g you The direct determination of haplotypes from extended regions of genomic DNA

Abstract

Background: One of the major obstacles to the exploitation of genetic variation in human medicine, veterinary medicine, and animal breeding is the difficulty in defining haplotypes in unrelated individuals.

Results: We have developed a Multiplex Double Amplification Refractory Mutation System combined with Solid Phase PCR on Fluorescently labelled beads. The process is inherently amenable to automation. It provides a high degree of internal Quality Control, as each PCR product is represented in duplicate on the bead array, and each SNP is tested against multiple partners. This technique can resolve very complex genotypes into their constituent haplotypes; it defined all the alleles at 60 SNP in exon 2 of the ovine DRB1 MHC locus in a sample of 109 rams. These 60 SNP formed 33 DRB1 exon 2 alleles; two of which had not been previously identified; although both of them have been independently confirmed.

Conclusion: This technique has the same resolution as allele specific sequencing. Sequencing has the advantage of identifying novel polymorphic sites but where all SNP sites have been identified this novel procedure can resolve all alleles and haplotypes and identify novel combinations of polymorphisms. This method is similar in price to direct sequencing and provides a low cost system for direct haplotyping of extended DNA sequences.

Figure 1

Consensus sequence for reported alleles.

Figure 2

Frequency chart of the 33 alleles in 109 Scottish Blackface rams.

Figure 3

A short summary of the haplotyping procedure.

Figure 4

Diagram of the typing procedure. In 4A, in the first phase, allele specific primers are shown in red and black. The sample is shown as wavy lines in grey and blue. There is a PCR reaction in the supernatant with forward and reverse allele-specific primers. The PCR amplicon is then kidnapped and attaches to one of the reverse primers on the solid phase. In the presence of nucleotides and polymerase a second chain is formed. In 4B, the kidnapped amplicon, which is only held by hydrogen bonding, is released by heating. Washing leaves a covalently bonded single strand. In 4C, a reverse primer is introduced which binds to the covalently attached amplicon. In reality we have all possible forward and reverse primers illustrated as 3 candidate SNP in a row in green, red and grey. In 4D, the results from the separate reactions can be combined to indicate the SNP haplotypes.