Multiple displacement amplification (MDA) of total genomic DNA from Meloidogyne spp. and comparison to crude DNA extracts in PCR of ITS1, 28S D2-D3 rDNA and Hsp90
2005
Skantar, Andrea | Carta, Lynn
Because the quantity of nematode specimens available for molecular analysis is often limited, the number of analyses possible is constrained by the availability of DNA. Multiple displacement amplification (MDA) was assessed for whole genome amplification of crude DNA from several Meloidogyne species. MDA produced microgram quantities of template that resulted in successful amplification of the ribosomal internal transcribed spacer (ITS1) and 28S D2-D3 expansion regions, producing PCR results that were comparable to template generated by the single nematode smash method. MDA greatly improved degenerate primer PCR of single-copy Hsp90, a gene which is more sensitive than multi-copy ribosomal genes to limited DNA template. MDA should expand the number of molecular analyses possible for single nematodes. MDA will also be useful for archiving DNA from valuable specimens and provide a way for laboratories to share identical genetic material for nematode diagnosis.
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