Genetic Bit Analysis: a solid phase method for typing single nucleotide polymorphisms

Abstract

A new method for typing single nucleotide polymorphisms in DNA is described. In this method, specific fragments of genomic DNA containing the polymorphic site(s) are first amplified by the polymerase chain reaction (PCR) using one regular and one phosphorothioate-modified primer. The double-stranded PCR product is rendered single-stranded by treatment with the enzyme T7 gene 6 exonuclease, and captured onto individual wells of a 96 well polystyrene plate by hybridization to an immobilized oligonucleotide primer. This primer is designed to hybridize to the single-stranded target DNA immediately adjacent from the polymorphic site of interest. Using the Klenow fragment of E. coli DNA polymerase I or the modified T7 DNA polymerase (Sequenase), the 3' end of the capture oligonucleotide is extended by one base using a mixture of one biotin-labeled, one fluorescein-labeled, and two unlabeled dideoxynucleoside triphosphates. Antibody conjugates of alkaline phosphatase and horseradish peroxidase are then used to determine the nature of the extended base in an ELISA format. This paper describes biochemical features of this method in detail. A semi-automated version of the method, which we call Genetic Bit Analysis (GBA), is being used on a large scale for the parentage verification of thoroughbred horses using a predetermined set of 26 diallelic polymorphisms in the equine genome.

Gold nanoparticles assisted characterization of amine functionalized polystyrene multiwell plate and glass slide surfaces

“…APTES functionalized PS plates and GS are used in the immobilization of antibodies for the detection of antigens. Similarly, oligonucleotide probes/DNA were used to detect target DNA sequences and single nucleotide polymorphisms (SNP) typing and assaying in the protein kinases activity (Dixit et al 2010;Qin et al 2007;Pack et al 2007;Hirayama et al 1996;Nikiforov et al 1994;Kim et al 2008).…”

Using high-throughput SNP technologies to study cancer

“…The SNPStream genotyping assay is based on the technique of single-base extension (SBE) (Nikiforov et al, 1994;Bell et al, 2002). Briefly, for each polymorphic site amplified by multiplex PCR, primers containing a unique 5 0 tag sequence are hybridized one base pair upstream of the SNP.…”

Single nucleotide polymorphism genotyping: biochemistry, protocol, cost and throughput

“…Most solid-phase-mediated methods require a clean-up because the substrates of the allele-specific reaction can produce false-positive signals and complicate genotype scoring. Examples are mass spectrometry, 12,[16][17][18][19][20][21] zip-code technology, 22,23 Illumina color beads, 24,25 Orchid SNP-IT technology 11 and all hybridization based methods. 26,27 Product detection and identification can be performed in many different ways.…”

“…To facilitate detection the terminators can have specific labels, of which fluorophores are the most popular. 6,[34][35][36][37] By detecting the fluorescence labels or other moieties 11 linked to the terminators, the genotype of a sample can be inferred. The major difference between SBE and DNA sequencing is that SBE identifies only one base (the polymorphic base) while sequencing can identify up to several hundreds bases and determine their relative order.…”