John M. S. Bartlett.pdf - Bio-Nica.info

Mutation Detection Using RT-PCR-RFLP 321
Fig. 1. G88A within IFNGR1 is detected by FokI digestion of the PCR product using a 1-bp
mismatch primer. The upstream primer contains a 1-bp mismatch just proximal to its 3′ end
such that the 164-bp amplified product incorporates a restriction site for FokI in the presence of
guanine at nucleotide 88 (a allele) but not in the presence of adenine (b allele). A PCR product
of genotype ab is cleaved by endonuclease FokI and shows three bands, 164, 130, and 34 bp, on
the ethidium bromide-stained 4% agarose gel.
Lowercase nucleotides represent the mismatch base. Underlined nucleotides represent the
polymorphism site. Line 1: Part of the registered IFNGR1 cDNA sequence; line 2: the upstream
primer for mismatch PCR; line 3: restriction enzyme Fok I recognition site.
4. Notes
We were searching for amino acid polymorphisms in cytokine receptors. At first
we used the RT-PCR SSCP method for cDNA sequence. After confirmation of the
base substitutions of SSCP-positive samples, we designed primers for population
screening by RFLP analysis. Although RT-PCR RFLP is an easy and sensitive method
for examining whether an already-known base substitution is present within the sample
cDNA, there are some disadvantages involved. Concerning RT-PCR, the various kinds
of amplicons resulting from alternative splicing could prove to be an obstacle for
analysis. If the genomic DNA sequence is available, then analysis using PCR products
templated with genomic DNA is recommended. Changing the primer setting position
would be another solution. Concerning RFLP, this analysis is based on the fact that
each restriction enzyme recognizes a specific DNA sequence, and incomplete digestion
leads to a false-negative result. The causes of incomplete digestion are low enzyme
activity (in cases with long-term usage of the restriction enzyme), an extremely
small quantity of the enzyme compared with that of the DNA sample, reaction under
the wrong conditions, and the use of the wrong reaction buffer. Performance of the