(Download) "Accuracy of Genotyping of Single-Nucleotide Polymorphisms by PCR-ELISA Allele-Specific Oligonucleotide Hybridization Typing and by Amplification Refractory Mutation System (Technical Briefs)" by Clinical Chemistry " eBook PDF Kindle ePub Free
eBook details
- Title: Accuracy of Genotyping of Single-Nucleotide Polymorphisms by PCR-ELISA Allele-Specific Oligonucleotide Hybridization Typing and by Amplification Refractory Mutation System (Technical Briefs)
- Author : Clinical Chemistry
- Release Date : January 01, 1999
- Genre: Chemistry,Books,Science & Nature,
- Pages : * pages
- Size : 220 KB
Description
Early methods for the detection of single-nucleotide Polymorphisms (SNPs) were based on restriction fragment length polymorphism analysis (1) and the effects of base-pair changes on DNA fragment melting temperatures (denaturing gradient gel electrophoresis) (2). More recently, detection techniques have utilized allele-specific oligonucleotide (A50) hybridization (3, 4), the single-stranded conformational polymorphism method (5), allele-specific priming of PCR (6, 7), primer-guided nucleotide incorporation assays (8), and oligonucleotide ligation assays (9). The technique of differential hybridization to A50 probes has been widely used. Under high stringency conditions, synthetic DNA probes will only anneal to their complementary target sequences in the sample DNA if they are perfectly matched, with a single base-pair mismatch sufficient to prevent formation of a stable probe-target duplex (10,11). PCR-amplified genomic DNA products are applied to nylon filters as a series of "dot blots" to which radiolabeled ASOs are hybridized (4). The reverse approach can also be used with A50 probes fixed to a membrane support (12) or in a microtiter plate format (13). A combination of PCR amplification with allele-specific hybridization in a microtiter plate format using colorimetric ELISA-based detection was developed (PCR-ELISA A50 typing) that provided highly sensitive and quantitative detection; this method was initially applied to HLA typing (14,15). The PCR-ELISA A50 typing method amplifies the number of copies of a DNA segment from a sample of genomic DNA by PCR with the incorporation of digoxigenin-11-dUTP. Samples are analyzed in a microtiter plate format by alkaline denaturation and hybridization to biotinylated allele-specific capture probes bound to streptavidin-coated plates. The hybridized DNA is detected by ELISA, using anti-digoxigenin horseradish peroxidase conjugate and the colorimetric substrate 2,2'-azino-di-(3-ethylbenzthiazolinsulfonate) (14).
 "Accuracy of Genotyping of Single-Nucleotide Polymorphisms by PCR-ELISA Allele-Specific Oligonucleotide Hybridization Typing and by Amplification Refractory Mutation System (Technical Briefs)" by Clinical Chemistry " eBook PDF Kindle ePub Free){kind=link}