Principles of QF-PCR

Fluorescently labeled marker specific primers are used for PCR amplification of individual markers and the copy number of each marker is indicative of the copy number of the chromosome. The resulting PCR products may be analyzed and quantified using an automated genetic analyzer.

The genetic markers/STRs may vary in length between individual chromosomes and subjects, depending on the number of repeated STRs. The relative copy number of each allele is determined by calculating the ratio of the peak areas or peak heights detected for each marker.

A normal diploid sample has the contribution of two of each of the investigated chromosomes. Two alleles of a chromosome specific marker are detected as two peaks in a 1:1 ratio when the marker is heterozygous and as one peak when the marker is homozygous (have alleles of same length). The detection of an additional allele as three peaks in a 1:1:1 ratio or as two peaks in a 2:1/1:2 ratio indicates the presence of an additional marker copy possibly corresponding to an additional chromosome, as in the case of trisomy. Subjects who are homozygous or monosomic for a specific marker will display only one peak.

The inability of STR marker analysis to distinguish subjects who are homozygous or monosomic is a major shortcoming when testing for sex chromosome abnormalities. When STRs specific for chromosome X are used, some samples from normal XX females may show homozygous QF PCR patterns, indistinguishable from those produced by samples with a single X, as in Turner syndrome. Incorporating additional X-chromosome STR markers into the analysis will reduce but not eliminate the likelihood of homozygosity. To facilitate the detection of monosomy X the Devyser QF PCR kits include X-chromosome counting markers for relative quantification of chromosome X to an autosomal chromosome.