Direct determination of haplotypes from single DNA molecules

Abstract

Determining the long-range haplotypes in a diploid individual is a major technical challenge. Here we report a method of molecular haplotyping by directly imaging multiple polymorphic sites on individual human DNA molecules simultaneously. We demonstrate the utility of this technology by accurately determining the haplotypes consisting of up to 16 single-nucleotide polymorphisms in genomic regions up to 50 kilobases.

Figure 1. Allele-specific labeling of SNPs and regeneration of dsDNA

(a) Two oligonucleotides, each labeled with a different-colored fluorophore, are designed with one of the SNP alleles at the 3′ end. Only the perfectly matched primers will be extended by DNA polymerase. Subsequently ssDNA is converted to dsDNA by primer extension. (b) Gel showing regenerated dsDNA from ssDNA. Long-range PCR products of four different DNA samples (lanes 9–12) were digested with lambda exonuclease to produce the corresponding ssDNA (lanes 5–8, respectively). After primer extension, dsDNA samples were regenerated (lanes 1–4, respectively). M, marker.

Figure 2. Molecular haplotyping of four SNPs across a 12.5 kb region

(a) Relative locations of four SNPs on a 12.5 kb DNA fragment. The arrows indicate the distance from the SNP site to one end of the DNA molecule. (b) The rescaled false-color composite image of labeled DNA fragments of DNA sample NA12156 labeled with YOYO-1 (blue) and Cy3 (green) or Cy5 (red). High-magnification images of two fully labeled fragments marked by boxes are shown in the insets. (c,d) Images of labeled DNA fragments of DNA sample NA010860 (c) and NA07357 (d). Fully labeled fragments are marked by arrows. Scale bars, 5 µm (b–d) and 2 µm (insets in b).

Figure 3. Molecular haplotyping of 16 SNPs across the 50 kb PLAT gene region

(a) Schematic of the region showing the positions of 16 SNPs and the overlapping fragments. The two alleles of each SNP are represented as the green (Cy3) and red (Cy5) circles on the DNA backbone. (b) Images from fully labeled DNA fragments for DNA sample NA12865. Six SNPs in fragment 1 were haplotyped in two groups. Two predominant haplotypes were found for the first (SNPs 5,7,9,10) and the second (SNPs 6,8,10) group. The two full haplotypes of the six SNPs comprising fragment 1 are T-G-T-C-T-T and G-C-C-G-G-C for SNPs 5,6,7,8,9,10. Haplotypes for the remaining four fragments are indicated, and the frequencies of the haplotypes are described in Supplementary Table 5.