Effect of freezing of the PCR buffer on the amplification specificity: allelic exclusion and preferential amplification of contaminating molecules.

Abstract

Department of Medical Genetics, Biomedical Center, University of Uppsala, S-751 47 Uppsala, Sweden During a study of allelic variation at HLA class II loci using PCR amplification and typing with sequence-specific oligonucleotides (SSO), we have come across two situations that can result in erroneous genetic typing. Amplification of the HLA-DQB1 locus was performed using the primers DB130 and DB131, (1~ and the amplification cycle previously found to amplify all known alleles (denaturation at 94~ anneal ing at 55~ and extension at 72~ using a final concentration of 1.5 mM MgC12). (1~ However, after typing of the amplified products, we found a significant excess of homozygotes for the DQBI*0301 as well as 0501 and 0502 alleles in the population examined. Because an excess was not evident for this populat ion at other class II loci and the typing of the DQB1 locus using other primers and other populations has not shown similar deviations, the result for the DQB1 is likely to reflect a bias in the amplification using the DB130 and DB131. Bugawan noted that there is a 1-to 2-bp mismatch between one of the primers (DB130) (1~ and some (0401, 0402, 0601, 0602) of the DQB1 alleles. (2~ However, no discrimination between alleles was found when 1.5 mM MgC12 and an anneal ing temperature of 55~ was used. (1~ When we performed a titration experiment of the MgC12 concentration in our PCR buffer, we found that when using 1.5 mM MgCl 2 and an anneal ing temperate of 55~ both the 0301 and 0601 alleles were amplified, whereas the 0301 allele was preferentially amplif ied in the 0301/0601 heterozygote when the MgCl 2 concentrat ion was below 1.0 mM (Fig. 1). The 10• PCR buffer used in our populat ion study included 500 mM KC1, 100 mM Tris-HC1 (pH 8.3), and 15 mM MgC12, and had been stored frozen ( -20~ for about 6 months. The titration experiment indicates that during storage the available MgC12 in the PCR buffer was reduced from 1.5 to about 0.9 mM, resulting in the preferential amplification of alleles completely matched to the PCR primer. In reexamining the individuals previously typed as 0301/0301 homozygotes using new buffer, a number were, in fact, found to be 0301/0601 heterozygotes. Although we are unable to exclude the possibility that other components of the PCR buffer may be responsible for the change in specificity, our titration clearly shows that the effect could be attributable entirely to the MgC12. Possibly, repeated freezing and thawing has resulted in the precipitation or accumulat ion of MgCl 2 in insoluble form. This is supported by observation that heating the buffer to 90~ for 10 m i n and vortexing restored the ability of the old PCR buffer to ampli fy all DQB1 alleles. In addition, when amplifications were performed with the MgC12 concentrations used in the titration on homozygotes for the 0601 allele, several individuals appeared during the interval 1.5-0.9 mM MgC12 to carry only the 0601 allele,