Quality Control Measures for Genotyping Polymorphic Genes

Reference #:

2005-088

Inventors/Contributors

Dennis J. O'Kane Ph.D.

Description

Polymorphic genes pose problems for genotyping measurements when PCR amplification is involved. Mutations under PCR primers may lead to allelic drop-out; mutations under probes may produce allelic drop-outs in detection. Quality control measures to diminish the frequency of allelic drop-out and genotyping miscalls have been developed. This includes globally the use of two sets of independent primers for each amplified portion of a gene. The primer sequences are then tested in the detection phase for presence indicating absence of drop-outs. The primers can be tenadem contiguous sequences, can be overlapping, or can be separated. Detection can be by size differences of the amplified DNA, or by probing for the presence of the primer sequences using solid phase or suspension microarrays. For highly polymorphic genes such as CYP2D6, additional quality control measures include subdividing the gene into different amplicons and confirming the concordance of haplotypes between the subparts of the amplified DNA; utilizing specific synonymous coding polymorphisms for checking the quality of the amplification, such as 1661G>C and 4180G>C to detect allelic drop-outs or to assist in determining the number of copies of alleles present.

Patent Status

None