Cloning, Sequencing and Site-directed Mutation of Lipoprotein Lipase Gene

The total RNA was isolated from the human fat tissues.According to the reported cDNA sequence of the lipoprotein lipase (LPL),primers were designed and synthesized.By means of RT\|PCR, one PCR fragment (1 6 kb) was obtained.The PCR products were cloned into pGEM\|T vector.Three clones were sequenced.Analysis of the nucleotide sequences showed that they were the same as that of the LPL cDNA.The polymorphisms of LPL gene were detected by denaturing high performance liquid chromatography(DHPLC) in coronary heart disease (CHD) patients, and a novel point mutation G830A within the fifth exon was found,which changed the 192 codon CGA into CAA and resulted in the substitution of glutamine for arginine.Site\|directed mutation based on PCR was used to produce the varied LPL cDNA,and another two primers surrounding the varied nucleotide were synthesized and the altered nucleotide was included in one of the primers.Two PCR fragments (843 bp and 811 bp)were ligated and their product was cloned into pGEM\|T vector,whose sequence was confirmed by DNA sequencing.