PCR bias in amplification of androgen receptor alleles, a trinucleotide repeat marker used in clonality studies

ABSTRACT Trinucleotide CAG repeats in the X-linked human an drogen receptor gene (HUMARA) have proved a useful means of determining X chromosome haplotypes, and when combined with methylation analysis of nearby cytosine residues permits identification of non-random X inactivation in tumors of women....

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Bibliographic Details
Published in:Nucleic acids research 1995-04, Vol.23 (8), p.1411-1418
Main Authors: Multer, George L., Boynton, Kevin A.
Format: Article
Language:English
Subjects:
Alleles
androgen receptors
Base Composition
Base Sequence
Deoxyguanine Nucleotides - metabolism
DNA - genetics
DNA - radiation effects
Female
Genetic Markers
haplotypes
Humans
HUMARA gene
Male
man
Molecular Sequence Data
Nucleic Acid Conformation
Nucleic Acid Denaturation
polymerase chain reaction
Polymerase Chain Reaction - methods
Receptors, Androgen - genetics
repeated sequence
Repetitive Sequences, Nucleic Acid - genetics
Spermatozoa
testosterone receptors
Ultraviolet Rays
Uterus
X chromosome
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Summary:ABSTRACT Trinucleotide CAG repeats in the X-linked human an drogen receptor gene (HUMARA) have proved a useful means of determining X chromosome haplotypes, and when combined with methylation analysis of nearby cytosine residues permits identification of non-random X inactivation in tumors of women. Co-amplification of two alleles In a heterozygote generates PCR products which differ in the number of CAG units, and thus their melting and secondary structure characteristics. We have shown that under optimal conditions amplification efficiency of two HUMARA alleles is near-equivalent, generating PCR products Ina ratio proportional to that of the genomic template. In contrast, reduction of template quantity, damage of template by ultraviolet irradiation or addition of monovalent salts (sodium chloride, sodium acetate or ammonium acetate) produces highly variable imbalances of allelic PCR products, with a strong tendency to preferentially amplify lower molecular weight alleles. Variability and biasing was diminished by substitution of 7-deaza-2'-dGTP for dGTP during amplification, an intervention which reduces stability of intramolecular and inter molecular GC base pairing. We conclude that DNA which is scanty,damaged or salt contaminated may display amplification bias of GC-rich PCR targets, potentially confounding accurate interpretation or reproducibility of assays which require co-amplification of alleles.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/23.8.1411