Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-06-26
2003-10-28
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S004000, C435S091100, C435S091200, C536S023100, C536S023500, C536S024300, C536S024330
Reexamination Certificate
active
06638719
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 genotypes.
BACKGROUND OF THE INVENTION
Obtaining genotype information on thousands of biallelic markers in a highly parallel fashion is increasingly becoming an important task in mapping disease loci, in indentifying quantitative trait loci, in diagnosing tumor loss of heterozygocity, and in performing association studies. A currently available method for simultaneously obtaining large numbers of biallelic marker genotypes 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 biallelic 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. Because of these limitations, there is a need in the art for more reliable and more quantitative methods to identify genotypes at biallelic markers.
SUMMARY OF THE INVENTION
It is an object of the invention to provide methods and compositions for analysis of variations in genomic DNA. These and other objects of the invention are provided by one or more of the embodiments described below.
One embodiment of the invention provides a method for determining the genotype of one or more individuals at a biallelic marker. The method comprises the step of amplifying a region of double stranded DNA comprising a biallelic marker to form an amplified DNA product using a first and a second pair of primers. The first pair of primers specifically amplifies a first allelic form of the biallelic marker and the second pair of primers specifically amplifies a second allelic form of the biallelic marker. Each pair of primers comprises an upstream and a downstream primer. Each upstream primer is complementary to a strand of the DNA which is opposite to a strand of the DNA to which the downstream primer is complementary. Each upstream primer is labeled with a color tag; the first upstream primer is labeled with a first color tag and the second upstream primer is labeled with a second color tag. The upstream primer of the first primer pair terminates in a 3′ nucleotide which is complementary to the first allelic form but not complementary to the second allelic form. The upstream primer of the second primer pair terminates in a 3′ nucleotide which is complementary to the second allelic form but not complementary to the first allelic form. The method further comprises the step of hybridizing the amplified DNA product to at least two probes which are immobilized to known locations on a solid support. A first probe is complementary to the first allelic form and a second probe is complementary to the second allelic form of the biallelic marker. A unique pattern of hybridization is formed on the solid support, which permits differentiation of and quantification of heterozygotes from homozygotes for the biallelic marker.
Another embodiment of the invention provides a set of primers for use in determining the genotype of an individual at a biallelic marker. The set of primers comprises a first pair of primers which specifically amplifies a first allelic form of the biallelic marker and a second pair of primers which specifically amplifies a second allelic form of the biallelic marker. Each pair of primers comprises an upstream and a downstream primer. Each upstream primer is complementary to a strand of the DNA which is opposite to a strand of the DNA to which the downstream primer is complementary. Each upstream primer is labeled with a color tag; the first upstream primer is labeled with a first color tag and the second upstream primer is labeled with a second color tag. The upstream primer of the first primer pair terminates in a 3′ nucleotide which is complementary to the first allelic form but not complementary to the second allelic form. The upstream primer of the second primer pair terminates in a 3′ nucleotide which is complementary to the second allelic form but not complementary to the first allelic form.
Still another embodiment of the invention provides a kit comprising in a single container two or more sets of primers as described in the preceding paragraph.
Yet another embodiment of the invention provides a kit comprising in a single container a set of primers as described above and a solid support comprising at least two probes which are immobilized to known locations on the solid support A first probe is complementary to a first allelic form and a second probe is complementary to a second allelic form of a biallelic marker.
The invention thus provides the art with methods and compositions for identification of genotypes in a DNA sample from one or more individuals.
REFERENCES:
patent: 5639611 (1997-06-01), Wallace et al.
patent: 5700637 (1997-12-01), Southern
patent: 5837832 (1998-11-01), Chee et al.
patent: 5981176 (1999-11-01), Wallace
patent: 6013449 (2000-01-01), Hacia et al.
patent: 6287778 (2001-09-01), Huang et al.
patent: 6368799 (2002-04-01), Chee
patent: 89/10977 (1989-11-01), None
Stratagen Catalog (1988).*
Tobe et al. “Single-well genotyping of diallelic sequence variations by a two-color ELISA-based eligonucleotide ligation assay” Nucleic Acids Research, 1996, vol. 24, No. 19.
Gunderson Kevin
Lockhart David J.
Affymetrix Inc.
Banner & Witcoff , Ltd.
Horlick Kenneth R.
Spiegler Alexander H.
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