Detect Hotspot Mutations In Deafness Genes Directly From Whole Blood By Pfu DNA Polymerase

Objective:To establish a simple accurate method to detect ten hotspot mutations in deafness genes utilizing the whole blood PCR and mutation sensitive "molecular switch", mediated by high-fidelity Pfu DNA polymerase and phosphorothioate modified allele specific primers.Methods:Peripheral blood samples from patients with deafness definitely diagnosed were collected. Genomic DNA were extracted and concentration were measured by spectrophotometer; DNA fragments of GJB2,SLC26A4,GJB3,mtDNA containing the ten hotspot mutations of each genomic DNA sample were amplified and the PCR products were directly sequenced. The genotype of the ten spot mutations in each sample was analyzed. The same PCR products were cloned in pMD19 vector and the corresponding mutant template were obtained by PCR mediated mutagenesis. These wild type and mutant cloned templates were used to test and optimize the allele specific primers for analyzing the hotspot mutations. Specific primer pairs for the hotspot mutations were designed according to NCBI nucleotide sequence database. In the high-fidelity DNA polyerase reaction system, only the template-matched phosphorothioate modified primers can extended and generate PCR products, while the mismatched primers would not extend and no product would be seen under the optimized PCR condition. The most specific primers were selected for mutation analysis with genomic DNA samples and the results were examined by direct sequencing. To eliminate DNA extraction, whole blood PCR were used as the template in PCR reaction and a mutant Pfu DNA polymerase and UNG-dUTP were included in the PCR syetem to establish a simple whole blood mutation detection method base on the AS-PCR technology.Results:PCR amplification products containing mutation hotspots of deafness genes were cloned and direct sequencing confirmed that no mutations were found in these samples. Corresponding mutations were obtained by mutagenesis based on these cloned wild type templates. Wild type and mutation specific primer pairs were tested and optimized. In the following mutation analysis of genomic DNA mediated by Pfu DNA polymerase, high specificity and efficiency were achieved. When using whole blood as template to do the mutation analysis, the PCR conditions to enhance the identification ability of the "molecular switch" were optimized. High specificity and sensitivity of whole blood mutation analysis were achieved. The results were confirmed by direct sequencing.Conclusions:This work successfully established whole blood PCR method with specific phosphorothioate modified primers mediated by Pfu DNA polymerase for detection of the ten hotspot mutations in deafness genes. This method is simple, fast and low cost without sacrifice specificity and sensitivity. It can be used in clininal genetic diagnostic lab and can be used for screening mutations carriers.