Use of a PCR-based amplification analysis as a substitute for the Southern blot method to determine the C4A and C4B genes

An investigation of the C4 gene arrangement in ethnic Chinese (Taiwanese)

“…We previously pointed out (Lee et al, 2006b) that the trimodular RCCX carries two TNXA genes with one adjacent to CYP21A1P and the other adjacent to CYP21A2 and these are specifically targeted by the respective primers for the CYP21A1P (primer 21P-in2) and CYP21A2 (primer 21-8bpR1) genes in PCR amplification. Therefore, C4 haplotype determination with one more Figure 2.…”

“…1). The PCR conditions and reaction mixture were as described previously (Lee et al ., 2006b). For amplification of the C4 fraction containing residues 1101–1106 adjacent to CYP21A1P , primers C4‐25exF1 (5′‐tct cca agg cta cat gcg gat‐3′, nt 96,634–96,614, GenBank accession no.…”

“…To determine the C4A and C4B genes, these two differential primary PCR products were individually used as templates in a secondary PCR. The previously described (Lee et al ., 2006b) paired primers, C4‐25inF (5′‐tgg gac aga gct ggt atg at‐3′, nucleotides 96,495–96,476; GenBank accession no. AL049547)/C4‐27inR (5′‐gtt cct gag gaa aaa ggg ag‐3′, nucleotides 95,959–95,978; GenBank accession no.…”

“…In this paper, we used the established differential polymerase chain reaction (PCR) method (Lee et al ., 2006a,b) to individually amplify C4 genes with residues 1101–1106 adjacent to the respective CYP21A1P and CYP21A2 genes, followed by Nla IV restriction digestion in a secondary PCR product to determine the C4A and C4B genes which were then used to categorize the haplotypes of the C4 gene arrangement from 100 individuals of our local Taiwanese population. In addition, analysis of the total C4 serum concentration for each haplotype is included.…”