DMSO Increases Mutation Scanning Detection Sensitivity of High-Resolution Melting in Clinical Samples.
DMSO Increases Mutation Scanning Detection Sensitivity of High-Resolution Melting in Clinical Samples.
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DOI：
10.1373/clinchem.2015.245357
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发表时间：
2015-11
影响因子：
9.3
通讯作者：
Makrigiorgos GM
中科院分区：
文献类型：
--
作者：
Song C;Castellanos-Rizaldos E;Bejar R;Ebert BL;Makrigiorgos GM
突变扫描提供了用于鉴定单个PCR扩增子上的DNA变异的最简单、最低成本的方法，并且可以在测序之前进行，以避免筛选无信息的野生型样品。高分辨率熔解（HRM）是最常用的突变扫描方法。然而，通过使用PCR-HRM，低于10%的突变仍然可能是临床显著的，可能经常被错过。因此，提高HRM检测灵敏度对突变扫描及其临床应用具有重要意义。我们使用含有TP 53外显子8突变的细胞系的系列稀释来证明在DMSO存在下常规-PCR-HRM的检测灵敏度的提高。我们还进行了全COLD-PCR以在HRM±DMSO之前进一步富集低水平突变，并采用液滴数字PCR来获得突变富集的最佳条件。使用常规-PCR-HRM和全-COLD-PCR-HRM ±DMSO对含有已知突变的癌症样本和含有未知突变的骨髓增生异常综合征样本中的TP 53外显子8进行突变扫描。其他基因的突变也进行了检测。在DMSO存在下，PCR-HRM扫描的检测灵敏度增加2-5倍，这也取决于突变类型和序列背景，并且通常可以检测约1%的突变丰度。当在使用全COLD-PCR的扩增期间应用突变富集，然后在DMSO存在下进行HRM时，可以检测到TP 53外显子8中具有0.2-0.3%突变丰度的突变。DMSO提高了HRM突变扫描灵敏度。当采用全COLD-PCR，然后DMSO-HRM时，与常规PCR-HRM相比，总体改善约20倍。
Mutation scanning provides the simplest, lowest cost method for identifying DNA variations on single PCR amplicons, and it may be performed prior to sequencing to avoid screening of non-informative wild type samples. High resolution melting (HRM) is the most commonly used method for mutation scanning. However, by using PCR-HRM mutations below ≈ 3–10% that can still be clinically significant may often be missed. Therefore, enhancing HRM detection sensitivity is important for mutation scanning and its clinical application. We used serial dilution of TP53 exon 8 mutation containing cell lines to demonstrate the improvement in detection sensitivity for conventional-PCR-HRM in the presence of DMSO. We also conducted full-COLD-PCR to further enrich low-level mutations prior to HRM±DMSO and employed droplet-digital PCR to derive the optimal conditions for mutation enrichment. Both conventional-PCR-HRM and full-COLD-PCR-HRM ±DMSO were used for mutation scanning in TP53 exon 8 in cancer samples containing known mutations and in myelodysplastic syndrome samples with unknown mutations. Mutations in other genes were also examined. The detection sensitivity of PCR-HRM-scanning increases 2–5-fold in the presence of DMSO, depending also on mutation type and sequence context, and can typically detect mutation abundance of about 1%. When mutation enrichment is applied during amplification using full-COLD-PCR and followed by HRM in the presence of DMSO, mutations with 0.2–0.3% mutation abundance in TP53 exon 8 can be detected. DMSO improves HRM mutation scanning sensitivity. When full-COLD-PCR is employed, followed by DMSO-HRM, the overall improvement is about 20-fold as compared to conventional PCR-HRM.
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影响因子：
3.9
作者：
Li, Jin;Milbury, Coren A.;Li, Cheng;Makrigiorgos, G. Mike
通讯作者：
Makrigiorgos, G. Mike
影响因子：
9.5
作者：
Pabinger S;Dander A;Fischer M;Snajder R;Sperk M;Efremova M;Krabichler B;Speicher MR;Zschocke J;Trajanoski Z
通讯作者：
Trajanoski Z
影响因子：
14.9
作者：
Chakrabarti, R;Schutt, CE
通讯作者：
Schutt, CE
影响因子：
9.3
作者：
Milbury CA;Correll M;Quackenbush J;Rubio R;Makrigiorgos GM
通讯作者：
Makrigiorgos GM
影响因子：
4.1
作者：
Castellanos-Rizaldos, Elena;Paweletz, Cloud;Makrigiorgos, G. Mike
通讯作者：
Makrigiorgos, G. Mike