Buccal cells DNA extraction to obtain high quality human genomic DNA suitable for polymorphism genotyping by PCR-RFLP and Real-Time PCR.
Buccal cells DNA extraction to obtain high quality human genomic DNA suitable for polymorphism genotyping by PCR-RFLP and Real-Time PCR.
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DOI：
10.1590/s1678-77572012000400013
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发表时间：
2012-07
期刊：
影响因子：
--
通讯作者：
Amorim LM
中科院分区：
文献类型：
--
作者：
Küchler EC;Tannure PN;Falagan-Lotsch P;Lopes TS;Granjeiro JM;Amorim LM
本研究的目的是评估，通过PCR-RFLP和Real-time PCR，从口腔细胞中收集的基因组DNA的产量和质量的漱口水后，在室温下不同的存储时间。 招募了一组志愿者，使用漱口水溶液收集口腔细胞。将收集的溶液分到3个试管中，一个试管用于立即提取，其余试管接受乙醇并在室温下保持4天和8天，然后进行DNA提取。采用分光光度法和电泳法测定DNA的浓度、纯度和完整性。还评价了三个孵育时间之间的DNA质量差异，通过PCR-RFLP对EGF +61 A/G（rs 4444903）多态性进行基因分型，并使用实时PCR对IRF 6多态性（rs 17015215）进行基因分型。 在三种不同的孵育时间之间，DNA产量（p=0.75）和纯度（p=0.86）没有显著差异。不同孵育时间的DNA均呈现高分子量。对所有DNA样品成功地进行了PCR-RFLP和真实的时间PCR反应，即使是孵育8天后提取的那些。所有通过实时PCR进行基因分型的样本均呈现IRF 6基因多态性的C等位基因（纯合子：CC;杂合子：CT），并且C等位基因用作Ct值的参考。在两种技术中，样品在不同时间呈现相同的基因型。 我们证明本文所述的方法简单且成本低，并且可以在室温下储存在漱口水溶液中后提取DNA并进行PCR扩增。
The aim of this study was to evaluate, by PCR-RFLP and Real-time PCR, the yield and quality of genomic DNA collected from buccal cells by mouthwash after different storage times at room temperature. A group of volunteers was recruited to collect buccal cells using a mouthwash solution. The collected solution was divided into 3 tubes, one tube were used for immediate extraction and the remaining received ethanol and were kept at room temperature for 4 and 8 days followed by DNA extraction. The concentration, purity and integrity of the DNA were determined using spectrophotometry and electrophoresis. DNA quality differences among the three incubation times were also evaluated for genotyping EGF +61 A/G (rs 4444903) polymorphism by PCR-RFLP and for IRF6 polymorphism (rs 17015215) using Real-Time PCR. There was no significant difference of DNA yield (p=0.75) and purity (p=0.86) among the three different incubation times. DNA obtained from different incubation times presented high-molecular weight. The PCR-RFLP and Real time PCR reactions were successfully performed for all DNA samples, even those extracted after 8 days of incubation. All samples genotyped by Real-Time PCR presented C allele for IRF6 gene polymorphism (homozygous: CC; heterozygous: CT) and the C allele was used as a reference for Ct values. The samples presented the same genotype for the different times in both techniques. We demonstrated that the method described herein is simple and low cost, and that DNA can be extracted and PCR amplified after storage in mouthwash solution at room temperature.
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