Genetic Bit Analysis: a solid phase method for typing single nucleotide polymorphisms (original) (raw)

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Center for Advanced Biotechnology and Medicine

679 Hoes Lane, Piscataway, NJ 08854

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Chemical and Physical Sciences, Research and Development, Du Pont Merck Pharmaceutical Company

PO Box 80353, Wilmington, DE 19880, USA

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Revision received:

07 September 1994

Accepted:

07 September 1994

Published:

11 October 1994

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Theo T. Nikiforov, Robert B. Rendie, Philip Goelet, Yu-Hui Rogers, Michael L. Kotewicz, Stephen Anderson, George L. Trainor, Michael R. Knapp, Genetic Bit Analysis: a solid phase method for typing single nucleotide polymorphisms, Nucleic Acids Research, Volume 22, Issue 20, 11 October 1994, Pages 4167–4175, https://doi.org/10.1093/nar/22.20.4167
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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 doublestranded 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-la 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.

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