High Annealing Specificity of Mitochondrial DNA Primers Towards ±0.1°C Temperature Differences

INTRODUCTION

Mitochondrial DNA is widely used in population studies and forensic human identification through matrilineal lineage. Therefore, the development of mitochondrial DNA typing methodologies has been an interest among researchers recently. Optimization of Polymerase Chain Reaction (PCR) forms the basic platform in DNA typing methodologies that requires optimum annealing temperature along with setting the proper concentration of ions and PCR components for high product yield. Nevertheless, usually, the concentration of ions and volume of PCR components could be followed from the manufacturer’s protocol yet the determination of optimal annealing temperature for best yield is cumbersome. Many scholars suggested running gradient PCR 3-5°C below the melting temperature of primers to determine the optimum annealing temperature. In this study, we implemented the same concept but noted high specificity of mtDNA primers even between ±0.1°C differences in annealing temperature during PCR optimization which could widely influence the amplification of the target region¹.

The mtDNA target regions chosen in this study contain SNPs for the development of allele-specific PCR among the Malay population^2-4. This stage is classified as first-round PCR and is focused on this study⁵. The selected SNPs are located in both the control and coding regions to increase the discrimination power. The primers were designed using NCBI primer blast (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). The length of the target region was set from 250-450 bp to assist in forensic cases involving degraded samples. The parameter for primers was left in default settings except the Tm was set at 40-65°C.

For optimization purposes, only one human buccal swab sample was collected using FTA card. The 1.2 mm DNA containing FTA card was punched out and then purified using FTA purification reagent and TE buffer. The disc was left to dry completely. The DNA containing disc was added with PCR components such as ddH₂O (18.75 μL), 10x Taq buffer (2.5 μL), dNTP (0.5 μL), 10 μM forward primer (1 μL), 10 μM reverse primer (1μL) and Taq DNA polymerase (0.25 μL). The PCR amplification was carried out in T100 Thermocycler at pre-denaturation (94°C at 5 min), complete denaturation (94°C at the 30 sec), annealing (55°C at 30 sec), extension (72°C at 1 min) and final elongation (72°C at 7 min). The PCR amplification was run for 30 cycles. After completion, the products were separated through gel electrophoresis using 1.2% agarose gel and visualized under UV light.

The results of gel observation under UV light were summarized in Table 1. The pattern of annealing temperature of mtDNA primers lies between 49.5-51.3°C. The designed primers successfully amplified the target region within a narrow annealing temperature range.

The current focus to determine the optimum binding of primers is according to where the mtDNA primers bind to the template without the formation of secondary structures and high PCR product yield observed from the bright intensity of the DNA band on the gel. Initially, the annealing temperature of primers has been tried 1-2°C below the melting temperature of primers as suggested by the Vazyme manufacturer. Nevertheless, the primers failed to amplify in the suggested temperature range. After a series of amplification, the annealing temperature range of primers was determined from 3-5°C below the melting temperature of mtDNA primers. Besides that, significant changes in presence of DNA bands or their intensity were observed at ±0.1°C differences in annealing temperature during PCR optimization as indicated in Table 1.

In a previous study on the optimization of first round PCR, the amplified fragments were seen in lower quantity This might be due to optimizing primer annealing temperature in integers forms only. For instance, there was the presence of a DNA band (target region 903-1227) at Ta of 50°C but not when it is adjusted to 49 or 51°C. Even though, the amplification occurred at 50°C but the low intensity of the band shows low product yield. Thus, in this study, the annealing temperature was tested between the intervals of 49.8-50.6°C. It was proven that 49.9°C is the best optimal temperature for amplification of mtDNA target region from 903-1227 since non-specific binding did not occur and the DNA band on gel appeared the brightest compared to other temperature intervals.