The role of single nucleotide polymorphisms (SNPs) has been implicated in a number of complex diseases including Alzheimer’s disease (1
), osteoporosis (2
), Crohn’s disease (4
) and obesity (5
). More recently, they have played a fundamental role in the emerging field of pharmacogenomics and adverse drug reactions (6
). As such, it is becoming clear that SNPs have the potential to act as informative markers for the discovery and characterization of genes in complex disease (8
). This will be made possible, in part, due to greater knowledge of the human genome sequence (9
) and the increasing number of SNPs in databases (11
). The estimated number of SNPs required to be screened genome-wide in order to detect a disease-causing gene could be as high as 500 000 (12
) but this number may be lowered with the ongoing ascertainment of the extent of linkage disequilibrium (13
With this increase in knowledge, there has been an associated explosion in the number of technologies that offer higher and higher throughput SNP genotyping on an industrial scale (16
). These assays offer up to 500
000 genotypes a day at reagent costs as low as 1 c/SNP. However, with this throughput comes an increasing demand for DNA template on which to carry out these large numbers of reactions. For example, if 10 ng of DNA is required per reaction and 500 000 SNPs were to be screened then 5 mg of DNA is required. This would heavily deplete a valuable genomic DNA resource.
Two similar approaches have employed total genomic amplification to produce non-specific uniform amplification of DNA. By using a degenerate primer, a representation of the genome can be produced by means of the polymerase chain reaction (PCR) to act as a template for subsequent typing efforts. First, primer extension preamplification (PEP), which was originally developed to amplify DNA from a single cell (17
), has been applied to HLA typing from mouth swabs (18
) by generating a mixture of PEP reactions carried out in quadruplet to gain sufficient genome coverage. The primer for this approach is totally degenerate, made up simply of 5′-NNNNNNNNNNNNNNN-3′.
A more recognized approach for complete genome coverage in one reaction is the use of degenerate oligonucleotide primed PCR (DOP-PCR) which employs a more specific primer: 5′-CCGACTCGAGNNNNNNATGTGG-3′ (19
). This approach has been successfully modified and applied to genomic DNA in order to carry out microsatellite genotyping (20
). Although there was 100% success in amplification of each marker and correct assignment of genotypes, it was noticed that there was some preferential amplification of the shorter allele. A similar observation was made with microsatellites, Alu insertions and variable-length segments of the lipoprotein lipase gene (LPL) when using long DOP-PCR on rare archival anthropological samples (21
). As microsatellite typing is based on the length of a repeat, the issue of band intensity is not of great importance. However, if the same approach is to be employed for SNP typing, it is vital that uniform amplification of alleles occurs for current technologies to call the genotype with confidence. The use of a DOP-PCR-amplified template has been applied previously to a small cohort and the typing of two SNPs (22
). Another emerging application of this procedure in the context of SNP typing is the use of DOP-PCR for the reduction of genome complexity (23
). In addition, the first paper published on a non-PCR-based approach was published only a few months ago using multiple displacement amplification (24
In this work, we analyze the application of a DOP-PCR-amplified template in a high-throughput setting and the impact on quality. In addition, the role of DOP-PCR storage conditions is assessed.