Direct molecular haplotyping of long-range genomic DNA with M1-PCR

Abstract

Haplotypes, combinations of several phase-determined polymorphic markers, are extremely valuable for studies of disease association and chromosome evolution. Here we describe a technique called M1-PCR (M for "multiplex" and 1 for "single-copy DNA molecules") that enables direct molecular haplotyping of several polymorphic markers separated by as many as 24 kb. A genomic DNA sample first is diluted to approximately single-copy. The haplotype is directly determined by simultaneously genotyping several polymorphic markers in the same reaction with a multiplex PCR and base extension reaction. This approach does not rely on pedigree data and does not require previous amplification of the entire genomic region containing the selected markers.

Fig. 1.

Multiplex genotyping of single-copy DNA molecules for haplotype analysis. Traditional genotyping methods using 5 ng of genomic DNA (≈1,600 copies of genomic templates) yield the genotypes of each individual SNP marker, but the phases of these SNPs are not determined (Upper Right). Simultaneous genotyping of several markers using multiplex assays with single-copy DNA molecules allows haplotyping analysis, because the two alleles can be physically separated at extremely dilute DNA concentrations (Lower Right; please refer to Materials and Methods for details of haplotyping and genotyping). In contrast to other molecular haplotyping methods, the entire haplotype block is not amplified in this approach. Instead, only ≈100 bp around each individual SNP is amplified for genotyping, resulting in very high efficiency of PCR amplification from single-copy DNA molecules. The SNP markers can be as far apart as desired, as long as physical breaks between the SNP markers exist only in a small proportion of the genomic DNAs. Peaks identified as P are from unextended primers.

Fig. 2.

Effect of genomic DNA concentration on haplotyping efficiency. Approximately 3, 5, and 9 pg (or 1, 1.6, and 3 copies of genomic templates) were used for haplotyping of three SNP markers (GenBank SNP ID: rs289741, rs289742, and rs289744; Table 1) in the cholesteryl ester transfer protein region. The DNA copy number in a specific reaction is estimated by the Poisson distribution. The haplotyping result can be a failed assay, successful haplotyping, the presence of both alleles (no phase determination for the markers), or an incomplete multiplex. Except for incomplete multiplexes, values are percentages from 54 to 144 individual multiplex assays (see Materials and Methods for details of the calculation), followed by predicted values (*) determined by using the Poisson distribution.

Fig. 3.

Overlapping multiplex genotyping assays with single-copy DNA molecules. Seven SNP markers (A, rs289744; B, rs2228667; C, rs5882; D, rs5880; E, rs5881; F, rs291044; and G, rs2033254) from an 8-kb genomic region of the cholesteryl ester transfer protein locus were chosen (Table 1). Two five-plex genotyping assays were designed for these seven markers, and the overlapping heterozygous SNPs were used to obtain the entire haplotype of the markers. Assays on individual 6 were used to demonstrate how this approach was carried out. Multiplex assay 1 determined the haplotype of five SNPs as AGAGT and CGGGC. Multiplex assay 2 determined the other haplotype of five SNPs as GGGCT and AGGTT. Then, the genotypes of the overlapping SNPs (SNPs C, E, and F) were used to combine the two haplotypes of five SNPs into a haplotype of seven SNPs covering the entire region under investigation.

Fig. 4.

Haplotyping of three SNP markers spanning 24 kb in the chromosome 5q31 region. The SNPs chosen were IGR2150a_1, IGR2175a_2, and IGR2198a_1 (Table 1). The results from Centre d'Etude du Polymorphisme Humain (CEPH)/French Pedigree 66 are shown. (a) Relative positions and distances of the three SNP markers are shown. (b) Pedigree genotypes and haplotypes. Genotypes of the paternal grandfather (individual in upper left corner) in the figure are G/C for SNP IGR2150a_1, G/A for SNP IGR2175a_2, and C/G for SNP IGR2198a_1. The two haplotypes for the same individual are GGC and CAG. The son of that individual is homozygous for all three SNPs. Thus, his haplotype is GGC for both alleles.