Submitted to: Plant and Animal Genome Conference Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: December 20, 2002
Publication Date: N/A
Single base extension (SBE) is a commonly used method of single nucleotide polymorphism (SNP) detection. Here we describe a rapid, high throughput and relatively inexpensive system for the analysis of single base extension products using flow cytometry. SNP-containing fragments are amplified from genomic DNA using multiplex PCR. Sets of 15-20 PCR products are treated with shrimp alkaline phosphatase and exonuclease. The SNP-containing fragments are then queried at the polymorphic base by polymerase extension with a biotin-labeled dideoxynucleotide of a SBE oligonucleotide specific to the sequence directly 3' of the SNP. The 5' end of the SBE oligo contains a 21 base 'ZipCode' sequence to allow capture and demultiplexing of the reaction products using fluorescent polystyrene microspheres to which the complementary 21 base 'anti-ZipCode' sequences are attached. A streptavidin-phycoerythrin conjugate is bound to the biotinylated extension products. Up to 100 fluorescently labeled microspheres with their corresponding extension products are analyzed simultaneously in each well of a 96-well microtiter plate using a flow cytometer. One laser of the flow cytometer excites the two fluorophores of the microsphere to identify the single base extension product (locus) while a second laser excites the phycoerythrin to allow detection of the presence or absence of a labeled extension product. To date, 171 SBE primers have been designed using ArrayDesigner2 software. Of these, 39 (23%) failed to unambiguously distinguish genotypes carrying alternative SNP alleles. After reselection of these 39 SBE primers only 12 (7% of the original 171 loci) did not provide unambiguous allele discrimination.