1. The reagent blank should not show any bands unless there is cross contamination. The growth hormone gene internal control should show a distinct band of 434 bp for all samples analyzed (see Fig. 1). The APC PCR product should show a distinct band of 97 bp. For a normal individual, the 97 bp PCR product will only be detected when the wild type PCR mix is used. Similarly, an individual who is heterozygous for the I1307K mutation will have the 97 bp band detected when either the wild type or mutant PCR mix is used.
2. The internal control is used to confirm the presence of amplifiable DNA in the sample and to monitor the efficiency of the amplification. In an individual who is a heterozygote for the mutation, the internal control bands in both wild type and mutant PCR mixes should be of equal intensities, and the same is true for the APC bands. If the internal control bands are of equal intensities but the APC bands in the wild type and mutant PCR mixes show a significant difference in intensity, one has to be cautious about the possibility of nonspecific amplification. DNA samples may need to be purified further before being subjected to
Fig. 1. I1307K mutation detection by allele specific PCR. Samples from four different patients (lanes 2 to 5) were subjected to PCR detection using either the wild type (W) or mutant (M) master mixes. Lanes 2 and 3 are blood samples and lanes 4 and 5 are buccal swabs. Lane 1 is a reagent blank control, with TE buffer substituted for DNA. A 123 bp DNA marker (MA) was used, and 2.5% agarose gel electrophoresis was performed to separate the PCR products. Growth hormone gene amplification shows a 434 bp band whereas APC gene amplification shows a 97 bp band. Primer-dimer running in front of the 97 bp band can be seen in some lanes. Patients 2 and 5 are heterozygous for the I130K mutation, whereas patients 3 and 4 do not carry the mutation.
amplification. Occasionally, for various reasons, the internal control bands for the two different mixes may not show the same intensity, and it is important to consider such differences when interpreting the results for the APC amplification.
3. Occasionally, primer-dimers may appear in the gel. These primer-dimers migrate in front of the 97 bp APC band and should not be confused with the APC band. A higher percentage of agarose gel may be used to achieve a better separation.
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