Whole genome amplification using a degenerate oligonucleotide primer allows hundreds of genotypes to be performed on less than one nanogram of genomic DNA

Abstract

Genetic analysis of limiting quantities of genomic DNA play an important role in DNA forensics, paleoarcheology, genetic disease diagnosis, genetic linkage analysis, and genetic diversity studies. We have tested the ability of degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) to amplify picogram quantities of human genomic DNA for the purpose of increasing the amount of template for genotyping with microsatellite repeat markers. DNA was uniformly amplified at a large number of typable loci throughout the human genome with starting template DNAs from as little as 15 pg to as much as 400 ng. A much greater-fold enrichment was seen for the smaller genomic DOP-PCRs. All markers tested were amplified from starting genomic DNAs in the range of 0.6-40 ng with amplifications of 200-to 600-fold. The DOP-PCR-amplified genomic DNA was an excellent and reliable template for genotyping with microsatellites, which give distinct bands with no increase in stutter artifact on di-, tri-, and tetranucleotide repeats. There appears to be equal amplification of genomic DNA from 55 of 55 tested discrete microsatellites implying near complete coverage of the human genome. Thus, DOP-PCR appears to allow unbiased, hundreds-fold whole genome amplification of human genomic DNA for genotypic analysis.

Whole genome amplification for CGH analysis: Linker‐adapter PCR as the method of choice for difficult and limited samples

“…For either amplification method, DNA amounts from 300 ng to 20 pg resulted in reliable experiments, with identical results obtained from undiluted and diluted DNA samples. These data are in good agreement with previous studies in which the minimal DNA amounts for successful DOP-PCR-amplification and CGH were described to range between 12.5-50 pg (5,21,23). At lower amounts of target DNA, equivalent to one or a few diploid cells, we found LA-PCR to be clearly superior as compared to DOP-PCR.…”

“…Both, DOP-PCR and LA-PCR (also termed SCOMP) have been suggested to be suitable for analysis of very minor amounts of DNA, equivalent to one or a few cells (9,16,17,(21)(22)(23). We compared the sensitivity and efficiency of both amplification procedures in a series of DNA dilution experiments.…”

Multiple Mutation Analyses in Single Tumor Cells with Improved Whole Genome Amplification

“…22 The latter turned out to be more effective for DNA amplification and can be done in at least two different ways. One approach is degenerate oligonucleotide primer-PCR (DOP-PCR), 23,24 often used as the first step in in situ hybridization with flow-sorted chromosomes, 24,25 microdissected chromosomes, and, more recently, for comparative genomic hybridization. 26,27 DOP primers have defined sequences at the 5Ј end and the 3Ј end.…”

Multiplex Loss of Heterozygosity Analysis by Using Single or Very Few Cells

“…20,21 When starting with low copy numbers, WGA may increase the copy number of the entire or major portion of the human genome for 3000 to 100,000-fold 21 so that a limited amount of genomic DNA can be amplified to a sufficient amount for genetic analysis with a relatively large number of markers. The major difference between WGA and multiplex PCR is that WGA raises the copy number of the entire or the major portion of the genome but its product cannot be used for direct analysis.…”