PCR products were 310 bp in size and were represented by an intense band when visualised with ethidium bromide (Figure 4). Secondary banding of size approximately 420 bp was present in some samples, while other samples had faint bands or no bands at all (Figure 5). These samples were repeated with varied DNA and magnesium concentration and annealing temperature until a good PCR product was obtained.
The A1 allele is represented by an uncut 310 bp fragment of DNA. The A2 allele is represented by two fragments of 130 bp and 180 bp. Polyacrylamide gel electrophoresis of the digested PCR products resolved these fragments (Figure 6).
Four individuals showed intense A1 bands, indicating homozygosity for A1, however they also had faint A2 bands (Figure 7). PCR for these subjects was repeated. Two of the subjects were then clearly seen to be heterozygotes, indicating that the TaqI enzyme had failed to cleave all the DNA. A third was homozygous for A1 with no A2 bands detected. The fourth subject continued to show an intense A1 band with faint A2 bands, indicating possible contamination.
Figure 4. Results of agarose gel electrophoresis of PCR products. Lanes 1 - 8 contain PCR product from eight individuals, represented by a strong 310 bp band. Lane 9 contains the results of a blank reaction with no DNA. Lane 10 contains the pUC19/HpaII molecular weight marker.
Figure 5. Results of agarose gel electrophoresis of problematic DNA PCR products. Lane 1 contains the pUC19/HpaII molecular weight marker. Lanes 2 - 6 and 8 and 9 contain PCR product that also contains a larger secondary product of approximately 420 bp in size. Lane 7 contains the result of a PCR that did not work. Lane 10 contains the result of the blank reaction.
Figure 6. An example polyacrylamide gel. Lanes 4, 9 and 14 contain DNA from individuals homozygous for A1, which is represented by an intense band of 310 bp. Lanes 2, 3, 5, 7 and 10 contain DNA from individuals homozygous for A2, represented by two bands of 130 bp and 180 bp. Lanes 1, 6, 8, 11, 12 and 13 contain DNA from individuals who are heterozygous A1A2. They have all three bands. Lane 15 is the pUC19/HpaII marker lane
Figure 7. An example of a gel with faint secondary banding. Lane 3 contains an intense A1 band, but also faint A2 bands, indicating incomplete cleavage by TaqI or contamination.
