Hexaprimer Amplification Refractory Mutation System PCR for Simultaneous Single-Tube Genotyping of 2 Close Polymorphisms

Tetraprimer amplification refractory mutation system PCR (T-ARMS-PCR) is a simple and inexpensive genotyping method for differentiating both alleles of a polymorphism/mutation (both single-nucleotide polymorphisms and small insertions/deletions) with a single-tube PCR (1). In T-ARMS-PCR, a pair of common (outer) primers produces a non–allele-specific control amplicon and in combination with 2 allele-specific (inner) primers (designed to anneal in the opposite orientation) produces 2 allele-specific amplicons. These allele-specific amplicons have different sizes because the polymorphism/mutation is asymmetrically located with respect to the common primers. Thus, the amplicons can be separated by standard gel electrophoresis. T-ARMS-PCR has also been designed in a multiplex fashion to genotype more than one polymorphism/mutation by a single-tube PCR (2). We describe a modified multiplex T-ARMS-PCR, the hexaprimer ARMS-PCR (H-ARMS-PCR), which is for when 2 polymorphisms are close in the sequence. H-ARMS-PCR uses only 6 primers and provides direct information about haplotype structure. The CTLA4 gene (cytotoxic T-lymphocyte–associated protein 4; also known as CD152) is a negative regulator of T-cell function (3). The CTLA4 polymorphisms −318 C>T (rs5742909) and +49 A>G (rs231775) are associated with susceptibility to autoimmune diseases and cancer (3)(4). To genotype these 2 polymorphisms, which are only 365 bp apart in the 5′ region of the CTLA4 gene, we designed an H-ARMS-PCR that combines a single pair of common primers and 2 pairs of allele-specific primers in the same tube (Fig. 1A⇓ ). Figure 1. Polymorphism genotyping by H-ARMS-PCR. (A), H-ARMS-PCR for CTLA4 −318 C>T (rs5742909) and …