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Home / Equine Research Bank / Nucleic Acids Res / Genetic Bit Analysis: a solid phase method for typing single...
Nucleic acids research1994; 22(20); 4167-4175; doi: 10.1093/nar/22.20.4167

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.
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Publication Date: 1994-10-11 PubMed ID: 7937143PubMed Central: PMC331910DOI: 10.1093/nar/22.20.4167Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research article presents a novel method, Genetic Bit Analysis (GBA), for typing single nucleotide polymorphisms (SNPs) in DNA. Specific fragments of genomic DNA are amplified, rendered single-stranded, captured, and sensed to determine the nature of the resolved nucleotides. Applying this technique, the researchers successfully verified the parentage for thoroughbred horses.

Methodology and Techniques

  • Genomic DNA containing the polymorphic sites was amplified using the Polymerase chain reaction (PCR). PCR is a standard molecular biology technique used to make multiple copies of a particular DNA segment from a less quantity. Special primers including a phosphorothioate-modified primer were used. A primer is a short DNA or RNA segment that attaches to the target DNA to start the replication process.
  • The molecular technique was implemented to transform the double-stranded amplified DNA into single strands. The T7 gene 6 exonuclease enzyme was used, which selectively digests double-stranded DNA starting from the ends followed by capturing on a plate.
  • The capture onto the wells of a plate was done using immobilized oligonucleotide primer. Oligonucleotide primers are small pieces of single-stranded DNA that are designed to bind to the start of the section of DNA to be amplified or studied.
  • The extension of the oligonucleotide is carried out using a mixture of labeled and unlabeled dideoxynucleoside triphosphates and either the Klenow fragment of DNA polymerase I or modified T7 DNA polymerase known as Sequenase. Dideoxynucleoside triphosphate is a molecule similar to those used to construct DNA but, when incorporated into a growing DNA chain, it causes chain termination.
  • To determine the nature of the extended base, ELISA (enzyme-linked immunosorbent assay), a method used to detect the presence of antibodies in a liquid sample, was implemented using enzymes such as alkaline phosphatase and horseradish peroxidase. These enzymes were conjugated to the antibodies for detection purposes.

Applications of Genetic Bit Analysis (GBA)

  • This research utilized the developed GBA method for the task of parentage verification of thoroughbred horses. The process was semi-automated and employed a prior set of 26 diallelic polymorphisms from the equine genome.
  • Diallelic polymorphisms represent the simplest form of polymorphisms where there are only two possible alleles, which refer to alternative forms of a gene that arise due to mutation.
  • By using the GBA method, the researchers could accurately match offspring to parents based on their genetic information, which is crucial in the practice of breeding horses.
  • The successful implementation of GBA in this context implies its potential application in other areas such as human genetic diseases studies, population genetics, and forensic analysis where detecting single nucleotide polymorphisms is critical.

Cite This Article

APA
Nikiforov TT, Rendle RB, Goelet P, Rogers YH, Kotewicz ML, Anderson S, Trainor GL, Knapp MR. (1994). Genetic Bit Analysis: a solid phase method for typing single nucleotide polymorphisms. Nucleic Acids Res, 22(20), 4167-4175. https://doi.org/10.1093/nar/22.20.4167

Publication

Nucleic acids research
ISSN: 0305-1048
NlmUniqueID: 0411011
Country: England
Language: English
Volume: 22
Issue: 20
Pages: 4167-4175

Researcher Affiliations

Nikiforov, T T
  • Molecular Tool, Inc., Alpha Center, Hopkins Bayview Research Campus, Baltimore, MD 21224.
Rendle, R B
    Goelet, P
      Rogers, Y H
        Kotewicz, M L
          Anderson, S
            Trainor, G L
              Knapp, M R

                MeSH Terms

                • Autoanalysis
                • Base Sequence
                • Biotin
                • Colorimetry
                • DNA / analysis
                • DNA / chemistry
                • DNA Polymerase I / metabolism
                • DNA Primers
                • DNA, Single-Stranded
                • DNA-Directed DNA Polymerase / metabolism
                • Enzyme-Linked Immunosorbent Assay
                • Escherichia coli / enzymology
                • Exodeoxyribonucleases / metabolism
                • Fluorescein
                • Fluoresceins
                • Fluorescent Dyes
                • Indicators and Reagents
                • Molecular Sequence Data
                • Nucleic Acid Hybridization
                • Polymerase Chain Reaction
                • Polymorphism, Genetic
                • Templates, Genetic

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