Enhanced discrimination of single nucleotide polymorphism in genotyping by phosphorothioate proofreading allele-specific amplification

2007 ◽  
Vol 369 (1) ◽  
pp. 54-59 ◽  
Author(s):  
Ya Jun Hu ◽  
Zong Fang Li ◽  
Alan M. Diamond
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Allele Specific ◽  
Specific Amplification

scholarly journals Homogeneous Assays for Single-Nucleotide Polymorphism Typing Using AlphaScreen

2001 ◽  
Vol 11 (4) ◽  
pp. 600-608
Author(s):  
Lucille Beaudet ◽  
Julie Bédard ◽  
Billy Breton ◽  
Roberto J. Mercuri ◽  
Marcia L. Budarf
Keyword(s):  
Single Nucleotide Polymorphism ◽  
High Throughput ◽  
Genomic Dna ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Specific Hybridization ◽  
Donor And Acceptor ◽  
Close Proximity ◽  
Allele Specific ◽  
Specific Amplification

AlphaScreen technology allows the development of high-throughput homogeneous proximity assays. In these assays, signal is generated when 680 nm laser light irradiates a donor bead in close proximity to an acceptor bead. For the detection of nucleic acids, donor and acceptor beads are brought into proximity by two bridging probes that hybridize simultaneously to a common target and to the generic oligonucleotides attached covalently to the beads. This method allows the detection of as little as 10 amole of a single-stranded DNA target. The combination of AlphaScreen with allele-specific amplification (ASA) and allele-specific hybridization (ASH) has allowed the development of two homogenous single-nucleotide polymorphism (SNP) genotyping platforms. Both types of assay are very robust, routinely giving accurate genotyping results with < 2 ng of genomic DNA per genotype. An AlphaScreen validation study was performed for 12 SNPs by using ASA assays and seven SNPs by using ASH assays. More than 580 samples were genotyped with accuracy >99%. The two assays are remarkably simple, requiring no post-PCR manipulations. Genotyping has been performed successfully in 96- and 384-well formats with volumes as small as 2 μL, allowing a considerable reduction in the amount of reagents and genomic DNA necessary for genotyping. These results show that the AlphaScreen technology can be successfully adapted to high-throughput genotyping.

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