Site-directed mutagenesis of long QT syndrome KCNQ1 gene in vitro

Abstract

To construct a polymerase chain reaction (PCR) site-directed mutagenesis of the long QT syndrome KCNQ1 gene in vitro, two sets of primers were designed according to the sequence of KCNQ1 cDNA and a mismatch was introduced into primers. Mutagenesis was performed in a two-step PCR. The amplified fragments from the third PCR which contained the mutation site were sub-cloned into the T-vector pCR2.1. Then, the fragments containing the mutation site was obtained from pCR2.1 using restriction enzymes digestion and inserted into the same restriction site of pIRES 2 -EGFP-KCNQ1. The sequencing analysis shows that the mutation site was correct. Mutation from A to G in site 983 of KCNQ1 cDNA was found. Using the Effectene transfection reagent, pIRES 2 -EGFP-KCNQ1 (G983A) was transfected into HEK cells successfully. These results may shed light on further functional study of KCNQ1 gene.

A novel missense mutation in the ALPL gene causes dysfunction of the protein

“…Next, the recombinant vectors were transformed into competent Escherichia coli cells. The transformation procedures and colony PCR were performed as previously described ( 12 ). The recombinant plasmids were extracted using Plasmid Preparation kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol.…”