Development Of Assays For Single Nucleotide Changes And Their Application In DNA Pool Analysis

Aims: To develop assays for several single nucleotide changes using mutation-sensitiveon/off switch and to apply these assays in DNA pool analysis.Methods: DNA was extracted from blood samples of 300 blood donors usingphenol/chloroform method. Five ml blood was drew from the median cubital vein andthe blood was prevented from coagulation with EDTA treatment. All blood sampleswere stored at -20℃until use. The integrity of DNA samples was assessed with 1%agarose gel analysis, followed by quantitative determination with UV spectrometer, andaliquots stored at -20℃. The basic DNA pool unit was consisted of DNA samples from30 individuals. Ten basic units made up the DNA pool covering 300 individuals. Thetargets to be analyzed included: 3 Y chromosomal SNPs, 1 X chromosomal pointmutation, 2 mitochondrial point mutations, 1 pair of sex specific amplicons of X and Ychromosomes. All allele specific primers and their paring primers are phosphorothioatemodified at the 3' termini. To test the sensitivity and specificity of the assays thatrequired template concentrations higher than the maximal template concentrations ofnatural genomic DNA could reach, artificial templates were thus prepared using 1 roundof PCR for the wild type template and 2 rounds of PCR for the mutated templates. Theartificial templates were quantitatively measured and their copy numbers were calculated.The mixture of genomic DNA and the artificial templates were prepared in a series ofconcentrations for sensitivity and specificity analysis using the primers targetingmutations with the Real-Time PCR. The SNP and mutation detection assays were furtherapplied in the analysis of the DNA pool of 300 individuals to determine the sex ratio andto screen rare mutation frequencies.Results: The electrophoresis data demonstrated that the on/off switch mediated assayswere highly sensitive and specific for the 3 Y chromosomal SNPs. Real-Time PCR ofthese SNPs showed more than 3-5 cycles in the Ct1/2 between the on-switch andoff-switch mediated amplifications, indicating their practice value in SNP analysis.Similar to analysis of Y chromosomal SNP, electrophoreses data illustrated sensitive and specific discrimination of mitochondrial and X chromosomal point mutations. TheReal-Time PCR showed significant different Ct1/2 with at least differed from each otherby 3 cycles. These mutation assays can be used to screen rare mutations with allelefrequencies as low as 5%. Finally, with the quantitative analysis of X and Y specificamplicons, the sex ratio of the 300 individuals in the DNA pool was approached.Conclusion: The present study illustrated the diagnostic value of the mutation-sensitiveon/off switch consisting high fidelity DNA polymerase and phosphorothioate-modifiedallelic specific primers. Based on the analysis with Y chromosomal SNP, mitochondrialand X chromosomal point mutations, and sex specific amplicons, these newly developedassays were relative fast, less equipment-dependent, cost-effective. When combinedwith DNA pool, they are expected to be powerful tool in screening rare mutations relatedto mitochondrial and X chromosomal or other mutations.