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Title: Determination of gene dosage by a quantitative adaptation of the polymerase chain reaction (gd-PCR): Rapid detection of deletions and duplications of gene sequences

Abstract

Screening methods based on the polymerase chain reaction (PCR), such as denaturing gradient gel electrophoresis, single-stranded conformational polymorphism, and heteroduplex analysis, are powerful tools for the detection of point mutations as well as small deletions and insertions, but are unable to detect heterozygous deletions or duplications of exons, genes, or chromosomes. The authors now report a PCR-based approach, designated gene dosage-PCR (gd-PCR), that allows rapid screening for heterozygous deletions and duplications of genes or exons. Gene dosage-PCR is a quantitative method in which two in vitro-synthesized DNA internal standards are coamplified with the genomic DNA sample, one corresponding to the gene of interest (test sequence) and the other to a reference (disomic) gene (reference sequence). Both internal standards are designed to be amplified with the same primer pairs and with efficiencies similar to those of their genomic DNA counterparts, yielding PCR products slightly smaller than those derived from genomic DNA. Amplification of approximately equimolar amounts of the two internal standards and genomic DNA, in the presence of [[sup 32]P]dCTP, results in four radiolabeled PCR products; after electrophoresis and quantification of the products, gene dosage is easily calculated. For validation, genomic DNA from 56 subjects, 28 with cytogenetically documented Down syndromemore » (trisomy 21) and 28 controls that were disomic for chromosome 21, was assayed. Using the [beta]-amyloid precursor protein gene (APP: Chromosome 21q21) as the test sequence, control subjects had an adjusted mean gene dose of 2.00 [+-] 0.29, while subjects with Down syndrome had a mean gene dose of 3.05 [+-] 0.27. There was a clear separation of all of the samples between the two groups. The authors also successfully used gd-PCR to detect allelic deletions by screening pertinent regions of the insulin receptor gene. 48 refs., 5 figs., 2 tabs.« less

Authors:
; ;  [1];  [2];  [3];  [4]
  1. National Institute of Health, Bethesda, MD (United States) Johns Hopkins Univ. School of Medicine, Baltimore, MD (United States)
  2. Univ. of Maryland School of Medicine, Baltimore, MD (United States)
  3. Johns Hopkins Univ. School of Medicine, Baltimore, MD (United States)
  4. National Institute of Health, Bethesda, MD (United States)
Publication Date:
OSTI Identifier:
6872537
Resource Type:
Journal Article
Journal Name:
Genomics; (United States)
Additional Journal Information:
Journal Volume: 21:2; Journal ID: ISSN 0888-7543
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; GENE AMPLIFICATION; DETECTION; GENE MUTATIONS; HEREDITARY DISEASES; POLYMERASE CHAIN REACTION; DISEASES; MUTATIONS; 550400* - Genetics

Citation Formats

Celi, F S, Roth, J, Schuldiner, A R, Cohen, M M, Antonarakis, S E, and Wertheimer, E. Determination of gene dosage by a quantitative adaptation of the polymerase chain reaction (gd-PCR): Rapid detection of deletions and duplications of gene sequences. United States: N. p., 1994. Web. doi:10.1006/geno.1994.1270.
Celi, F S, Roth, J, Schuldiner, A R, Cohen, M M, Antonarakis, S E, & Wertheimer, E. Determination of gene dosage by a quantitative adaptation of the polymerase chain reaction (gd-PCR): Rapid detection of deletions and duplications of gene sequences. United States. https://doi.org/10.1006/geno.1994.1270
Celi, F S, Roth, J, Schuldiner, A R, Cohen, M M, Antonarakis, S E, and Wertheimer, E. 1994. "Determination of gene dosage by a quantitative adaptation of the polymerase chain reaction (gd-PCR): Rapid detection of deletions and duplications of gene sequences". United States. https://doi.org/10.1006/geno.1994.1270.
@article{osti_6872537,
title = {Determination of gene dosage by a quantitative adaptation of the polymerase chain reaction (gd-PCR): Rapid detection of deletions and duplications of gene sequences},
author = {Celi, F S and Roth, J and Schuldiner, A R and Cohen, M M and Antonarakis, S E and Wertheimer, E},
abstractNote = {Screening methods based on the polymerase chain reaction (PCR), such as denaturing gradient gel electrophoresis, single-stranded conformational polymorphism, and heteroduplex analysis, are powerful tools for the detection of point mutations as well as small deletions and insertions, but are unable to detect heterozygous deletions or duplications of exons, genes, or chromosomes. The authors now report a PCR-based approach, designated gene dosage-PCR (gd-PCR), that allows rapid screening for heterozygous deletions and duplications of genes or exons. Gene dosage-PCR is a quantitative method in which two in vitro-synthesized DNA internal standards are coamplified with the genomic DNA sample, one corresponding to the gene of interest (test sequence) and the other to a reference (disomic) gene (reference sequence). Both internal standards are designed to be amplified with the same primer pairs and with efficiencies similar to those of their genomic DNA counterparts, yielding PCR products slightly smaller than those derived from genomic DNA. Amplification of approximately equimolar amounts of the two internal standards and genomic DNA, in the presence of [[sup 32]P]dCTP, results in four radiolabeled PCR products; after electrophoresis and quantification of the products, gene dosage is easily calculated. For validation, genomic DNA from 56 subjects, 28 with cytogenetically documented Down syndrome (trisomy 21) and 28 controls that were disomic for chromosome 21, was assayed. Using the [beta]-amyloid precursor protein gene (APP: Chromosome 21q21) as the test sequence, control subjects had an adjusted mean gene dose of 2.00 [+-] 0.29, while subjects with Down syndrome had a mean gene dose of 3.05 [+-] 0.27. There was a clear separation of all of the samples between the two groups. The authors also successfully used gd-PCR to detect allelic deletions by screening pertinent regions of the insulin receptor gene. 48 refs., 5 figs., 2 tabs.},
doi = {10.1006/geno.1994.1270},
url = {https://www.osti.gov/biblio/6872537}, journal = {Genomics; (United States)},
issn = {0888-7543},
number = ,
volume = 21:2,
place = {United States},
year = {1994},
month = {5}
}