Author
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Skantar, Andrea |
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Carta, Lynn |
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Submitted to: Biotechniques
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 9/1/2000 Publication Date: N/A Citation: N/A Interpretive Summary: Plant-parasitic nematodes are microscopic worms that annually cause agricultural losses of ten billion dollars in the United States. Regulatory agencies that monitor the safety of imported and exported crops must rely on swift and accurate identification of plant-parasitic nematodes from quarantine samples. New molecular diagnostics, particularly ones based upon behavioral or developmental traits of nematodes, should help to alleviate problems encountered when there is insufficient material for microscopic diagnosis. One such nematode trait, the response to stress, appears to be governed by heat shock protein (Hsp) genes. Therefore, we set out to develop Hsp90 genes as novel diagnostic molecules for selected nematodes. We modified the polymerase chain reaction (PCR) to create a new method called ramped annealing (RAN) PCR to allow ample production of desired nematode Hsp90 genes while eliminating undesirable contaminants. RAN-PCR successfully copied Hsp90 genes from genomic DNA of soybean cyst, root knot, and lesion nematodes. In addition to diagnostic applications, further characterization of Hsp90 genes should increase our fundamental understanding of how nematodes respond to environmental stressors, such as temperature. The RAN-PCR strategy we developed eliminated the need for time-consuming and expensive PCR optimization procedures. This technique may be used by scientists in situations when other methods have failed to produce the desired PCR products. Technical Abstract: Procedures for using PCR to amplify novel members of gene families from genomic DNA frequently involve the use of degenerate primers. Primer design protocols and software are improving, but finding the optimal amplification conditions often involves a tremendous amount of time and expense. Of key importance is the ability to produce ample quantities of specific PCR product, while minimizing or eliminating unspecific side reactions. Ramped annealing (RAN) PCR is an adaptation of the principle in touchdown PCR, but differs in that the annealing temperature is reduced within each PCR cycle rather than between cycles. Each annealing step starts at a relatively stringent temperature above the primer melting temperature (Tm), then is reduced gradually and held for several seconds at a temperature well below the primer Tm. RAN-PCR successfully amplified Hsp90 sequences from purified nematode genomic DNA and from crude single worm DNA extracts. This modification may be broadly applicable in degenerate or multiplex PCR when other methods have failed to produce the desired PCR products. It may also provide a flexible starting point for PCR optimization of uncharacterized DNA templates. |
