Screening of small variants of F8 and F9 by conformation sensitive gel electrophoresis (CSGE)

MARCHIONE, VANINA DANIELA; RADIC, CLAUDIA PAMELA; ROSSETTI, LILIANA CARMEN

World Federation for Hemophilia Genetics Laboratory manual.

World Federation of Hemophilia

Año: 2022;

Conformation-sensitive gel electrophoresis (CSGE) is a non-radioactive method for screening of small variants (i.e., small insertions/deletions INDELs and single nucleotide variants SNVs) for large (multi-exonic). CSGE was developed to screen large and complex genes, such as the F8 or BRCA1, using a single electrophoretic condition taking advantage of heteroduplex analysis obtained by mixing PCR products of a sample and a control (1). CSGE is based on the differential migration of species of double strand DNA (dsDNA) with mismatched bases (heteroduplex DNA) as compared with species of uninterrupted dsDNA with perfect matching (homoduplex DNA) using polyacrylamide gel electrophoresis (PAGE) under mildly denaturing conditions (1). Colloidal silver staining allows a sensitive detection of samples with abnormal CSGE patterns as a result of heteroduplex DNA formation. The detection of SNV by CSGE is strongly dependent on the physical setting of the electrophoresis (the longer run, the better resolution) (e.g., 40 cm long and 0.8 mm thick CSGE associate with the highest resolution), the nature of the mismatch in the heteroduplex (purine/purine mismatches generate more anomalous CSGE pattern than pyrimidine/pyrimidine) and its relative location and the quality/quantity of the loaded PCR product (mismatches at 40 bp or less from the end of the PCR product are hardly detected). Heteroduplex DNA segments (i.e., F8/F9 PCR products) showing anomalous CSGE pattern have to be subjected to DNA Sanger sequencing to characterize the eventually altered sequence. Williams et al (1998) applied the method of CSGE to the analysis of F8 and detected hemophilia A (HA) causative variants with high efficiency proving that CSGE screening can be successfully used to detect SNV and INDEL in the F8 and F9 PCR amplification sets (2). After that, Rossetti et al (2007) improved the described F8 amplification set for CSGE screening (3) and Radic et al (2013) reported a PCR amplification set to analyze the F9 that was adjusted to detect small variants in hemophilia B (HB) by CSGE (4). Module 3 and 4 from this series present, respectively, F8 and F9 PCR amplification sets, both specifically designed for heteroduplex analysis by CSGE.