Digital genotyping and haplotyping with polymerase colonies

Abstract

Polymerase colony (polony) technology amplifies multiple individual DNA molecules within a thin acrylamide gel attached to a microscope slide. Each DNA molecule included in the reaction produces an immobilized colony of double-stranded DNA. We genotype these polonies by performing single base extensions with dye-labeled nucleotides, and we demonstrate the accurate quantitation of two allelic variants. We also show that polony technology can determine the phase, or haplotype, of two single- nucleotide polymorphisms (SNPs) by coamplifying distally located targets on a single chromosomal fragment. We correctly determine the genotype and phase of three different pairs of SNPs. In one case, the distance between the two SNPs is 45 kb, the largest distance achieved to date without separating the chromosomes by cloning or somatic cell fusion. The results indicate that polony genotyping and haplotyping may play an important role in understanding the structure of genetic variation.

Figure 1

Polony haplotyping. 1, Polymerize dilute sample of patient DNA into a polyacrylamide gel on a glass microscope slide. 2, Amplify single chromosomes by PCR with two primer pairs flanking the SNPs of interest (Inset). 3, Genotype the first SNP by performing an SBE reaction and scan the slide using a laser scanner. 4, Genotype the second SNP, scan the slide, and 5, merge the images to detect overlapping polonies. The haplotype is determined directly from the overlapping polonies.

Figure 2

Amplification of two polonies from a single DNA molecule. We cut plasmid DNA (C) with EcoRI, leaving locus A and locus B on the same template molecule, or with EcoRI and NcoI, separating the loci. We performed polony amplification with two pairs of primers designed to amplify loci A and B and hybridized cy5-labeled probe (red, locus A) and cy3-labeled probe (green, locus B) to the polonies. Eighty-six percent of locus A polonies overlapped a locus B polony (and 80% of locus B polonies overlapped a locus A polony) when the plasmid was singly cut (A), but <2% of locus A polonies significantly overlapped a locus B polony when the plasmid was doubly cut (B).

Figure 3

Determining the allelic ratios using polony genotyping. We purified genomic DNA from two inbred strains of mouse with different alleles of the creatine kinase gene. We mixed the DNA at various ratios and performed polony genotyping. The observed allelic ratios correlate strongly to the predicted ratios (r = 0.99, error bars are shown at a 95% confidence level).

Figure 4

Polony haplotyping of SNPs in the CFTR gene. We performed polony amplification on the SNPs DK438 and DK445-2. First, we genotyped DK438 by SBE (A), stripped the extended primers from the gel, and genotyped DK445-2 (B). Twenty-two overlapping polonies were identified computationally and have been circled in A and B. All 22 polonies indicated that the “c” variant at SNP DK438 was in cis with the “c” variant at DK445-2 (LOD = 26.2).

Figure 5

Phasing of SNPs separated by a genomic distance of 45 kb. We amplified and genotyped SNPs DK331 and DK445–2 by SBE. One hundred fifty-three polonies were genotyped at the DK331 locus (A), and 175 polonies were genotyped at the DK445-2 locus (B). We identified 34 overlapping polonies (circled in the above figure), 32 of which indicated that the A variant at SNP DK 331 is in cis with the T variant of SNP DK445–2 (LOD = 20.3).