Compositions and methods for determining genotypes

Abstract

The present invention provides methods for determining the genotype of a selected gene present in at least two alleles in a sample. The methods involve amplifying DNA from the sample with a first pair of flanking primers that hybridize to nucleic acid sequences flanking a variant-specific gene sequence, the presence of which indicates the presence of a first gene variant, and the absence of which indicates the presence of a second gene variant. The DNA is also amplified with a third primer that specifically binds to the variant-specific sequence and together with one of the flanking primers forms a second pair of primers. Detection of one or more nucleic acid products of the amplification reaction is indicative of the genotype present in the sample.

1 . A method of determining the presence or absence of an insertion in a gene of interest in a sample obtained from a subject, comprising:
 amplifying DNA in a single amplification reaction from the sample with a first pair of flanking primers that hybridize to nucleic acid sequences flanking an insertion sequence, and a third primer that specifically binds to said insertion sequence and together with one of the flanking primers forms a second pair of primers; and   identifying a homozygous genotype lacking said insertion by detecting the production of one amplification product;   identifying a homozygous genotype having said insertion by detecting the production of two amplification products, and   identifying a heterozygous genotype by detecting the production of three amplification products.   
     
     
         2 . The method of  claim 1 , wherein said amplification reaction is by polymerase chain reaction. 
     
     
         3 . The method of  claim 1 , wherein the sample is a human sample. 
     
     
         4 . The method of  claim 1 , wherein the DNA is un-degraded DNA. 
     
     
         5 . The method of  claim 3 , wherein the sample is a tissue sample. 
     
     
         6 . The method of  claim 3 , wherein the sample is selected from the group consisting of blood, cultured cells, cells derived from amniotic fluid, and cells derived from chorionic villi. 
     
     
         7 . The method of  claim 3  wherein the sample is blood. 
     
     
         8 . The method of  claim 3 , wherein the DNA sample is from a source selected from the group consisting of: the endothelium of blood vessels, epithelial cells, blood mononuclear cells, macrophages, male germinal cells, and a biological fluid. 
     
     
         9 . The method of  claim 1 , wherein the gene of interest is selected from the group consisting of genes encoding cytochrome P450 enzymes, insulin receptors, neurofibromatosis type 1, plaktoglobin, and dipeptidyl carboxypeptidase-1. 
     
     
         10 . The method of  claim 1 , wherein said insertion is a single nucleotide polymorphism or a multiple nucleotide polymorphism. 
     
     
         11 . The method of  claim 1 , wherein said DNA is cDNA. 
     
     
         12 . The method of  claim 1 , wherein one of said first pair of flanking primers comprises a detectable label. 
     
     
         13 . The method of  claim 12 , wherein said detectable label is a fluorescent label. 
     
     
         14 . The method of  claim 1 , wherein said third primer comprises a detectable label. 
     
     
         15 . The method of  claim 14 , wherein said detectable label is a fluorescent label. 
     
     
         16 . The method of  claim 1 , wherein said sample is used to identify a subject at risk for a disease selected from the group consisting of essential hypertension, diabetic neuropathy, renal disease, congestive cardiomyopathies and myocardial infarction. 
     
     
         17 . The method of  claim 1 , wherein said sample is used to identify in said subject an altered responsiveness to pharmaceutical intervention. 
     
     
         18 . The method of  claim 17 , wherein said pharmaceutical intervention is selected from the group consisting of an ACE inhibitor and an angiotensin II type 1 receptor antagonist. 
     
     
         19 . The method of  claim 18 , wherein said ACE inhibitor is selected from the group consisting of benazepril, captopril, cilazapril, enalapril, enalaprilat, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril, and trandolapril. 
     
     
         20 . The method of  claim 18 , wherein said angiotensin II type 1 receptor antagonist is selected from the group consisting of irbesartan, losartan, valsartan, telmisartan, camdesartam, and eprosartan.