A real-time polymerase chain reaction assay for quantification of allele ratios and correction of amplification bias.
Allele-specific epigenetic modifications are crucial for several important biological functions, including genomic imprinting and X-inactivation in mammals. Consequently, an ever increasing number of investigations requires accurate quantification of the relative abundance of parental alleles of a specific sequence in a DNA sample. Here, combining the use of polymorphic restriction sites with real-time polymerase chain reaction (PCR) amplification, we describe a simple and quantitative assay to measure allele ratios. The efficiency of the assay was assessed on genomic DNA for several polymorphic restriction sites located in the mouse Igf2/H19 imprinted locus. The assay was also successfully applied to quantify allele ratio in cDNA samples. In addition, we provide an experimental procedure for detection and correction of potential PCR amplification bias which significantly extends the range of application of the assay.
Weber
Michaël
M
Institut de Génétique Moléculaire, UMR 5535 CNRS-Université Montpellier 2, IFR 24, 34293 Montpellier Cedex 5, France.
Hagège
Hélène
H
Lutfalla
Georges
G
Dandolo
Luisa
L
Brunel
Claude
C
Cathala
Guy
G
Forné
Thierry
T
D003627:Data Interpretation, Statistical; D018628:Gene Dosage; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA