Using the 2100 Bioanalyzer to Optimize the PCR Amplification of Mitochondrial DNA Sequences

In polymerase chain reaction (PCR) product analysis, a comparison of target amplicon yield to such PCR artifacts as primer-dimers and misprimed secondary products is essential in PCR assay optimization. This process requires accurate and reproducible measurement of amplified DNA products over a wide range of sizes and concentrations. The 2100 Bioanalyzer is ideally suited to provide rapid quantitative analysis of all products and byproducts in a PCR reaction, thereby facilitating the development of an optimized PCR amplification. Introduction Optimization of PCRs can often be a tedious and time-consuming process, particularly when the amplified segments contain long stretches of high or low melting temperature sequences. The objective of the optimization process is to minimize secondary products, such as primer-dimers and mispriming events, while maximizing the yield of the target amplicon. Analysis of the amplification products by gel electrophoresis is not particularly helpful because it is difficult to obtain accurate quantitative information about secondary PCR products. The absence of such quantitative information can delay and confound the optimization process and can even result in the final PCR Using the 2100 Bioanalyzer to Optimize the PCR Amplification of Mitochondrial DNA Sequences Application reaction being run under conditions that do not produce the maximum selectivity and yield. The 2100 Bioanalyzer is an ideal tool to address problems of PCR process optimization. Not only can secondary products be accurately and reproducibly measured, but secondary products that are similar in size to the desired target can also be resolved and quantified [1]. The ability to quantitate all of the reaction products provides the user with the opportunity to accurately characterize the mathematical relationship between specific reaction parameters and the overall efficiency of the PCR reaction. These relationships can then be used to facilitate the identification of the optimal reaction conditions. Mitochondrial DNA (mtDNA) is a good example of a target sequence that can present a number of PCR amplification difficulties. Human mtDNA is a small (16,569 bp) circular genome containing 37 genes found in cellular cytoplasm. The mitochondrial genome contains a noncoding region of 1100 bp known as a D-loop or control region. Within the D-loop is found two sections of highly variable DNA sequence. These regions, hypervariable region 1 (HV1) and hypervariable region 2 (HV2), contain sufficient sequence variation to be useful in human identity testing [2, 3]. When a sufficient amount of intact genomic DNA (gDNA) cannot be extracted from a sample, mtDNA becomes the next analysis tool because the cytoplasm of a single cell may contain up to 1000 copies of mtDNA. MtDNA is often extracted from small or badly degraded samples such as bone, teeth, or hair and amplified by PCR. The amplified product is used for DNA sequence analysis. Genomics