Multiplex polymerase chain reaction (PCR) is a variant of PCR in which two or more loci are simultaneously amplified in the same reaction. Two examples of XRCC1 and XRCC3 genotyping by multiplex PCR-RFLP are provided below.

What is XRCC1?

XRCC1 is a scaffold protein that plays an important role in base excision repair (BER). The XRCC1 protein complexes with OGG1, APE1/APEX1, polyadenosine diphosphate-ribose polymerases 1 and 2, DNA polymerase ß and  DNA ligase III to repair single strand breaks left during BER. Shen et al. identified three coding polymorphisms in the XRCC1 gene at codons 194 (Arg to Trp), 280 (Arg to His) and 399 (Arg to Gln).

What is XRCC3?

XRCC3 is a member of the Rad51 DNA-repair gene family. It functions in the homologous recombination repair pathway by repairing double-strand breaks.

Polymorphism analysis for XRCC1 codons 194/399:

● Genomic DNA is isolated from whole blood by means of the QIAamp Blood Mini/Midi Kit (QIAGEN).

● Two different sets of primers are used, which encompass the Arg194Trp and Arg399Gln polymorphic sites within the XRCC1 gene.

● Multiplex PCR yields a 491 and a 615 bp product, respectively. 3h digestion at 37°C with the MspI restriction enzyme results in the following fragments:
- for codon 194: a 178 bp fragment due to an invariant MspI site; 293 and 20 bp products for the Arg allele, and a 313 bp product for the Trp allele.
- for codon 399: 375 and 240 bp products for the Arg allele, and an undigested 615 bp fragment for the Gln allele.

● After 2.5h 120 V electrophoresis through a 3% Metaphor agarose gel, DNA fragments are visualised with ethidium bromide.

Polymorphism analysis for XRCC1 codon 280 and XRCC3 codon 241:

● Genomic DNA is isolated from whole blood by means of the QIAamp Blood Mini/Midi Kits (QIAGEN).

● Two different sets of primers are used, which encompass the Arg280His and Thr241Met polymorphic sites of XRCC1 and XRCC3 genes, respectively.

● A 280 bp fragment of XRCC1 and a 336 bp fragment of XRCC3 are amplified by multiplex PCR. The PCR products are digested 4h at 37°C with the restriction enzymes RsaI and NlaIII respectively, resulting in the following fragments:

- for codon 280 of XRCC1: a 140 bp fragment for the Arg allele, and a 280 bp fragment for the His allele.

- for codon 241 of XRCC3: an undigested 336 bp fragment for the Thr allele, and a 231 bp fragment for the Met allele. In addition, a 105 bp NlaIII restriction product is seen in all samples.

● After 1h30 120 V electrophoresis on a 3% agarose gel, DNA fragments are visualised with ethidium bromide.