Full COLD-PCR Protocol for Noninvasive Prenatal Diagnosis of Genetic Diseases

Abstract

To the Editor: After the discovery of fetal DNA in maternal plasma, investigators reported different strategies for the noninvasive prenatal diagnosis of genetic diseases (1). Despite the advances in improving the analytical sensitivity of methods, distinguishing between fetal and maternal sequences remains very challenging, and the field of noninvasive prenatal diagnosis of genetic diseases has yet to attain a routine application in clinical diagnostics. An innovative strategy that has been reported is based on COLD-PCR (coamplification at a lower denaturation temperature PCR),1 which exploits melting temperature ( T m) differences between variant or mismatched sequences and wild-type sequences. This approach uses a critical denaturation temperature ( T c) lower than the T m to selectively amplify minority mutated alleles (2). We have developed assays for the identification of fetal paternally inherited mutations in maternal plasma. These assays use full COLD-PCR for the detection of IVSI.110 (G>A) and Cd39 (C>T) HBB (globin, beta) gene mutations that cause β-thalassemia. Full COLD-PCR is based on the generation of heteroduplexes between mutant and wild-type sequences. These heteroduplexes melt at lower temperatures than the wild-type homoduplexes. They are selectively denatured at the T c and then subsequently amplified. Given that both the fetal and maternal DNA content in maternal plasma has been demonstrated to vary from pregnancy to …