Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-10-19
2001-09-11
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S005000, C435S091100, C435S091200, C435S069700, C536S026100, C536S026120, C536S023100, C536S024330
Reexamination Certificate
active
06287778
ABSTRACT:
FIELD OF THE INVENTION
The invention is related to the area of genome analysis. In particular it is related to the field of identification of bases at particular locations in a nucleic acid molecule.
BACKGROUND OF THE INVENTION
Obtaining genotype information on thousands of polymorphisms in a highly parallel fashion is becoming an increasingly important task in mapping disease loci, in identifying quantitative trait loci, in diagnosing tumor loss of heterozygosity, and in performing association studies. A currently available method for simultaneously evaluating large numbers of genetic polymorphisms involves hybridization to allele-specific probes on high density oligonucleotide arrays. In order to practice that method, redundant sets of hybridization probes, typically twenty or more, are used to score each allelic marker. A high degree of redundancy is required to reduce noise and achieve an acceptable level of accuracy. Even this level of redundancy is insufficient to unambiguously score heterozygotes or to quantitatively determine allele frequency in a population.
The technique of allele-specific polymerase chain reaction (ASPCR) can be applied to allele identification and quantitative analysis of allele frequency. However, this technique suffers from cross reactivity between amplified products when hybridizing to probes which differ by only a single nucleotide base. A partial solution to the cross-reactivity problem has been achieved by the addition of sequence tags to the ASPCR primers. The incorporation of tags in ASPCR primers can itself interfere with the identification of the amplification products because unreacted primers or partially extended products can compete with full products for hybridization to the probes. Thus, there is a further need in the art for methods and materials which permit the accurate determination of polymorphic loci without interference from incompletely reacted products.
SUMMARY OF THE INVENTION
It is an object of the invention to provide methods and compositions for the identification of nucleotides at a polymorphic locus in a nucleic acid sequence. This and other objects of the invention are provided by one or more of the embodiments described below.
In one embodiment of the invention, a method is provided to aid in detecting a selected allele of a gene in a sample. A region of single or double stranded DNA in the sample is amplified using one or a pair of amplification primers to form an amplified DNA product. The region comprises a polymorphic locus of the selected allele of the gene. An extension primer is labeled in the presence of the amplified DNA product, which serves as the template for the labeling reaction. The extension primer comprises a 3′ portion which is complementary to the amplified DNA product and a 5′ portion which is not complementary to the amplified DNA product. The extension primer also terminates in a 3′ nucleotide at the polymorphic locus of the selected allele. At least one labeled nucleotide is coupled to the 3′ terminal nucleotide of the extension primer to form a labeled extension primer. The labeled extension primer is hybridized to a probe on a solid support. All or a portion of the probe is complementary to the 5′ portion of the extension primer.
Another embodiment of the invention provides another method to aid in detecting a selected allele of a gene in a sample. A region of single or double stranded DNA in the sample is specifically amplified using one or a pair of amplification primers to form an amplified DNA product. The region comprises a polymorphic locus of the selected allele of the gene. An amplification primer terminates in a 3′ nucleotide at the polymorphic locus of the selected allele. An extension primer is labeled in the presence of the amplified DNA product, which serves as the template for the labeling reaction. The extension primer comprises a 3′ portion which is complementary to the amplified DNA product and a 5′ portion which is not complementary to the amplified DNA product. The extension primer also terminates in a 3′ nucleotide at the polymorphic locus of the selected allele. At least one labeled nucleotide is coupled to the 3′ terminal nucleotide of the extension primer to form a labeled extension primer. The labeled extension primer is hybridized to a probe on a solid support. All or a portion of the probe is complementary to the 5′ portion of the extension primer.
Yet another embodiment of the invention is a kit which comprises in a single container a set of primers for use in detecting a selected allele of a gene. The set of primers includes a pair of primers which amplify a region of the gene comprising a polymorphic locus and an extension primer which terminates in a 3′ nucleotide which is the polymorphic locus of the selected allele. A 3′ portion of the extension primer is complementary to the selected allele, and a 5′ portion of the extension primer is complementary to all or a portion of a probe on a solid support but not complementary to the amplified region of the gene.
Still another embodiment of the invention is a kit which comprises in a single container a set of primers for use in detecting an allele. The set of primers includes a pair of primers which specifically amplify a selected allele and an extension primer. The pair of primers comprises a first and a second primer. The first and second primers are complementary to opposite strands of a DNA target. The first primer and the extension primer each terminate in a 3′ nucleotide which is a polymorphic locus of the selected allele. A 3′ portion of the extension primer is complementary to the selected allele, and a 5′ portion of the extension primer is complementary to all or a portion of a probe on a solid support but not complementary to the amplified region of the DNA target.
Still another embodiment of the invention provides another method to aid in detecting a selected allele of a gene in a sample. A region of single or double stranded DNA in the sample comprises a polymorphic locus of the selected allele of the gene. An extension primer is labeled in the presence of the region of DNA which serves as the template for the labeling reaction. The extension primer comprises a 3′ portion which is complementary to the region of DNA and a 5′ portion which is not complementary to the region of DNA. The extension primer also terminates in a 3′ nucleotide at the polymorphic locus of the selected allele. At least one labeled nucleotide is coupled to the 3′ terminal nucleotide of the extension primer to form a labeled extension primer. The labeled extension primer is hybridized to a probe on a solid support. All or a portion of the probe is complementary to the 5′ portion of the extension primer.
The invention thus provides the art with sensitive and specific methods and compositions for identification of polymorphic nucleotides in a DNA sample which may be from one or more individuals.
REFERENCES:
patent: 5455169 (1995-10-01), Mullan
patent: 5700637 (1997-12-01), Southern
patent: 6004744 (1999-12-01), Goelet et al.
patent: 6013439 (2000-01-01), Lishanski et al.
patent: 89/10977 (1989-11-01), None
Huang Xiaohua
Kaplan Paul
Ryder Tom
Affymetrix Inc.
Banner & Witcoff , Ltd.
Jones W. Gary
Taylor Janell E.
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