Double-stranded conformational polymorphism analysis

Details Double-stranded conformational polymorphism analysis Double-stranded conformational polymorphism analysis

1996-11-01

2001-02-13

Myers, Carla J. (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200, C435S810000, C536S024300

Reexamination Certificate

active

06187532

ABSTRACT:

TECHNICAL FIELD
The field of this invention is detecting mutations in DNA.
BACKGROUND
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, &bgr;-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.
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.
Numerous techniques have been developed to identify differences between a known and target sequence.
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.
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.
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.
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.
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
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
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
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.
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.
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.
Nucleic acids of the desired length can be achieved, particularly with DNA, by restriction, use of PCR and

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