Researchers often face the problem of limited or insufficient DNA quantity for downstream analysis. QIAGEN's whole genome amplification technology solves this problem by providing high yields of whole genomic DNA from small or precious samples, including single cells. REPLI-g Kits and Service accurately copy the original source DNA without bias and the amplified DNA can be directly used in a range of genetic analyses.
What is whole genome amplification?
Whole genome amplification was developed in 1992 as a way of increasing the amount of limited DNA samples. This is particularly useful for forensics and genetic disease research, as well as new technologies such as next-generation sequencing and array CGH (comparative genomic hybridization), where DNA quantities are limited but many analyses are required. Various WGA techniques have been developed, which differ both in their protocols and their replication accuracy (see WGA techniques).
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WGA techniques — PCR vs. Multiple Displacement Amplification (MDA)
A number of WGA techniques have been developed; however, these techniques vary in their amplification accuracy and ease of use. QIAGEN's REPLI-g Kits and Service utilize a WGA technique called Multiple Displacement Amplification, which provides unbiased and accurate amplification of whole genomes (see figure
Unbiased amplification with Phi 29 polymerase).
PCR-based WGA
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). Phi 29 polymerase does not dissociate from the genomic DNA template, allowing the generation of DNA fragments up to 100 kb without sequence bias. The enzyme has a 3’→5’ exonuclease proofreading activity and delivers up to 1000-fold higher fidelity compared to
Taq DNA polymerase-based methods (see PCR-based WGA, above). Supported by the unique, optimized REPLI-g buffer system, Phi 29 polymerase easily solves secondary structures such as hairpin loops, thereby preventing slipping, stoppage, and dissociation of the polymerase during amplification. This enables the generation of DNA fragments up to 100 kb without sequence bias (see figure
Unbiased amplification with Phi29 polymerase).
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).
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WGA applications
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