Genomics Laboratory, Department of Genetic Engineering, SRM Institute of Science and Technology, Kattankulathur 603 203, Tamil Nadu, India.
Genome. 2021 Mar;64(3):165-171. doi: 10.1139/gen-2019-0221. Epub 2020 May 20.
ITS2 marker is highly efficient in species discrimination but its application in DNA barcoding is limited due to huge variations in the PCR success rate. We have hypothesized that higher GC content and the resultant secondary structures formed during annealing might hinder the PCR amplification of ITS2. To test this hypothesis, we selected 12 species from 12 different families in which ITS2 was not amplified under standard PCR reaction conditions. In these samples, DMSO, formamide, betaine, and 7-deaza-dGTP were evaluated for their ability to improve the PCR success rate. The highest PCR success rate (91.6%) was observed with 5% DMSO, followed by 1 M betaine (75%), 50 μM 7-deaza-dGTP (33.3%), and 3% formamide (16.6%). The one sample that did not amplify with DMSO was amplified by adding 1 M betaine. However, combining DMSO and betaine in the same reaction did not improve the PCR. Therefore, to achieve the highest PCR success rate for ITS2, it is recommended to include 5% DMSO by default and substitute it with 1 M betaine only in the case of failed reactions. When this strategy was tested in 50 species from 43 genera and 29 families, the PCR success rate of ITS2 increased from 42% to 100%.
ITS2 标记在物种鉴别中非常有效，但由于 PCR 成功率的巨大差异，其在 DNA 条形码中的应用受到限制。我们假设较高的 GC 含量和退火过程中形成的二级结构可能会阻碍 ITS2 的 PCR 扩增。为了验证这一假设，我们从 12 个不同科的 12 个物种中选择了一些样本，这些样本在标准 PCR 反应条件下无法扩增 ITS2。在这些样本中，评估了 DMSO、甲酰胺、甜菜碱和 7-脱氮-dGTP 提高 PCR 成功率的能力。添加 5% DMSO 可获得最高的 PCR 成功率（91.6%），其次是 1 M 甜菜碱（75%）、50 μM 7-脱氮-dGTP（33.3%）和 3%甲酰胺（16.6%）。有一个样本在添加 DMSO 后仍无法扩增，但添加 1 M 甜菜碱后可以扩增。然而，将 DMSO 和甜菜碱同时添加到同一反应中并不能提高 PCR 效果。因此，为了获得最高的 ITS2 PCR 成功率，建议默认添加 5% DMSO，仅在反应失败的情况下用 1 M 甜菜碱替代。当将该策略应用于 43 个属和 29 个科的 50 个物种时，ITS2 的 PCR 成功率从 42%提高到 100%。
