Rapid detection of expansions by PCR and non-radioactive hybridization: application for prenatal diagnosis of myotonic dystrophy

Abstract

The mutation specific for myotonic dystrophy (DM) is an unstable expanded CTG repeat located in the 3'-untranslated region of the myotonin protein kinase gene. Expansion of the CTG repeat shows a positive correlation with the severity of the disease and increases in successive generations of DM patients. Children with the congenital form of DM show the most severe phenotype and have large expansions, usually > 1000 repeats. For pregnant women with DM, prenatal diagnosis of DM may be offered. To reduce the time between chorionic villus sampling or amniocentesis and final results of DNA analysis in these cases, a fast and efficient method has been developed. This method combines direct PCR analyses for normal alleles with a nested PCR system followed by non-radioactive hybridization with a single-stranded probe.

Limited somatic mosaicism for Friedreich's ataxia GAA triplet repeat expansions identified by small pool PCR in blood leukocytes

“…For comparing repeat instability of expanded Friedreich's ataxia (FRDA) and myotonic dystrophy (MDY) alleles, PCR analysis were performed with 50 ng DNA. Products were separated on 1.0% agarose gels, transfered to Nylon membranes and hybridized with a DIG‐labelled (GAA) 9 or (CAG) 65 probe (13) using the DIG Luminescent Detection Kit (Roche).…”

“…To enhance the resolution of expanded alleles, we combined small pool PCR analyses (12) with Southern hybridization. An altered procedure, that is very efficient for myotonic dystrophy (13), was introduced in routine FRDA diagnosis to prevent uncertainty in analyses of heterozygous samples.…”

Southern Analysis after Long-range PCR: Clinical Application in Korean Patients with Myotonic Dystrophy 1

“…Especially for the prenatal genetic diagnosis, the reliability and speed is critical. 10) Currently, several molecular diagnostic methods are available for the genetic diagnosis of DM1; however, the accurate determination of expansion size using a single method is frequently problematic. Therefore, combined testing, including both PCR and Southern blot, has been recommended for routine genetic testing.…”