Single nucleotide polymorphisms (SNPs) that occur on the average once every kilobase pair in the human genome are the most abundant form of genetic variation (1
). As a consequence of the Human Genome Project and other large SNP discovery efforts, information on more than four million SNPs is available in public databases. The great interest in SNPs originates in their potential use as markers for whole genome linkage disequilibrium mapping to elucidate genes underlying complex, multifactorial disorders (2
). Recent studies on linkage disequilibrium patterns in the human genome indicate that very dense SNP maps with hundreds of thousands, or even millions of markers may be needed in genome wide association studies (3
). Even if most of the currently used genotyping techniques rely on amplification of the genomic DNA by the polymerase chain reaction (PCR) prior to genotyping (5
), the amount of DNA obtainable from patient or population samples would be an obstacle for SNP mapping studies on this scale. Given the large efforts involved in the collection of DNA samples from well characterized patient or population cohorts, it is desirable that the collected samples could serve as a long-lasting resource for future genetic studies. The amount of DNA is often limiting also in SNP genotyping studies on a more modest scale when the only available source of DNA are biobanked tumor or other tissue samples, buccal swabs or blood stains collected on filter paper.
One approach for creating an infinite source of DNA for current and future SNP studies is to immortalize the cell samples by transformation with Epstein–Barr virus (6
). The transformation procedure is, however, labor intensive, and therefore expensive to apply on a large scale. Moreover, it is not applicable to already existing biobanked DNA sample collections. A technically more feasible approach for increasing the amount of DNA is to use a whole genome amplification (WGA) procedure such as primer extension preamplification with random femtomers (PEP) (7
) or degenerate oligonucleotides (DOP–PCR) (8
) as primers in PCR. The PEP and DOP–PCR procedures were originally designed for analysis of single cells or very small DNA samples. However, imbalanced amplification of microsatellite (9
) and SNP alleles (10
) as well as incomplete coverage of the genome in the amplification products (12
) has been observed. A second concern related to these WGA methods is the possible introduction of artificial sequence variation into the amplification products via the degenerate PCR primers used.
An isothermal procedure for rolling circle amplification of DNA templates using the DNA polymerase from the Φ29 bacteriophage was first introduced as an amplification method for circularized DNA (13
). The method has later been adapted for amplification of linear templates, and it is a promising alternative to PEP and DOP–PCR (12
). This isothermal multiple displacement amplification (MDA) procedure uses random hexamers containing phosphorothioate-modified nucleotides as primers, and relies on the high processivity, high fidelity and strand displacement ability of the enzyme (14
). According to quantitative real time PCR analysis, MDA using the Φ29 DNA polymerase provided a less biased representation of different genomic loci than PEP or DOP–PCR (12
). When using WGA to increase the amount of DNA for SNP genotyping, a more critical requirement than balanced amplification of different genomic loci is balanced amplification of both alleles of each SNP at the same genomic loci. MDA products have been genotyped successfully at a few microsatellite and SNP loci (12
), but only a few markers were analyzed and no attempt to evaluate the robustness of the genotyping results has been made.
We present here a systematic, quantitative evaluation of WGA with PEP and MDA for generating DNA templates for SNP genotyping. In this evaluation, we applied an improved protocol for PEP (17
), in which a high fidelity PCR system is used in combination with thermo cycling conditions slightly modified from the original protocol. We genotyped a panel of 45 SNPs located in different genomic regions using multiplex, four-color fluorescent minisequencing in a microarray format. The results from PEP and MDA templates were compared to those obtained from genomic DNA. The minisequencing method is particularly useful for this evaluation because it is based on the high sequence-specificity of nucleotide incorporation by a DNA polymerase and therefore allows accurate, quantitative determination of the ratio between two SNP alleles (18
). Thus, it also facilitates detection and determination of the magnitude of possible imbalanced amplification of the alleles of a SNP during the WGA procedures. The performance of the WGA methods were evaluated with respect to genotyping success, signal-to-noise ratios, power of discrimination between homozygous and heterozygous SNP genotypes, yield and authenticity of allele representation in the WGA product by the quantitative minisequencing method.