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Real-time polymerase chain reaction-based genotyping assay for single nucleotide polymorphism

a polymorphism and genotyping technology, applied in biochemistry, sugar derivatives, organic chemistry, etc., can solve the problems of allelic discrimination and insufficient single base pair difference at the 3′ end of the primer, and achieve the effect of preventing the amplification of non-matching primers

Inactive Publication Date: 2006-06-22
UNIV OF TENNESSEE RES FOUND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0012] Using PCR growth curves, the assay disclosed herein accurately determined hetero- and homozygosity for C3435T and G2677T. Genotype assignments based on PCR growth curve, melt-curve analysis, agarose gel electrophoresis, and direct DNA sequencing results of PCR products were in perfect agreement. Thus, the present invention provides a rapid MDR1 genotyping method that can be used to assess the contribution of MDR1*2 to pharmacokinetic and pharmacodynamic variability of P-gp substrates.

Problems solved by technology

However, a single base pair difference at the 3′ end of the primer is insufficient, in most cases, to achieve allelic discrimination.

Method used

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  • Real-time polymerase chain reaction-based genotyping assay for single nucleotide polymorphism
  • Real-time polymerase chain reaction-based genotyping assay for single nucleotide polymorphism
  • Real-time polymerase chain reaction-based genotyping assay for single nucleotide polymorphism

Examples

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example 1

MDR1 Single Nucleotide Polymorphisms Genotyping

[0029] The present example describes a real-time PCR assays for the rapid detection of the MDR1 single nucleotide polymorphisms C3435T and G2677T. These methods can be readily applied to investigate the effect of MDR1 polymorphic expression on pharmacokinetic and pharmacodynamic variability of P-gp substrates.

example 2

Primer Design

[0030] PCR primers are listed in Table 1. Oligonucleotide primers were designed based on the published MDR1 sequence (Genbank #AC005068) using the online program Primer3. Hairpin structures and primer-dimers were predicted with Oligo Toolkit™. The primers were synthesized by Integrated DNA Technologies (Coralville, Iowa). Expected amplicon lengths were 216 base pairs (bp) and 134 bp for G2677T and C3435T, respectively. Discrimination between wild type and mutant alleles was achieved using PCR amplification of specific alleles modified to prevent non-Watson Crick base pairing (Okimoto & Dodgson, 1996; Sommer et al., 1992; Bottema et al., 1993; Newton et al., 1989). Briefly, the first nucleotide difference (C or T) between sense primers (3435W and 3435M) used to discriminate between wild type and mutant 3435 alleles is located at the 3′ terminal base. The second primer base change (A to G) located 3 bases from the 3′ end generates an internal primer / template mismatch, a...

example 3

Real-Time PCR Amplification

[0031] The Smart Cycler (Cepheid, Sunnyvale, Calif.) was used to monitor PCR amplification using SYBR™ Green I (Molecular Probes, Eugene, Oreg.), a nonspecific double-stranded DNA intercalating fluorescent dye. Thus, to achieve allelic discrimination between wild type and mutant alleles, two physically separate PCR reactions containing either wild type or mutant-specific primers were performed. All reactions were carried out in a total volume of 25 μL. Reaction conditions were identical for G2677T and C3435T except where noted. Each reaction mixture contained a 1:12,500 dilution of SYBR™ Green I nucleic acid gel stain 10,000× in dimethyl sulfoxide (DMSO) (Molecular Probes); 0.2 mM of dATP, dCTP, dGTP, and dTTP; 200 nM of both forward and reverse primers; 1.0 U of Taq DNA polymerase (Promega, Madison, Wis.); 6% DMSO; and 20 to 120 ng of genomic DNA in 1×PCR buffer (pH 8.3, 10× solution containing 100 mM Tris-HCl, 500 mM KCl, 15 mM MgCl2 and 0.01% gelatin)...

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Abstract

The present invention provides fluorescence-based real-time PCR assays for the rapid detection of single nucleotide polymorphisms (SNPs). The genotyping assay can be used to detect SNPs of a number of genes of interest that include, but are not limited to, the human multidrug resistance gene (MDR1) single nucleotide polymorphisms C3435T and G2677T, and cytochrome P-450 3A5 single nucleotide polymorphisms CYP3A5*3 (A22893G) and CYP3A5*6 (G30597A).

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This is a divisional application of non-provisional application U.S. Ser. No. 10 / 809,757, filed Mar. 25, 2004 which claims benefit of provisional U.S. Ser. No. 60 / 457,512, filed Mar. 25, 2003, now abandoned.BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] The present invention relates generally to the field of single nucleotide polymorphism genotyping. More specifically, the present invention provides a real-time polymerase chain reaction-based genotyping assay for the detection of single nucleotide polymorphisms. [0004] 2. Description of the Related Art [0005] P-glycoprotein (P-gp), a member of the large adenosine triphosphate-binding (ATP-binding) cassette superfamily of transport proteins also called traffic ATPases, is the product of the human multidrug resistance gene (MDR1). P-glycoprotein is highly expressed on the apical (luminal) surface of organs that have excretory functions, such as the bile canalicular me...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68C07H21/04C12P19/34C12Q
CPCC12Q1/6883C12Q2600/156
Inventor YATES, CHARLESMILLER, DUANEGOURLEY, DICKSONG, PENGFEI
Owner UNIV OF TENNESSEE RES FOUND
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