Objective A method combining the mutagenic primers PCR and restriction enzyme digestion was designed to facilitate the detection of gene mutation in familial defective apolipoprotein B 100 R3500W. Methods A pair of p...Objective A method combining the mutagenic primers PCR and restriction enzyme digestion was designed to facilitate the detection of gene mutation in familial defective apolipoprotein B 100 R3500W. Methods A pair of primer was designed and a mismatch nucleotide was introduced in its upstream primer. A segment of target DNA including the possibly mutated nucleotide was amplified by PCR and the products were digested by restriction enzyme Nco l. To overcome the potential false negative results due to improper digestion conditions, a segment of DNA with Nco1 cut size was added as reference. Results The target sequence was successfully amplified by PCR, producing a 144 bp DNA fragment as expected. When incubated with Nco1, the enzyme could digest the DNA, producing a 114 bp segment, only if it was amplified from the mutated gene, but not from the normal allele. This difference in length of DNA could be separated by electrophoresis on a 2%agarose gel. Thus we successfully detected two carriers of heterozygous FDB R3500W in 162 hypercholesterolemic patients. Conclusions Mutagenic primers PCR can be used to detect the gene mutation of apo B 100 R3500W, two cases were detected among 162 patients with hypercholesterolemia. It suggests that this mutation is not rare in China's Mainland.展开更多
文摘Objective A method combining the mutagenic primers PCR and restriction enzyme digestion was designed to facilitate the detection of gene mutation in familial defective apolipoprotein B 100 R3500W. Methods A pair of primer was designed and a mismatch nucleotide was introduced in its upstream primer. A segment of target DNA including the possibly mutated nucleotide was amplified by PCR and the products were digested by restriction enzyme Nco l. To overcome the potential false negative results due to improper digestion conditions, a segment of DNA with Nco1 cut size was added as reference. Results The target sequence was successfully amplified by PCR, producing a 144 bp DNA fragment as expected. When incubated with Nco1, the enzyme could digest the DNA, producing a 114 bp segment, only if it was amplified from the mutated gene, but not from the normal allele. This difference in length of DNA could be separated by electrophoresis on a 2%agarose gel. Thus we successfully detected two carriers of heterozygous FDB R3500W in 162 hypercholesterolemic patients. Conclusions Mutagenic primers PCR can be used to detect the gene mutation of apo B 100 R3500W, two cases were detected among 162 patients with hypercholesterolemia. It suggests that this mutation is not rare in China's Mainland.
