Whole Genome Amplification (WGA) With Repli-g Mini Kit From Qiagen

Certain DNA samples that have very low concentrations or are critical to an analysis can be problematic. The ability to amplify such DNA is important for labs that obtain very little starting material or have consumed most of a critical sample during analysis. The Repli-g Mini Kit from Qiagen uses a Multiple Displacement Amplification approach where random primers attach to template DNA which allows a highly processive DNA polymerase (phi29) to add complementary base pairs to the initial strand while displacing the other DNA strand. Secondary priming is initiated on the displaced DNA strand resulting in multiple branches. Phi29 is a “high processivity” polymerase in that it remains attached to template DNA far longer than typical Taq polymerases. Additionally, accurate loci and allele representation is maintained due to 3’→5’ exonuclease proofreading activity.

Although the kit is geared towards amplification of human genomic DNA, the application in our lab is for SNP-typing and VNTR (variable number tandem repeat) analysis of bacterial pathogens such as Bacillus anthracis and Yersinia pestis. Providing large quantities of pathogen DNA is hampered by the requirement to grow sufficient quantities of the pathogen while working under high containment conditions. Additionally, older protocols that use chloroform and isoamyl alcohol to separate nucleic acids in solution may not be optimized for every bacterial pathogen. Since this kit was designed for such a different application, I had several reservations about using it. I was concerned about whether or not the ratio of chromosomal to plasmid copy number would be maintained. We also wanted to see if heat-lysed DNA extractions would yield similar results to those obtained when using highly purified DNA (chloroform extractions or QIAamp DNA Mini Kit).

We typically run all of the reagents used for WGA (whole genome amplification) through our clean room which alleviates concerns of exogenous DNA finding its way into the reaction and subsequently being amplified with the intended target. Interestingly, no template “blanks” exhibited extension of random primers. These samples were analyzed on 0.7% agarose gels and showed the typical smear indicative of high molecular weight DNA. We also noted spurious amplification of this randomly generated “template” with B. anthracis specific primers although sequence analysis of these amplicons revealed no homology to any known B. anthracis genome. This phenomenon went away with the addition of as little as 10 pg of template DNA. For B. anthracis, genomic DNA prepared by either chloroform separation or by QIAamp extraction performed significantly better in downstream VNTR analysis by capillary electrophoresis than heat-lysed material with this kit. Crude lysates still have a significant amount of protein, polysaccharides, and cell wall debris not typically found in purified extracts and this probably accounts for the difference. Yields of WGA material were also better using a more purified B. anthracis extract. WGA of Y. pestis gave much better results and the starting material appeared to have little effect on downstream analyses. Lastly, preliminary results from Taqman® Real-Time PCR assays indicate that the ratio of plasmid copy number to chromosome is maintained during the WGA process.

This kit is somewhat scaleable for high throughput analysis. Reactions are performed in 50 µl volumes and a typical 96-well PCR plate can certainly handle this. The bottle neck comes in arraying a 96-well culture cluster with your samples. However, once this has been achieved, there are many DNA extraction protocols and kits amenable to a 96-well platform and the Repli-g kit can be adapted for this.