Alkaline-mediated differential interaction (AMDI): a simple automatable single-nucleotide polymorphism assay

Abstract

The key requirements for high-throughput single-nucleotide polymorphism (SNP) typing of DNA samples in large-scale disease case-control studies are automatability, simplicity, and robustness, coupled with minimal cost. In this paper we describe a fluorescence technique for the detection of SNPs that have been amplified by using the amplification refractory mutation system (ARMS)-PCR procedure. Its performance was evaluated using 32 sequence-specific primer mixes to assign the HLA-DRB alleles to 80 lymphoblastoid cell line DNAs chosen from our database for their diversity. All had been typed previously by alternative methods, either direct sequencing or gel electrophoresis. We believe the detection system that we call AMDI (alkaline-mediated differential interaction) satisfies the above criteria and is suitable for general high-throughput SNP typing.

Figure 1

Effect of pH on negative and positive PCR products. The conditions are as described in Methods, except that the pH of the CABS buffer has been adjusted as indicated. The oligonucleotides used were the sense 283 (5′-GATCGTTCGTGTCCCCACAG-3′) and antisense 314 (5′-CTGGTACTCCCCCAGGTCA-3′) (15). The first point at pH 8.4 is the result of a reaction at the normal pH of the PCR reaction and in the absence of CABS buffer. Each point is the mean of six reactions with bars indicating the standard deviation.

Figure 2

Effect of 0.2 M CABS (pH 12.7) on the background fluorescence. The black columns represent specific PCR product and the shaded columns represent no specific product. Conditions are as described in Methods. The oligonucleotides used were sense 68 (5′-GTTTCTTGGAGTACTCTACGT-3′ and antisense 104 (5′-CCCGCCTGTCTTCCAGGAA-3′) (15). The height of the column is the average of five readings, and the bar extension is the magnitude of the standard deviation.

Figure 3

Effect of individual constituents of the PCR on background fluorescence in the presence or absence of CABS. Each column represents the result of three PCRs performed: 1, in the absence of Taq Gold polymerase, but in the presence of oligonucleotides and template; 2, in the absence of Taq Gold polymerase and template, but in the presence of oligonucleotides; 3, in the absence of Taq Gold polymerase, template, and one oligonucleotide but in the presence of one oligonucleotide; 4, in the absence of primers and template, but in the presence of Taq Gold polymerase; 5, in the absence of template, but in the presence of both primers and polymerase; 6, as in case 3, except in the presence of the alternative primer of the primer combination; 7, in which specific amplicon is produced; and 8, in which no amplicon is expected. Conditions 1, 2, and 6 show the effect of CABS in reducing the background fluorescence caused by the single-strand oligonucleotides. The reaction with CABS in conditions 5 and 8 shows the residual effect caused by primer/dimers, in the case of 8 with additional template DNA.

Figure 4

HLA-DR typing of a lymphoblastoid cell line by using the AMDI assay. Thirty-two primer pairs, including one positive control, are arranged in triplicate on a 96-well plate. The heights of the columns indicate the actual reading at a given position on the plate. The only positives are the positive control and those for the DRB1*0701 and DRB*40101/2/3 oligonucleotide pairs. These are the only combinations expected to react from the known HLA type of the lymphoblastoid cell line.

Figure 5

HLA-DR typing reactions for three different DNA samples. The conditions for the assays are as described in Methods, and the 32 primer pairs are as described in Fig. 3. Each column is the mean of triplicate readings, and in each case the clear positive reactions are those expected from the known HLA type of the cell line. Primer pair 10 gives a consistently high background, probably because of crossreaction with other loci, and is negative in each case.