COLD-PR-PCR结合两核苷酸合成测序（SDBA）研究低丰度基因突变

To meet the demand of high sensitive detection and analysis of low-abundance mutations, we aim to develop a coamplification at lower denaturation temperature proofreading PCR (COLD-PR-PCR) and subsequently combine it with sequencing with di-base addition (SDBA) to sensitively and accurately enrich and detect the low-abundance mutations. The main works in this project include: 1. The project develops a novel form of PCR, COLD-PR-PCR, which enables to selectively enrich known and unknown mutations. 2. Based on the developed COLD-PR-PCR, the project will design and fabricate a new DNA library fabrication approach for next-generation sequencing (NGS), which will meet requirements of medical applications and biological researches in low inputting DNA and less sequencing errors resulted from base misincorporation by PCR polymerases during library fabrication. 3. The project will combine COLD-PR-PCR with SDBA (we call it COLD-PR-PCR/SDBA) to qualitatively and quantitatively detect and identify low-abundance mutations present at malignant melanoma so as to study the potential of COLD-PR-PCR/SDBA in detection of low-abundance mutations. COLD-PR-PCR/SDBA is expected to discriminate mutations down to ～0.0001% abundance, reaching the limit of mutation detection in clinical samples, and it will establish a comprehensive technology for personalized medicine that enables not only to significantly enrich low-abundance mutations but also to greatly increase the detection sensitivity.

本项目面向低丰度基因突变分析检测的需求，旨在发展一种低变性温度下的校读PCR（COLD-PR-PCR）技术，并将该技术与高灵敏度的两核苷酸合成测序（SDBA）结合（简称“COLD-PR-PCR/SDBA”技术），实现低丰度基因突变的准确富集与高灵敏检测。主要工作包括：①发展COLD-PR-PCR技术，以富集低丰度基因突变；②基于COLD-PR-PCR建立一种高通量测序文库制备方法，以期减少测序模板起始量、降低聚合酶错误导致的测序背景噪声，满足临床检测和生命科学研究的需求；③将COLD-PR-PCR与SDBA结合，以黑素瘤BRAF基因为例，从定性到定量不断深入研究COLD-PR-PCR/SDBA技术在低丰度基因突变检测中的应用潜能。COLD-PR-PCR/SDBA技术有望实现～0.0001%的检测限，达到突变样本的检测极限，为个性化医疗建立一种集突变富集与超高灵敏度检测为一体的综合技术。

低丰度基因突变在包括癌症、产前诊断、感染性疾病、器官移植和法医学等医学领域具有重要的影响，检测低丰度基因突变在个性化医疗中尤为重要。本项目建立了一种COLD-PR-PCR技术，并将其与两核苷酸合成测序技术相结合，为肿瘤等疾病相关的低丰度基因突变临床检测提供新的技术手段。本项目首先验证了COLD-PR-PCR技术的技术原理，并对其NS浓度、起始DNA模板量、关键变性温度Tc等反应条件进行优化；其次，将其与两核苷酸合成测序技术结合，研究了不同NS浓度、不同扩增方式、以及不同测序方式对突变富集效果的影响。此外，通过选择特异性的两核苷酸加入方式，准确地实现同时识别恶性黑素瘤样本中BRAF 基因6种最常见的突变类型；最后，针对临床恶性黑素瘤样本BRAF基因的6种常见突变，编写了BRAF基因6种常见突变的自动定量软件，研究了在不同扩增方式和不同下游测序方式下低丰度基因突变的检测限，以及临床黑素瘤样本中BRAF基因突变的识别和定量，将COLD-PR-PCR结合两核苷酸合成测序技术的检测限降低至0.005%。