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Which factors are mostimportant to achievinghigh genotyping accuracy?DNA quality. Purity is the number one factor.Molecular weight is also important. However, while itmay be important, it has become less of an issuetoday, especially since we're moving from RFLPs toSSTRs and now to SNPs. The region of the genomethat we're interrogating is actually getting smaller andsmaller, thus there's really not the requirement to haveextremely high molecular weight DNA samples likethere were when we were doing Southern blots.— David DugganThe number one factor isDNA quality. If you start withpoor quality DNA, you'regoing to get a poor-qualityresult. As these reactions arebecoming more highlymultiplexed, the quality ofthe starting material isbecoming more and moreimportant. We have qualitycontrol steps in our process where we thoroughlycheck the DNA before we put it in the reactions tomake sure that it is high quality.We do make some DNA quality recommendationsto our clients, though we don't recommend a specifictechnology for DNA extraction. We recommend theA260/280 ratio being 1.8-2.2 and the A260/230ration being 1.6-2.4. We also recommend that theDNA is high molecular weight and available foramplification. This is a key point, the DNA may bepresent and quantifiable by OD but it may notamplify. To check this we recommend using a realtimePCR system such as SYBR green to check DNAactivity and normalize the concentration.“We believe that the more factorstaken into consideration forimproving accuracy, the higheraccuracy will be achieved.”We also recommend that there is minimal EDTAin the elution buffers, because the EDTA caninterfere, particularly when it's at high concentration.If [EDTA] is up around 0.5 mM, then it can interferewith the activity of some of the enzymes that we use,so we recommend that they should elute in eitherwater, 10mM TRIS or reduced TE.— Darryl IrwinThe whole process of genotyping (from organism togenotype calling) should bemade as routine and— Huanming Yangstreamlined as possible.Some form of liquid-handlingrobot is essential for anythingother than modest amountsof genotyping, and willmarkedly reduce errorsintroduced by manualpipetting. Reducing thenumber of pipette steps andsample movement among plates is also important tominimize the possibility of contamination across wells.To minimize handling of our valuable DNA samples,using our liquid-handling robot we create manyreplicate sets of aliquoted DNA plates. Each set ofplates holds the entire panel of DNA samples, andeach well represents one sample, and containssufficient DNA for a single PCR reaction. The DNAaliquot plates are then dried and stored at -80ºC untilPCR. In this way we eliminate the need to repeatedlyfreeze/thaw DNA, and radically streamline the processof setting up PCR reactions.— Stuart Macdonald(continued on p.18)Genome Technology SNP Genotyping Tech Guide 11