SWGA allows for whole genome enrichment and sequencing of field collected samples without the need for a cold chain or laborious field processing methods. The method has been used on whole blood samples mixed with nucleic acid preservation solutions and dried blood spots (Sundararaman, 2016, Guggisberg, 2016). However, SWGA results are improved with optimal sample handling and storage.
DNA size and quality
SWGA works best with good quality high molecular weight DNA. I recommend that the average DNA length be at least 25-30 kb (the typical size range generated by column based purification kits). I also try to store my extracted DNA in ways that minimize damage from oxidation, hydrolysis, UV exposure, and heat to maintain DNA quality.
The reason for doing this is the ensure that the DNA is long enough for phi29 to perform multiple displacement amplification. This requires multiple binding sites per ssDNA molecule. The more fragmented the DNA, the fewer binding sites per molecule and the less total amplification (Figure 1). This also means that SWGA does does not work on very small genomes such as full-length cDNA from HIV.
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| Figure 1: Effect of template length on SWGA. Schematic of SWGA reactions are shown on a long ssDNA template (red line) and fragmented ssDNA template (blue lines) that represent the same total length of DNA. Primers (grey dashes) bind both templates and DNA synthesis is initiated (thick black line). In the red template, synthesis reaches complementary DNA and displaces that DNA at multiple points. In the blue templates, synthesis reaches the end of the molecule and only 1 very short fragment of DNA is displaced. The newly displaced ssDNA in the red template serves as a template for new primer binding (green dashes) and further DNA synthesis. This does not occur in the blue template as there are two few primer binding sites per fragment to produce displacement. |
Improving DNA quality prior to SWGA
References
