Fluorescent allele-specific PCR (FAS-PCR) improves the reliability of single nucleotide polymorphism screening.

The detection of single nucleotide polymorphisms (SNPs) is becoming increasingly important in both the evaluation of candidate gene polymorphisms and fine mapping studies (4,7). Many techniques exist for the detection of SNPs within random sequences, such as single-strand conformation polymorphism (SSCP) (5,6), heteroduplex analysis and direct sequencing, but few techniques specifically identify the different alleles efficiently enough to use in a population screen. Neither SSCP (5,6) nor heteroduplex analyses detect all polymorphisms. Restriction fragment-length polymorphisms (RFLPs) can only be used with genetic variations that either create or abolish a restriction enzyme site. Direct sequencing of polymerase chain reaction (PCR) products can become time-consuming and expensive if analyzing a large population. DNA chip technology (3,7) is evolving rapidly, but is currently too expensive and is not available to the majority of investigators. Other allelespecific techniques, such as allele-specific oligonucleotide hybridization and oligonucleotide ligation assay combined with PCR (1), are very specific but require a large amount of time to perform an analysis with multiple polymorphisms. The most specific and potentially most efficient technique is allele-specific PCR (AS-PCR) (8). AS-PCR depends on the observation that if the 3′ nucleotide of a primer does not match exactly, then the amplification will not be successful. Allele specificity is determined by selecting alternative primers that are identical except for the 3′ base and performing two amplification reactions. In this situation, DNA from heterozygotes will produce product in both reaction tubes, while DNA from homozygotes will produce product in only a single tube. It is possible to incorrectly score DNA from a heterozygote as a homozygote, if one of the PCRs fails. A control for successful amplification can be added, but, for technical reasons, the optimal conditions for this control might be different from the product of interest. In an effort to evaluate the Q576R polymorphism in the Interleukin-4 receptor (IL-4R) gene (2), we have modified the standard method for AS-PCR in a manner that controls for the absence of amplification and adapts the methodology to Models 310/373/377 DNA Analysis Systems (PE Biosystems, Foster City, CA, USA). With this technique, the allele-specific primers are modified so that the two distinct products, produced in a single PCR, can be identified on a standard sequencer lane. A second advantage of this new method is that it decreases the amount of Taq DNA polymerase required to detect the two alleles. Allele-specific primers were designed for the IL-4R Q576R polymorphism from the IL-4R mRNA sequence (GenBank Accession No. X52425) and previously published sequences (2). Two forward primers were designed with different fluorescent labels. These primers differed in the number of nucleotides on the 5′ end and in the allelespecific nucleotide on the 3′ end. The differences between the two forward primers are underlined. The primer specific for the Q576 allele was tet-5′GGCCCCCACCAGTGGCTATCA-3′ and the primer specific for the R576 allele was fam-5′-CCCCCACCAGTGGCTATCG-3′. The same reverse primer, 5′-CCAGTCCAAAGGTGAACA-