PCR Amplification of Nematode rDNA

Polymerase Chain Reaction

Polymerase chain reaction (PCR) (Mullis & Faloona 1987) has revolutionized molecular systematic and population genetic studies (Avise 1994). This technique, uses a thermostabile bacterial polymerase, Taq, to replicate DNA. Repeated cycling of the replication event produces millions of copies of a specific genomic region.

PCR reduces the amount of DNA required, and specific primers permit amplification of specific regions of the genome. Integration of PCR with RFLP (PCR-RFLP) provides a robust tool for molecular population genetic analysis. PCR-RFLP has been used for several population genetic studies, including screwworm fly (Taylor et al. 1996), salmon (Cronin et al. 1993) and humans (Torroni et al. 1994).

Random primers for PCR is another method for studying genetic variation. This method, known as random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR), can be used to detect genetic polymorphisms within and among species (Black et al. 1992, Kambhampati et al. 1992). Unlike PCR-RFLP, no information about the amplified fragments is available with RAPD-PCR (Black 1993). Thus, contamination of the DNA with another organism may cause misinterpretation of the fragment pattern.

A. PCR reagents: 
For 25 ul PCR reaction

X ul dd-H2O |

Y ul juvenile smash or cyst DNA (X+Y), plus distilled water = 25 ul
It is easiest to make a master mix of the reagents and alloquot to the individual PCR tubes.

C. Verify PCR reaction

  • Pour a 1% agarose gel
  • Load 4ul of the PCR product from each sample.
  • Load size marker (use a 100-1500, or 50-1000 bp marker).

If PCR reaction went well, expect to see a single band (amplicon) for each sample. Faint bands can occur in addition to the primary ampicon, these are usually artifacts and are usually not a problem.
B. Thermocycler reaction
Program the thermocycler for the following method:

Thirty five cycles of:

    • 94C for 45 sec
    • 50C for 1 min
    • 72C for 1 min
    1. Start thermocycler method and allow to heat up to 94C, then pause the program.
    2. Place PCR tubes in thermocycler heat block.
    3. Keep method paused for one minute after tubes are added, then start method.

After method is complete, store PCR products at 4C.



Here is an agarose gel from several Tylenchid nematode genera.