucleic acid. The hybridizing medium will provide for mild to low
stringency, to ensure that substantially all of the target nucleic
acid is cross-linked. Generally, the stringency will be equivalent
to a temperature in the range of about 25-70.degree. C., frequently
40-70.degree. C., more usually 30-50.degree. C., with 0.05-1.5 M
sodium, more usually 0.25-1 M sodium ion or 0-20% formamide. With
RNA, guanidinium thiocyanate may be added in an amount of 0.1 to
6M. Other denaturants besides formamide include urea and
dimethylsulfoxide. The hybridization conditions are selected to
afford the maximum amount of hybridization between the probe and
target-sequence for both the matched and mismatched nucleic acid
sequences. The time for the hybridization will be sufficient to
form a detectable amount of double-stranded nucleic acid, will be
dependent upon the conditions of the hybridization, and the
sensitivity with which the label can be detected. Times will
usually be at least 5 minutes and not more than 6 hours, more
usually about 10 minutes -1 hour.
[0024] After the hybridization has occurred, the probe-containing
double-stranded nucleic acid may be cross-linked. The light will be
at or greater than 300 nm to avoid naturally occurring
cross-linking of nucleic acid. Generally, the light will be in the
range of 300-400 nm using a light source in conjunction with a
Pyrex filter. While chemical activation may be employed, normally
photolytic activation is more convenient and will be the method of
choice. The irradiation time will generally be in the range of
about 1 minute to 2 hours, more usually in the range of about 5
minutes to 1 hour, depending upon the size of the sample, the power
of the irradiating source, the desired amount of product, and the
like. If desired, an aliquot of the sample will be taken and
electrophoresed to determine whether a sufficient amount of
cross-linking has occurred.
[0025] After the irradiation, the sample may then be treated, such
as by heating, or combined with a front-running dye, glycerol,
sucrose, formamide, etc. for loading purposes. These techniques are
well known in the art and do not require elaboration here.
[0026] The electrophoresis is carried out using polyacrylamide gel,
generally 5-23% acrylamide and a ratio of 10-30:1 of acrylamide to
bis-monomer. Denaturing conditions are used so as to remove any
non-cross-linked nucleic acid from the region of the cross-linked
nucleic acid. Other denaturants may be used in place of urea. Any
of the typical running buffers may be employed such as
Tris-borate-EDTA. The electrophoresis is carried out under
conventional conditions to allow for separation between mismatched
and matched sequences. Having an appropriately matched or
mismatched standard in one of the lanes, one can compare the band
in the sample lane with the standard band. The differences between
the standard and the sample will indicate whether the target
sequence is different from the standard sequence. By using the
appropriate label, or staining the gel, one can detect the presence
or absence of any mismatches between the target and probe
sequences.
[0027] If one wishes further corroboration, one can use an
adaptation of the ASO technique, determining the degree of duplex
formation. However, providing for cross-linking substantially
diminishes the criticality of the conditions employed. In this
procedure, the temperature will generally be in the range of about
50-70.degree. C., while the sodium ion concentration will generally
be in the range of about 50-500 mM, more usually about 100-400 mM.
For each target sequence and probe, one would optimize the
conditions so as to obtain the greatest difference in the degree of
duplex formation between mismatched sequences and matched
sequences. Desirably, there should be at least about 2, usually at
least about a 5-fold ratio, between the amount of cross-linked
matched sequences and cross-linked, non-matched sequences. In this
process, higher stringency conditions are employed. Otherwise, the
conditions will be substantially the same as the conditions
employed for the differences in migration. The amount of
cross-linked DNA can be readily determined by measuring the signal
obtained from the band associated with the matched standard and the
probe and target sequence. Where the signal is substantially less,
this would indicate that the sequences are mismatched. Where the
signal is about the same as the standard, this would indicate that
the sequences are matched.
[0028] For convenience of the user, kits may be provided comprising
one or more, usually 2 or more probes, particularly a pair of
probes, where one probe is complementary to a sequence, which may
be referred to as the "wild-type" sequence, and the other probe may
be referred to as the "mutant" sequence. However, it should be
understood that these designations are arbitrary, since in many
situations one may only wish to know whether the target sequence is
the same or different from the probe sequence, without there being
the concept that one sequence is common or wild-type and the other
sequence is uncommon or mutant. For example, one may wish to know
which of two MHC alleles are present which differ by one or two
mismatches. The pair of probes will usually have not more than 5,
more usually not more than 3 differences. Depending upon the target
sequence, there may be a plurality of probes, particularly pairs of
probes, usually not more than about 12 pairs, where the target
sequence has a plurality of potential mutations, which may be
spread through the gene. Ancillary materials may be provided, such
as dyes, labeled antibodies, where a ligand is used as a label,
labeled primers for use with PCR, etc.
[0029] The following examples are offered by way of illustration
and not by way of limitation.
EXPERIMENTAL
EXAMPLE 1
[0030] Detection of A Single-Base Mismatch by (A) Allele-Specific
Hybridization and Cross-Lining and (B) DSCP Analysis
[0031] An oligonucleotide (oligo #1) comprising nucleotides 374-403
of the E6 gene of human papilloma virus type 16 was synthesized by
the phosphoramidite method of DNA synthesis and labeled with
.sup.32P at the 5' end. A second .sup.32P-labeled oligonucleotide
(oligo #2) containing the same sequence as oligo #1 except for a
single G->A base change at position 388 was also prepared.
[0032] Oligo #1: 5'-CAA TAC AAC AAA CCG TTG TGT GAT TTG TTA-3'
[0033] Oligo #2: 5'-CAA TAC AAC AAA CCA TTG TGT GAT TTG TTA-3'
[0034] A 20-mer DNA probe (oligo #3) containing the photoactive
cross-linking group, 3-O-(7-coumarinyl) glycerol (denoted by X in
the sequence) was prepared. This DNA sequence of this probe is
fully complementary to oligo #1 but would hybridize with oligo #2
to form a duplex containing an A/C mismatch.
[0035] Oligo #3: 3'-TTG TTT GGC AAC ACA CTA XA-5'
[0036] Oligo #1/#3 duplex:
[0037] 5'-CAA TAC AAC AAA CCG TTG TGT GAT TTG TTA-3'
[0038] 3'-TTG TTT GGC AAC ACA CTA XA-5'
[0039] Oligo #2/#3 duplex:
[0040] 5.ident.-CAA TAC AAC AAA CCA TTG TGR GAT TTG TTA-3'
[0041] 3'-TTG TTT GGC AAC ACA CTA XA-5'
[0042] Oligo #3 (20 pmole) was incubated in the presence of 2 pmole
of either 32P-5'end-labeled oligo #1 or oligo #2 in 0.15 mL samples
at the temperatures and NaCl concentrations summarized below:
1 Sample Oligonucleotides Temp., .degree. C. NaCl conc., mM 1 1 + 3
45 150 3 1 + 3 45 300 2 2 + 3 45 150 4 2 + 3 45 300 5 1 + 3 50 150
7 1 + 3 50 300 6 2 + 3 50 150 8 2 + 3 50 300 9 1 + 3 55 150 11 1 +
3 55 300 10 2 + 3 55 150 12 2 + 3 55 300
[0043] After 20 minutes incubation, the solutions were irradiated
under UV-A wavelength light for 45 minutes. Upon completion of the
irradiation step, one-tenth of the samples (0.015 mL) was removed
and mixed with an equal volume of formamide-bromophenol blue dye
mix and heated to 70.degree. C. for 3 minutes. The samples were
cooled on ice and loaded onto a 15% polyacrylamide gel (19:1
acrylamide/bisacrylamide) containing 7 M urea and electrophoresed
at 300 V until the bromophenol blue dye reached the bottom of the
gel. The gel was taken down and exposed to X-ray film overnight at
-80.degree. C.
[0044] Method 1: Allele specific hybridization and
cross-linking
[0045] By carrying out the experiment under a range of
hybridization temperatures (45-55.degree. C.) and NaCl
concentration (150-300 mM), it was possible to define conditions
that led to appreciable cross-link formation between the
complementary oligonucleotides #1 and #3 but not the mismatched
oligonucleotides #2 and #3. To determine the best conditions for
mismatch discrimination the radioactive bands were excised from the
gel, quantified by scintillation counting and the percent yield of
cross-linked product measured (relative to unreacted
.sup.32P-labeled oligonucleotide). The results are shown below:
2 NaCl Sample Oligonucleotides Temp., .degree. C. conc., mM
cross-linking, % 1 1 + 3 45 150 46 2 2 + 3 45 150 44 3 1 + 3 45 300
49 4 2 + 3 45 300 51 5 1 + 3 50 150 40 6 2 + 3 50 150 22 7 1 + 3 50
300 48 8 2 + 3 50 300 44 9 1 + 3 55 150 39 10 2 + 3 55 150 3 11 1 +
3 55 300 43 12 2 + 3 55 300 18
[0046] From the data in the above table, it can be determined that
the optimal conditions for discriminating the complementary and
mismatched duplexes are those used in samples 9 and 10 (55.degree.
C., 150 mM NaCl); under these conditions the complementary
oligonucleotide pair yields 13-fold more cross-linked product than
the mismatched pair (39% vs. 3%).
[0047] Method 2: DSCP Analysis (Double-Stranded Conformational
Polymorphism Analysis)
[0048] Analysis of the autoradiogram for the samples (3 and 4) run
under the least stringent hybridization conditions (45.degree. C.,
300 mM NaCl) clearly showed that the product obtained from
cross-linking between the mismatched oligonucleotides #2 and #3
migrated slower through the gel than the product obtained from
cross-linking the complementary oligonucleotides #1 and #3 (the
DSCP effect).
[0049] The results obtained from this experiment highlighted two
advantages of the DSCP method over the more conventional method of
developing hybridization conditions to detect single base
mismatches:
[0050] 1. The DSCP method is simple and does not require the
careful optimization of hybridization conditions to distinguish
matched from mismatched sequences. The DSCP method uses
non-stringent hybridization conditions.
[0051] 2. By using non-stringent conditions the cross-link yield
and hence the signal in the assay is higher than when the
hybridization stringency method is employed; under the conditions
used for DSCP analysis (45.degree. C., 300 mM NaCl) the cross-link
yield for the reaction between the complementary oligonucleotides
#1 and #3 was 49%, however under the conditions that led to the
best mismatch discrimination with the hybridization stringency
method (55.degree. C., 150 mM NaCl), the cross-linking efficiency
was 39%. Thus the DSCP method resulted in 26% greater signal.
EXAMPLE 2
[0052] Detection of Normal (.beta..sup.A) and Sickle Cell
(.beta..sup.S) .beta.-Globin Alleles by DSCP Analysis
[0053] Two 56 base oligonucleotides comprising a portion of the
sequence of either the normal human .beta.-globin gene
(.beta..sup.A-target) or the sickle cell p-globin gene
(.beta..sup.S-target) were synthesized by the phosphoramidite
method of DNA synthesis and labeled with .sup.32P at their 5' ends.
The .beta..sup.S-globin target sequence differs from the
.beta..sup.A-target by a single A->T mutation that gives raise
to a mutant .beta.-globin protein that contains valine instead of
glutamic acid.
[0054] .beta..sup.A-target: 5'-TGA CTC CTG AGG AGA AGT CTG CCG TTA
CTG CCC TGT-GGG GCA AGG TGA ACG TGG AT-3'
[0055] .beta..sup.S-target: 5'-TGA CTC CTG TGG AGA AGT CTG CCG TTA
CTG CCC TGT-GGG GCA AGG TGA ACG TGG AT-3'
[0056] Two probes complementary to either the .beta..sup.A-target
sequence (.beta..sup.A-probe) or the .beta..sup.S-target
(.beta..sup.S-probe) were also synthesized. These probes were
modified with the photoactive cross-linking group,
3-O-(7-coumarinyl) glycerol (denoted by X in the sequence):
[0057] .beta..sup.A-probe: 3'-TGA GGA CTC CTC TTC AXA-5'
[0058] .beta..sup.S-probe: 3'-TGA GGA CAC CTC TTC AXA-5'
[0059] Hybridization and cross-linking experiments were carried out
to show that DSCP analysis with the two .beta.-globin probes could
be used to detect and distinguish the presence of either the
.beta.-globin targets when the target molecules were present
individually or together in a 1:1 mixture (as would be found in a
heterozygous individual). The experiments summarized below were
carried out:
3 Sample .beta.-Globin probe .beta.-Globin target UV-A irradiation
1 .beta..sup.A .beta..sup.A - 2 .beta..sup.S .beta..sup.S - 3
.beta..sup.A .beta..sup.A + 4 4.beta..sup.A .beta..sup.S + 5
.beta..sup.S .beta..sup.S + 6 .beta..sup.S .beta..sup.A + 7
.beta..sup.A .beta..sup.A/.beta..sup.S + 8 .beta..sup.S
.beta..sup.A/.beta..sup- .s +
[0060] Each 0.05 mL sample contained 10 pmole of the relevant probe
and 0.2 pmole of .sup.32P-labeled target (samples 7 and 8 contained
0.2 pmole of each target molecule). The NaCl concentration of the
solutions was 0.75 M.
[0061] Hybridization was carried out at 35.degree. C. for 20
minutes at which time the samples were irradiated with a UV-A light
source for 60 minutes. One-fifth of the samples (0.010 mL) was
removed and mixed with an equal volume of formamide-bromophenol
blue dye mix and heated to 70.degree. C. for 3 minutes. The samples
were cooled on ice and loaded onto a 10% polyacrylamide gel (19:1
acrylamide/bisacrylamide) containing 7 M urea and electrophoresed
at 300 V until the bromophenol blue dye reached the bottom of the
gel. The gel was taken down and exposed to X-ray film overnight at
-80.degree. C.
[0062] The data obtained in the experiment showed that by using
either of the two cross-linker-modified probes, it was possible to
employ DSCP analysis to detect and distinguish the two
.beta.-globin alleles. The major cross-linked products obtained
from reaction between the fully complementary .beta..sup.A-probe
and .beta..sup.A-target (sample 3) and the .beta..sup.S-probe and
.beta..sub.S-target (sample 5) migrated through the gel
significantly faster than the products obtained after cross-linking
between the mismatched probes and targets (samples 4 and 6).
Furthermore, analysis of the reactions carried out in the presence
of both the .beta..sup.A- and .beta..sup.S-targets (samples 7 and
8), showed that the two probes were able to detect and distinguish
both alleles simultaneously. This finding is clinically relevant
since individuals who are carriers of sickle-cell anemia possess
both the .beta..sup.A- and .beta..sup.S-alleles in their DNA.
[0063] It is evident from the above results, that a simple, and
accurate technique is provided which can readily detect single base
mismatches. The methodology is convenient, the assay can be rapidly
carried out, and is not subject to error due to minor changes in
control of the conditions.
[0064] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
[0065] The invention now being fully described, it will be apparent
to one of ordinary skill in the art that many changes and
modifications can be made thereto without departing from the spirit
or scope of the appended claims.
* * * * *