What is whole genome amplification?
WGA techniques — PCR vs. Multiple Displacement Amplification (MDA)
There are 2 main PCR-based WGA techniques. These are Degenerate Oligonucleotide PCR (DOP-PCR) (1) and Primer Extension Preamplification (PEP) (2). The main difference between the techniques is that PEP utilizes random primers and a low PCR annealing temperature, while DOP-PCR uses semi-degenerate oligonucleotides (i.e., CGACTCGAGNNNNNNATGTGG) and an increasing annealing temperature. The use of Taq DNA polymerase in both techniques limits the fragment lengths to 3 kb (average fragment sizes are 400–500 kb) and also introduces a number of errors into the sequence. Furthermore, these techniques have been found to exhibit incomplete genome coverage and amplification bias — where a sequence is overrepresented in the amplified DNA due to preferential binding of the primers to specific regions (see figure Highly representative amplification using MDA).
Multiple Displacement Amplification WGA
REPLI-g uses isothermal genome amplification, termed Multiple Displacement Amplification (MDA), which involves the binding of random hexamers to denatured DNA followed by strand displacement synthesis at a constant temperature using the enzyme Phi 29 polymerase. Additional priming events can occur on each displaced strand leading to a network of branched DNA structures (see figure REPLI-g MDA Technology).
Advantage of MDA versus PCR-based WGA methods
Traditional methods of genomic DNA amplification include the time-consuming process of creating EBV-transformed cell lines, followed by whole genome amplification using random or degenerate oligonucleotide-primed PCR. Also, PCR-based methods (e.g., DOP-PCR and PEP), as generally used by other suppliers, can produce nonspecific amplification artifacts and give incomplete coverage of loci. In several cases, DNA less than 1 kb long may be generated that cannot be used in many downstream applications. In general, the resulting DNA is generated with a much higher mutation rate due to the use of the low-fidelity enzyme Taq DNA polymerase, which can lead to error-prone amplification that results in, for example, single base-pair mutations, STR contractions, and expansions. In contrast to these disadvantages, REPLI-g provides highly uniform amplification across the entire genome, with minimal locus bias and minimized mutation rates during amplification (see figures Unbiased amplification from a single cell, Highly representative amplification using MDA and Consistent and accurate whole genome amplification).
REPLI-g amplified genomic DNA can be used in a variety of downstream applications, including:
- SNP genotyping with TaqMan primer/probe sets
- qPCR- and PCR-based mutation detection
- Next-generation sequencing
- STR/microsatellite analysis
- Sanger sequencing
- RFLP and Southern blot analysis
- Array technologies, such as comparative genomic hybridization