DNA polymorphic analysis from extremely small amounts of DNA samples using a semi-nested PCR

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Since jeffreys et al. reported that DNA with hypervariable minisatellite loci could be used for identification of individuals, DNA typing systems have been developed. Large amounts(micrograms)of gemonic DNA must be analyzed in these methods, so application of these methods to forensic science samples, consisting of degraded or very small amounts of DNA, is not always possible. The polymerase chain reaction(PCR)procedure has provided a solution to this problem, enabling the amplification of a target DNA region by 100,000 times. However, one finds that the large number of PCR cycles required to amplify extremely small amounts of DNA such as a single sperm cell makes for even less efficiency of the reaction, often resulting in insufficient amounts of PCR products. The nested PCR method is known to increase the amplification sensitivity and can be applied to various DNA analyzes.In the present study, we have improved the procedure of DNA purification or PCR to minimize the inhibition of the contaminated substances. Furthermore, we devised a new PCR system with a semi-nested set of primers in the second PCR to improve the amplification sensitivity of HLA-DQAI gene.Using the semi-nested PCR, more than 2 or 3 pg of template DNA could be amplified and typed correctly. The semi-nested PCR technique was found to enhance the sensitivity and efficiency of the amplification reaction and allowed the successful typing of the HLA-DQA1 gene. This is helpful for genotyping from samples with extremely small amounts of DNA, such as forensic or ancient DNA samples.