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

Francesco S. Celi, Maimon M. Cohen, Styuanos E. Antonarakis, Efrat Wertheimer, Jesse Roth, Alan R. Shuldiner*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

49 Scopus citations


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. We 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 [32P]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 β-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. We also successfully used gd-PCR to detect allelic deletions by screening pertinent regions of the insulin receptor gene (IR; chromosome 19p13.2-p13.3) in three unrelated patients with genetic syndromes of extreme insulin resistance-known heterozygotes for deletions of either exon 3, exon 14, or exons 17-22. Gene dosage-PCR is a versatile, rapid, and sensitive method for screening for duplications and deletions of exons, genes, or chromosomes, with broad application in both clinical and research settings.

Original languageEnglish
Pages (from-to)304-310
Number of pages7
Issue number2
StatePublished - 15 May 1994
Externally publishedYes


FundersFunder number
Eunice Kennedy Shriver National Institute of Child Health and Human DevelopmentP01HD024605


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