CN107119331A - The construction method of tumour radiotherapy virulent gene mutated library - Google Patents
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Abstract
Description
技术领域technical field
本发明涉及一种用于高通量测序检测的肿瘤放射治疗毒性基因突变文库的构建方法。The invention relates to a method for constructing a tumor radiotherapy toxicity gene mutation library for high-throughput sequencing detection.
背景技术Background technique
恶性肿瘤是我国及全球主要的公共健康问题。在我国肿瘤死亡占全部死因的1/4。放射治疗对多种恶性肿瘤均有治疗作用，是常用的治疗方法之一。越来越多的患者通过放射治疗减轻了病痛，延长了生命。大约70％的恶性肿瘤患者，在其病程的某一时期需要不同目的的放射治疗参与，包括根治性放疗、辅助性放疗或姑息性放疗。放疗杀伤肿瘤细胞的机制是通过放射线作用于生物体产生刺激电子引电离损伤DNA分子，或通过射线与生物组织内水分子的作用产生自由基损伤DNA分子，当损伤超出细胞的修复能力是即导致细胞死亡和组织破坏。Malignant tumors are a major public health problem in my country and the world. In my country, tumor death accounts for 1/4 of all deaths. Radiation therapy has a therapeutic effect on a variety of malignant tumors and is one of the commonly used treatment methods. More and more patients have alleviated their pain and prolonged their lives through radiation therapy. About 70% of patients with malignant tumors need radiation therapy for different purposes at a certain stage of their disease course, including radical radiotherapy, adjuvant radiotherapy or palliative radiotherapy. The mechanism of radiotherapy to kill tumor cells is to stimulate electrons to ionize and damage DNA molecules through the action of radiation on organisms, or to generate free radicals to damage DNA molecules through the action of radiation and water molecules in biological tissues. When the damage exceeds the repair ability of cells, it will cause Cell death and tissue destruction.
完美的肿瘤治疗方法最好是对肿瘤组织杀伤有效且无毒副作用。随着科学技术的发展，人们逐渐改善了传统放射治疗技术发展起能够更精准杀伤肿瘤组织的治疗方法，从而减少了正常组织在治疗中所受到的辐射剂量。但是临床实际肿瘤治疗中，放疗在杀伤恶性肿瘤细胞的同时，肿瘤附近的正常组织和器官也不可避免地受到放射性损伤，从而产生放射性治疗毒副作用。心脏毒性是最威胁患者生命的放疗毒副作用之一，放射诱发心脏病，属于晚期并发症,一般在放疗10-20年后出现；表现为心包炎、心肌病、心脏瓣膜病、传导异常和冠状动脉狭窄。在HD长期生存患者中，有2％-5％的患者最终死于心脏损伤性疾病而非原发疾病，心肌梗死则是这些肿瘤治愈后长期生存患者的主要死亡原因。放疗引起的毒副作用给患者带来了不同程度的痛苦，严重者还影响治疗的顺利进行。A perfect tumor treatment method should be effective in killing tumor tissue without toxic side effects. With the development of science and technology, people have gradually improved the traditional radiation therapy technology and developed a treatment method that can kill tumor tissue more accurately, thereby reducing the radiation dose received by normal tissues during treatment. However, in actual clinical tumor treatment, while radiotherapy kills malignant tumor cells, normal tissues and organs near the tumor are inevitably damaged by radiation, resulting in toxic and side effects of radiotherapy. Cardiotoxicity is one of the most life-threatening side effects of radiotherapy. Radiation-induced heart disease is a late complication that usually occurs 10-20 years after radiotherapy; manifested as pericarditis, cardiomyopathy, valvular heart disease, conduction abnormalities, and coronary heart disease. The arteries are narrowed. Among the long-term survivors of HD, 2%-5% of the patients eventually died of cardiac injury rather than the primary disease, and myocardial infarction was the main cause of death for these long-term survivors after the tumor was cured. The toxic and side effects caused by radiotherapy have brought different degrees of pain to patients, and in severe cases, it also affects the smooth progress of treatment.
据预测80％对临床治疗反应的个体差异是有患者相关的个体因素导致的，确定这些因素可以预测患者发展严重毒副反应的风险。因此识别患者个体从放疗获得的副反应的易感性是肿瘤个体化放疗的一个重要先决条件。通过发现与患者放疗毒副作用相关的风险因子，评估患者相关因子的表达从而预测患者的放疗毒副反应易感性，可以对正常组织耐受放疗的患者通过增加肿瘤放射剂量来扩大治疗窗口；另一方面对正常组织毒副反应具有高风险的患者可能更改治疗方案、换成手术或化疗干预改善症状等，从而为患者提供有效且毒副反应小的治疗方案，改善患者的生存情况。It is predicted that 80% of the individual variation in response to clinical treatment is due to patient-related individual factors, and the identification of these factors can predict the risk of patients developing serious toxic side effects. Therefore, identifying the susceptibility of individual patients to side effects from radiotherapy is an important prerequisite for individualized radiotherapy for tumors. By discovering the risk factors related to the side effects of radiotherapy in patients and evaluating the expression of patient-related factors to predict the susceptibility of patients to radiotherapy side effects, the treatment window can be expanded by increasing the tumor radiation dose for patients whose normal tissues are resistant to radiotherapy; another On the one hand, patients with high risk of normal tissue toxic side effects may change their treatment plan, switch to surgery or chemotherapy intervention to improve symptoms, etc., so as to provide patients with an effective and less toxic treatment plan and improve their survival.
发明内容Contents of the invention
针对现有技术中存在的上述不足，本发明提供了一种用于高通量测序检测的肿瘤放射治疗毒性基因突变文库的构建方法。Aiming at the above-mentioned deficiencies in the prior art, the present invention provides a method for constructing a tumor radiotherapy toxicity gene mutation library for high-throughput sequencing detection.
为了解决上述技术问题，本发明采用了如下技术方案：In order to solve the problems of the technologies described above, the present invention adopts the following technical solutions:
肿瘤放射治疗毒性基因突变文库的构建方法，覆盖人类基因XRCC1、TNFa、TXNRD2、ERCC2、TGFB1、VEGF、DNMT1、TP53、XRCC3、LIN28B、MTHFR、PON1、GSTP1、ATM、NOS2、APEX1、MLH1、IL8、CD44、LIG4、IL1A、TNF、CYP2C8、IL4、NEIL1、NBN、APC、NFKBIA、DEAD、Tpa、RAD51、NFE2L2、GSTA1、MGMT、TDG、GSK3B、FSHR、MYO3B、TGFBR2、5q31、HSPB1和TXN上的总共61种遗传性变异位点，具体包括如下步骤：The construction method of tumor radiotherapy toxicity gene mutation library, covering human genes XRCC1, TNFa, TXNRD2, ERCC2, TGFB1, VEGF, DNMT1, TP53, XRCC3, LIN28B, MTHFR, PON1, GSTP1, ATM, NOS2, APEX1, MLH1, IL8, Total on CD44, LIG4, IL1A, TNF, CYP2C8, IL4, NEIL1, NBN, APC, NFKBIA, DEAD, Tpa, RAD51, NFE2L2, GSTA1, MGMT, TDG, GSK3B, FSHR, MYO3B, TGFBR2, 5q31, HSPB1 and TXN 61 kinds of genetic variation sites, including the following steps:
(1)针对目的基因XRCC1、TNFa、TXNRD2、ERCC2、TGFB1、VEGF、DNMT1、TP53、XRCC3、LIN28B、MTHFR、PON1、GSTP1、ATM、NOS2、APEX1、MLH1、IL8、CD44、LIG4、IL1A、TNF、CYP2C8、IL4、NEIL1、NBN、APC、NFKBIA、DEAD、Tpa、RAD51、NFE2L2、GSTA1、MGMT、TDG、GSK3B、FSHR、MYO3B、TGFBR2、5q31、HSPB1和TXN上设计基本扩增引物组，该基本扩增引物组的正向引物和反向引物的5’端设有额外的2～5个T，且该2～5个T的靠近3’端的第一个T具有PNA修饰，同时该基本扩增引物组的Tm值相差不超过1℃；(1) For target genes XRCC1, TNFa, TXNRD2, ERCC2, TGFB1, VEGF, DNMT1, TP53, XRCC3, LIN28B, MTHFR, PON1, GSTP1, ATM, NOS2, APEX1, MLH1, IL8, CD44, LIG4, IL1A, TNF, Basic amplification primer sets were designed on CYP2C8, IL4, NEIL1, NBN, APC, NFKBIA, DEAD, Tpa, RAD51, NFE2L2, GSTA1, MGMT, TDG, GSK3B, FSHR, MYO3B, TGFBR2, 5q31, HSPB1 and TXN. The 5' end of the forward primer and reverse primer of the augmentation primer set is provided with an additional 2-5 Ts, and the first T of the 2-5 Ts near the 3' end has a PNA modification, and the basic amplification The Tm values of the primer sets differ by no more than 1°C;
(2)将模板、上述基本扩增引物组置于一含有RingCap-Taq酶的PCR反应体系中进行扩增，纯化后得扩增产物；将扩增产物、多对第一不对称连接探针、通用引物和上述RingCap-Taq酶混合进行PCR，纯化后获得文库产物；(2) Place the template and the above-mentioned basic amplification primer set in a PCR reaction system containing RingCap-Taq enzyme for amplification, and obtain the amplification product after purification; the amplification product, multiple pairs of the first asymmetric connection probe , universal primers and the above-mentioned RingCap-Taq enzyme are mixed for PCR, and the library product is obtained after purification;
(3)将文库产物组成所述用于高通量测序的基因变异文库；(3) Composing the library products into the gene variation library for high-throughput sequencing;
上述RingCap-Taq酶由Taq酶、DNA连接酶和DNA末端修饰酶组成。The aforementioned RingCap-Taq enzyme consists of Taq enzyme, DNA ligase and DNA end modification enzyme.
作为本发明的一种优选方案，所述基本扩增引物组包括FL-XRCC1-1-F、FL-XRCC1-1-R、FL-TNFa-1-F、FL-TNFa-1-R、FL-TXNRD2-1-F、FL-TXNRD2-1-R、FL-ERCC2-1-F、FL-ERCC2-1-R、FL-TGFB1-1-F、FL-TGFB1-1-R、FL-VEGF-1-F、FL-VEGF-1-R、FL-DNMT1-1-F、FL-DNMT1-1-R、FL-XRCC1-2-F、FL-XRCC1-2-R、FL-TP53-1-F、FL-TP53-1-R、FL-XRCC3-1-F、FL-XRCC3-1-R、FL-LIN28B-1-F、FL-LIN28B-1-R、FL-XRCC3-2-F、FL-XRCC3-2-R、FL-MTHFR-1-F、FL-MTHFR-1-R、FL-PON1-1-F、FL-PON1-1-R、FL-GSTP1-1-F、FL-GSTP1-1-R、FL-ATM-1-F、FL-ATM-1-R、FL-XRCC1-3-F、FL-XRCC3-1-R、FL-LIN28B-1-F、FL-LIN28B-1-R、FL-XRCC3-2-F、FL-XRCC3-2-R、FL-MTHFR-1-F、FL-MTHFR-1-R、FL-PON1-1-F、FL-PON1-1-R、FL-GSTP1-1-F、FL-GSTP1-1-R、FL-ATM-1-F、FL-ATM-1-R、FL-XRCC1-3-F、FL-XRCC1-3-R、FL-NOS2-1-F、FL-NOS2-1-R、FL-TGFB1-2-F、FL-TGFB1-2-R、FL-APEX1-1-F、FL-APEX1-1-R、FL-VEGF-2-F、FL-VEGF-2-R、FL-XRCC1-4-F、FL-XRCC1-4-R、FL-MLH1-1-F、FL-MLH1-1-R、FL-IL8-1-F、FL-IL8-1-R、FL-CD44-1-F、FL-CD44-1-R、FL-LIG4-1-F、FL-LIG4-1-R、FL-IL1A-1-F、FL-IL1A-1-R、FL-TNF-1-F、FL-TNF-1-R、FL-CYP2C8-1-F、FL-CYP2C8-1-R、FL-IL1A-2-F、FL-IL1A-2-R、FL-IL4-1-F、FL-IL4-1-R、FL-IL4-2-F、FL-IL4-2-R、FL-NEIL1-1-F、FL-NEIL1-1-R、FL-NBN-1-F、FL-NBN-1-R、FL-APC-1-F、FL-APC-1-R、FL-NFKBIA-1-F、FL-NFKBIA-1-R、FL-DEAD-1-F、FL-DEAD-1-R、FL-ATM-2-F、FL-ATM-2-R、FL-IL8-2-F、FL-IL8-2-R、FL-ATM-3-F、FL-ATM-3-R、FL-tPA-1-F、FL-tPA-1-R、FL-TGFB1-3-F、FL-TGFB1-3-R、FL-RAD51-1-F、FL-RAD51-1-R、FL-NFE2L2-1-F、FL-NFE2L2-1-R、FL-GSTA1-1-F、FL-GSTA1-1-R、FL-MGMT-1-F、FL-MGMT-1-R、FL-ATM-4-F、FL-ATM-4-R、FL-TDG-1-F、FL-TDG-1-R、FL-TDG-2-F、FL-TDG-2-R、FL-GSK3B-1-F、FL-GSK3B-1-R、FL-NOS2-2-F、FL-NOS2-2-R、FL-XRCC1-5-F、FL-XRCC1-5-R、FL-FSHR-1-F、FL-FSHR-1-R、FL-MYO3B-1-F、FL-MYO3B-1-R、FL-TGFBR2-1-F、FL-TGFBR2-1-R、FL-TGFBR2-2-F、FL-TGFBR2-2-R、FL-5q31-1-F、FL-5q31-1-R、FL-LIN28B-2-F、FL-LIN28B-2-R、FL-HSPB1-1-F、FL-HSPB1-1-R、FL-NBN-2-F、FL-NBN-2-R、FL-TXN-1-F和FL-TXN-1-R。As a preferred solution of the present invention, the basic amplification primer set includes FL-XRCC1-1-F, FL-XRCC1-1-R, FL-TNFa-1-F, FL-TNFa-1-R, FL -TXNRD2-1-F, FL-TXNRD2-1-R, FL-ERCC2-1-F, FL-ERCC2-1-R, FL-TGFB1-1-F, FL-TGFB1-1-R, FL-VEGF -1-F, FL-VEGF-1-R, FL-DNMT1-1-F, FL-DNMT1-1-R, FL-XRCC1-2-F, FL-XRCC1-2-R, FL-TP53-1 -F, FL-TP53-1-R, FL-XRCC3-1-F, FL-XRCC3-1-R, FL-LIN28B-1-F, FL-LIN28B-1-R, FL-XRCC3-2-F , FL-XRCC3-2-R, FL-MTHFR-1-F, FL-MTHFR-1-R, FL-PON1-1-F, FL-PON1-1-R, FL-GSTP1-1-F, FL -GSTP1-1-R, FL-ATM-1-F, FL-ATM-1-R, FL-XRCC1-3-F, FL-XRCC3-1-R, FL-LIN28B-1-F, FL-LIN28B -1-R, FL-XRCC3-2-F, FL-XRCC3-2-R, FL-MTHFR-1-F, FL-MTHFR-1-R, FL-PON1-1-F, FL-PON1-1 -R, FL-GSTP1-1-F, FL-GSTP1-1-R, FL-ATM-1-F, FL-ATM-1-R, FL-XRCC1-3-F, FL-XRCC1-3-R , FL-NOS2-1-F, FL-NOS2-1-R, FL-TGFB1-2-F, FL-TGFB1-2-R, FL-APEX1-1-F, FL-APEX1-1-R, FL -VEGF-2-F, FL-VEGF-2-R, FL-XRCC1-4-F, FL-XRCC1-4-R, FL-MLH1-1-F, FL-MLH1-1-R, FL-IL8 -1-F, FL-IL8-1-R, FL-CD44-1-F, FL-CD44-1-R, FL-LIG4-1-F, FL-LIG4-1-R, FL-IL1A-1 -F, FL-IL1A-1-R, FL-TNF-1-F, FL-TNF-1-R, FL-CYP2C8-1-F, FL-CYP2C8-1-R, FL-IL1A-2-F , FL-IL1A-2-R, FL-IL4-1-F, FL-IL4-1-R, FL-IL4-2-F, FL-IL4-2-R, FL -NEIL1-1-F, FL-NEIL1-1-R, FL-NBN-1-F, FL-NBN-1-R, FL-APC-1-F, FL-APC-1-R, FL-NFKBIA -1-F, FL-NFKBIA-1-R, FL-DEAD-1-F, FL-DEAD-1-R, FL-ATM-2-F, FL-ATM-2-R, FL-IL8-2 -F, FL-IL8-2-R, FL-ATM-3-F, FL-ATM-3-R, FL-tPA-1-F, FL-tPA-1-R, FL-TGFB1-3-F , FL-TGFB1-3-R, FL-RAD51-1-F, FL-RAD51-1-R, FL-NFE2L2-1-F, FL-NFE2L2-1-R, FL-GSTA1-1-F, FL -GSTA1-1-R, FL-MGMT-1-F, FL-MGMT-1-R, FL-ATM-4-F, FL-ATM-4-R, FL-TDG-1-F, FL-TDG -1-R, FL-TDG-2-F, FL-TDG-2-R, FL-GSK3B-1-F, FL-GSK3B-1-R, FL-NOS2-2-F, FL-NOS2-2 -R, FL-XRCC1-5-F, FL-XRCC1-5-R, FL-FSHR-1-F, FL-FSHR-1-R, FL-MYO3B-1-F, FL-MYO3B-1-R , FL-TGFBR2-1-F, FL-TGFBR2-1-R, FL-TGFBR2-2-F, FL-TGFBR2-2-R, FL-5q31-1-F, FL-5q31-1-R, FL -LIN28B-2-F, FL-LIN28B-2-R, FL-HSPB1-1-F, FL-HSPB1-1-R, FL-NBN-2-F, FL-NBN-2-R, FL-TXN -1-F and FL-TXN-1-R.
作为本发明的另一种优选方案：XRCC1基因引物名称：FL-XRCC1-1-F，序列信息TTTTCTGCTGCAGGACACGACA；FL-XRCC1-1-R，序列信息TTTTCGCGCTTGCGCACTTTA；As another preferred solution of the present invention: XRCC1 gene primer name: FL-XRCC1-1-F, sequence information TTTTCTGCTGCAGGACACGACA; FL-XRCC1-1-R, sequence information TTTTCGCGCTTGCGCACTTTA;
TNFa基因引物名称：FL-TNFa-1-F，序列信息TTTTCCCTCCCAGTTCTAGTTCTAT；FL-TNFa-1-R，序列信息TTTTCTTCTGGGCCACTGACTGATT；TNFa gene primer name: FL-TNFa-1-F, sequence information TTTTCCCCCCAGTTCTTAGTTCTAT; FL-TNFa-1-R, sequence information TTTTCTTCTGGGCCACTGACTGATT;
TXNRD2基因引物名称：FL-TXNRD2-1-F，序列信息TTTTCACATTGTCGTAGTCCATCAGA；FL-TXNRD2-1-R，序列信息TTTTGCAAAGGGTGATGGCATAGG；TXNRD2 gene primer name: FL-TXNRD2-1-F, sequence information TTTTCACATTGTCGTAGTCCATCAGA; FL-TXNRD2-1-R, sequence information TTTTGCAAAGGGTGATGGCATAGG;
ERCC2基因引物名称：FL-ERCC2-1-F，序列信息TTTTAAGACTCAGGAGTCACCAGGA；FL-ERCC2-1-R，序列信息TTTTTCTGTTCTCTGCAGGAGGATC；ERCC2 gene primer name: FL-ERCC2-1-F, sequence information TTTTAAGACTCAGGAGTCACCAGGA; FL-ERCC2-1-R, sequence information TTTTTCTGTTCTCTGCAGGAGGATC;
TGFB1基因引物名称：FL-TGFB1-1-F，序列信息TTTTGTCAGGCTGGGAAACAAGGTA；FL-TGFB1-1-R，序列信息TTTTGGTGCTCAGTAAAGGAGAGCAA；TGFB1 gene primer name: FL-TGFB1-1-F, sequence information TTTTGTCAGGCTGGGAAACAAGGTA; FL-TGFB1-1-R, sequence information TTTTGGTGCTCAGTAAAGGAGAGCAA;
VEGF基因引物名称：FL-VEGF-1-F，序列信息TTTTCCCAAATCACTGTGGATTTTG；FL-VEGF-1-R，序列信息TTTTCCCAAAAGCAGGTCACTCACT；VEGF gene primer name: FL-VEGF-1-F, sequence information TTTTCCCAATCACTGTGGATTTTG; FL-VEGF-1-R, sequence information TTTTCCCAAAGCAGGTCACTCACT;
DNMT1基因引物名称：FL-DNMT1-1-F，序列信息TTTTCCCAAACATAATCCCGGACTAT；FL-DNMT1-1-R，序列信息TTTTTAAGCATGTGCTTTGTTTCCTGT；DNMT1 gene primer name: FL-DNMT1-1-F, sequence information TTTTCCCAAACATAATCCCGGACTAT; FL-DNMT1-1-R, sequence information TTTTTAAGCATGTGCTTTGTTTCCTGT;
XRCC1基因引物名称：FL-XRCC1-2-F，序列信息TTTTCTGGGACCACCTGTGTTCT；FL-XRCC1-2-R，序列信息TTTTCCGCATCGTGCGTAAGGA；XRCC1 gene primer name: FL-XRCC1-2-F, sequence information TTTTCTGGGACCACCTGTGTTCT; FL-XRCC1-2-R, sequence information TTTTCCGCATCGTGCGTAAGGA;
TP53基因引物名称：FL-TP53-1-F，序列信息TTTTGGTTTTCTGGGAAGGGACAGA；FL-TP53-1-R，序列信息TTTTCGGACGATATTGAACAATGGTTC；TP53 gene primer name: FL-TP53-1-F, sequence information TTTTGGTTTTCTGGGAAGGGACAGA; FL-TP53-1-R, sequence information TTTTCGGACGATATTGAACAATGGTTC;
XRCC3基因引物名称：FL-XRCC3-1-F，序列信息TTTTCGCATCCTGGCTAAAAATACGA；FL-XRCC3-1-R，序列信息TTTTATTCCGCTGTGAATTTGACAG；XRCC3 gene primer name: FL-XRCC3-1-F, sequence information TTTTCGCATCCTGGCTAAAAATACGA; FL-XRCC3-1-R, sequence information TTTTATTCCGCTGTGAATTTGACAG;
LIN28B基因引物名称：FL-LIN28B-1-F，序列信息TTTTTGAATTAAAACATGTAGCTGCTGAAG；FL-LIN28B-1-R，序列信息TTTTAAGAAACCTCTTTAACTAGGCATCA；LIN28B gene primer name: FL-LIN28B-1-F, sequence information TTTTTGAATTAAAACATGTAGCTGCTGAAG; FL-LIN28B-1-R, sequence information TTTTAAGAAACCTCTTTAACTAGGCATCA;
LIN28B基因引物名称：FL-LIN28B-1-F，序列信息TTTTTGAATTAAAACATGTAGCTGCTGAAG；FL-LIN28B-1-R，序列信息TTTTAAGAAACCTCTTTAACTAGGCATCA；LIN28B gene primer name: FL-LIN28B-1-F, sequence information TTTTTGAATTAAAACATGTAGCTGCTGAAG; FL-LIN28B-1-R, sequence information TTTTAAGAAACCTCTTTAACTAGGCATCA;
XRCC3基因引物名称：FL-XRCC3-2-F，序列信息TTTTAGTGTCCACTGACGGATAACAGA；FL-XRCC3-2-R，序列信息TTTTCCTAATCAGCTGTCAAGGGTGAT；XRCC3 gene primer name: FL-XRCC3-2-F, sequence information TTTTAGTGTCCACTGACGGATAACAGA; FL-XRCC3-2-R, sequence information TTTTCCTAATCAGCTGTCAAGGGTGAT;
MTHFR基因引物名称：FL-MTHFR-1-F，序列信息TTTTCACTCCAGCATCACTCACTTTG；FL-MTHFR-1-R，序列信息TTTTGGGAGCTGAAGGACTACTACCT；MTHFR gene primer name: FL-MTHFR-1-F, sequence information TTTTCACTCCAGCATCACTCACTTTG; FL-MTHFR-1-R, sequence information TTTTGGGAGCTGAAGGACTACTACCT;
PON1基因引物名称：FL-PON1-1-F，序列信息TTTTATACTTGCCATCGGGTGAAATGT；FL-PON1-1-R，序列信息TTTTGCACTTTTATGGCACAAATGATCAC；PON1 gene primer name: FL-PON1-1-F, sequence information TTTTATACTTGCCATCGGGTGAAATGT; FL-PON1-1-R, sequence information TTTTGCACTTTTATGGCACAAATGATCAC;
GSTP1基因引物名称：FL-GSTP1-1-F，序列信息TTTTAGTGACTGTGTGTTGATCAGGC；FL-GSTP1-1-R，序列信息TTTTAGATGCTCACATAGTTGGTGTAGATG；GSTP1 gene primer name: FL-GSTP1-1-F, sequence information TTTTAGTGACTGTGTGTTGATCAGGC; FL-GSTP1-1-R, sequence information TTTTAGATGCTCACATAGTTGGTGTAGATG;
ATM基因引物名称：FL-ATM-1-F，序列信息TTTTACTCATTTTTACTCAAACTATTGGG；FL-ATM-1-R，序列信息TTTTCGAAGACAGCTGGTGAAAAATCC；ATM gene primer name: FL-ATM-1-F, sequence information TTTTACTCATTTTTACTCAAACTATTGGG; FL-ATM-1-R, sequence information TTTTCGAAGACAGCTGGTGAAAAATCC;
XRCC1基因引物名称：FL-XRCC1-3-F，序列信息TTTTCTACCACACCCTGAAGGATCTTC；FL-XRCC1-3-R，序列信息TTTTCTAATCTACTCTTTGTCTTCTCCAGT；XRCC1 gene primer name: FL-XRCC1-3-F, sequence information TTTTCTACCACCCCTGAAGGATCTTC; FL-XRCC1-3-R, sequence information TTTTCTAATCTACTCTTTGTCTTTCTCAGT;
NOS2基因引物名称：FL-NOS2-1-F，序列信息TTTTGGCTAGGAGTAGGACAACGGAA；FL-NOS2-1-R，序列信息TTTTTGGCCAGGTTTCCAGAAGAAAG；NOS2 gene primer name: FL-NOS2-1-F, sequence information TTTTGGCTAGGAGTAGGACAACGGAA; FL-NOS2-1-R, sequence information TTTTTGGCCAGGTTTCCAGAAGAAAG;
TGFB1基因引物名称：FL-TGFB1-2-F，序列信息TTTTGTAACCATCATGGGCCTTGTC；FL-TGFB1-2-R，序列信息TTTTTGTTCTGAGGACATGGGCAAA；TGFB1 gene primer name: FL-TGFB1-2-F, sequence information TTTTGTAACCATCATGGGCCTTGTC; FL-TGFB1-2-R, sequence information TTTTTGTTCTGAGGACATGGGCAAA;
APEX1基因引物名称：FL-APEX1-1-F，序列信息TTTTCTATTGATGCCTAATGCCTGAACT；FL-APEX1-1-R，序列信息TTTTCGAGTCAAATTCAGCCACAATCAC；APEX1 gene primer name: FL-APEX1-1-F, sequence information TTTTCTATTGATGCCTAATGCCTGAACT; FL-APEX1-1-R, sequence information TTTTCGAGTCAAATTCAGCCACAATCAC;
APEX1基因引物名称：FL-APEX1-1-F，序列信息TTTTCTATTGATGCCTAATGCCTGAACT；FL-APEX1-1-R，序列信息TTTTCGAGTCAAATTCAGCCACAATCAC；APEX1 gene primer name: FL-APEX1-1-F, sequence information TTTTCTATTGATGCCTAATGCCTGAACT; FL-APEX1-1-R, sequence information TTTTCGAGTCAAATTCAGCCACAATCAC;
VEGF基因引物名称：FL-VEGF-2-F，序列信息TTTTCACACCATCACCATCGACAGAA；FL-VEGF-2-R，序列信息TTTTGCAAGAAAAATAAAATGGCGAATC；VEGF gene primer name: FL-VEGF-2-F, sequence information TTTTCACACCATCACCATCGACAGAA; FL-VEGF-2-R, sequence information TTTTGCAAGAAAAATAAAATGGCGAATC;
XRCC1基因引物名称：FL-XRCC1-4-F，序列信息TTTTCGAGTCTAGGTCTCAACCCTAC；FL-XRCC1-4-R，序列信息TTTTCGGGACCTTAGAAGGTGACAGT；XRCC1 gene primer name: FL-XRCC1-4-F, sequence information TTTTCGAGTCTAGGTCTCAAACCCTAC; FL-XRCC1-4-R, sequence information TTTTCGGGACCTTAGAAGGTGACAGT;
MLH1基因引物名称：FL-MLH1-1-F，序列信息TTTTCAACCCACAGAGTTGAGAAATTTG；FL-MLH1-1-R，序列信息TTTTAAGAGCCAAGGAAACGTCTAGATG；MLH1 gene primer name: FL-MLH1-1-F, sequence information TTTTCAACCCACAGAGTTGAGAAATTTG; FL-MLH1-1-R, sequence information TTTTAAGGCCAAGGAAACGTCTAGATG;
IL8基因引物名称：FL-IL8-1-F，序列信息TTTTTGTTCTAACACCTGCCACTCTAGT；FL-IL8-1-R，序列信息TTTTCGGAGTATGACGAAAGTTTTCTTTGA；IL8 gene primer name: FL-IL8-1-F, sequence information TTTTTGTTCTAACACCTGCCACTCTAGT; FL-IL8-1-R, sequence information TTTTCGGAGTATGACGAAAGTTTTCTTTGA;
CD44基因引物名称：FL-CD44-1-F，序列信息TTTTGGTCCCTGGGAGGAAATTTGAA；FL-CD44-1-R，序列信息TTTTGTGTCTCCCAGAAGCATCTGAAAA；CD44 gene primer name: FL-CD44-1-F, sequence information TTTTGGTCCCTGGGAGGAAATTTGAA; FL-CD44-1-R, sequence information TTTTGTGTCTCCCAGAAGCATCTGAAAA;
LIG4基因引物名称：FL-LIG4-1-F，序列信息TTTTAAGAATCTAAAAATTCCCTGAAGTGTCT；FL-LIG4-1-R，序列信息TTTTTGCTTTACTAGTTAAACGAGAAGATTCAT；LIG4 gene primer name: FL-LIG4-1-F, sequence information TTTTAAGAATCTAAAAATTCCCTGAAGTGTCT; FL-LIG4-1-R, sequence information TTTTTGCTTTACTAGTTAAACGAGAAGATTCAT;
IL1A基因引物名称：FL-IL1A-1-F，序列信息TTTTCAGCCGTGAGGTACTGATCATT；FL-IL1A-1-R，序列信息TTTTCATTAATCTGCACTTGTGATCATGGTT；IL1A gene primer name: FL-IL1A-1-F, sequence information TTTTCAGCCGTGAGGTACTGATCATT; FL-IL1A-1-R, sequence information TTTTCATTAATCTGCACTTGTGATCATGGTT;
TNF基因引物名称：FL-TNF-1-F，序列信息TTTTGATGTGACCACAGCAATGGGTA；FL-TNF-1-R，序列信息TTTTCCCAGTGTGTGGCCATATCTTC；TNF gene primer name: FL-TNF-1-F, sequence information TTTTGATGTGACCACAGCAATGGGTA; FL-TNF-1-R, sequence information TTTTCCCAGTGTGTGGCCATATCTTC;
CYP2C8基因引物名称：FL-CYP2C8-1-F，序列信息TTTTTCAATTGGGAACAACAGAGTTAACTCC；FL-CYP2C8-1-R，序列信息TTTTTGGAATTAGTTGGAATTTACATG；CYP2C8 gene primer name: FL-CYP2C8-1-F, sequence information TTTTTCAATTGGGAACAACAGAGTTAACTCC; FL-CYP2C8-1-R, sequence information TTTTTGGAATTAGTTGGAATTTACATG;
IL1A基因引物名称：FL-IL1A-2-F，序列信息TTTTAGAAGCCAGTGGCTAAGTTTGG；FL-IL1A-2-R，序列信息TTTTTTCATTTGCTAAGAGTCTGGTGTTCT；IL1A gene primer name: FL-IL1A-2-F, sequence information TTTTAGAAGCCAGTGGCTAAGTTTGG; FL-IL1A-2-R, sequence information TTTTTTCATTTGCTAAGAGTCTGGTGTTCT;
IL4基因引物名称：FL-IL4-1-F，序列信息TTTTCCCAAGTGACTGACAATCTGGT；FL-IL4-1-R，序列信息TTTTCCCATTAATAGGTGTCGATTTGCAGT；FL-IL4-2-F，序列信息TTTTCCCAAACTAGGCCTCACCTGATA；FL-IL4-2-R，序列信息TTTTGTACAGGTGGCATCTTGGAAACT；IL4 gene primer name: FL-IL4-1-F, sequence information TTTTCCCAAGTGACTGACAATCTGGT; FL-IL4-1-R, sequence information TTTTCCCATTAATAGGTGTCGATTTGCAGT; FL-IL4-2-F, sequence information TTTTCCCAAACTAGGCCTCACCTGATA; FL-IL4-2-R, sequence information infoTTTTGTACAGGTGGCATCTTGGAAACT;
NEIL1基因引物名称：FL-NEIL1-1-F，序列信息TTTTGGCTTCTCAACTCATGGTCTCT；FL-NEIL1-1-R，序列信息TTTTGATGGCTTCCCAGGTATTTGGT；NEIL1 gene primer name: FL-NEIL1-1-F, sequence information TTTTGGCTTCTCAACTCATGGTCTCT; FL-NEIL1-1-R, sequence information TTTTGATGGCTTCCCAGGTATTTGGT;
NBN基因引物名称：FL-NBN-1-F，序列信息TTTTATCCTGAAACAAGCATTAAAGAGGGA；FL-NBN-1-R，序列信息TTTTCGTCCAATTGTAAAGCCAGAATATTTT；NBN gene primer name: FL-NBN-1-F, sequence information TTTTATCCTGAAACAAGCATTAAAGAGGGA; FL-NBN-1-R, sequence information TTTTCGTCCAATTGTAAAGCCAGAATATTTT;
APC基因引物名称：FL-APC-1-F，序列信息TTTTGCGATGGATATATACGCAGGTAATTTT；FL-APC-1-R，序列信息TTTTCTTTCTTTGGCTATTTTTGATATGCCT；APC gene primer name: FL-APC-1-F, sequence information TTTTGCGATGGATATATACGCAGGTAATTTT; FL-APC-1-R, sequence information TTTTCTTTCTTTGGCTATTTTTGATATGCCT;
APC基因引物名称：FL-APC-1-F，序列信息TTTTGCGATGGATATATACGCAGGTAATTTT；FL-APC-1-R，序列信息TTTTCTTTCTTTGGCTATTTTTGATATGCCT；APC gene primer name: FL-APC-1-F, sequence information TTTTGCGATGGATATATACGCAGGTAATTTT; FL-APC-1-R, sequence information TTTTCTTTCTTTGGCTATTTTTGATATGCCT;
NFKBIA基因引物名称：FL-NFKBIA-1-F，序列信息TTTTAGTGTGCAGTGTGGATATAAGTACAC；FL-NFKBIA-1-R，序列信息TTTTTTTCAGCTGCCCTATGATGACTG；NFKBIA gene primer name: FL-NFKBIA-1-F, sequence information TTTTAGTGTGCAGTGTGGATATAAGTACAC; FL-NFKBIA-1-R, sequence information TTTTTTTCAGCTGCCCTATGATGACTG;
DEAD基因引物名称：FL-DEAD-1-F，序列信息TTTTTCCTTGCTTCCAGTTTTTCCACT；FL-DEAD-1-R，序列信息TTTTGGACTTTATCCACTGAAATGTGATA；DEAD gene primer name: FL-DEAD-1-F, sequence information TTTTTCCTTGCTTCCAGTTTTTCCACT; FL-DEAD-1-R, sequence information TTTTGGACTTTATCCACTGAAATGTGATA;
ATM基因引物名称：FL-ATM-2-F，序列信息TTTTTGCGTGGCTAACGGAGAAAA；FL-ATM-2-R，序列信息TTTTGGGTCGCACACGACTGAATT；ATM gene primer name: FL-ATM-2-F, sequence information TTTTTGCGTGGCTAACGGAGAAAA; FL-ATM-2-R, sequence information TTTTGGGTCGCACACGACTGAATT;
IL8基因引物名称：FL-IL8-2-F，序列信息TTTTGCCTACTATAAATAACACTGTGGTA；FL-IL8-2-R，序列信息TTTTCCCTTGACCTCAGTTAGTTCTTTGTT；IL8 gene primer name: FL-IL8-2-F, sequence information TTTTGCCTACTATAAATAACACTGTGGTA; FL-IL8-2-R, sequence information TTTTCCCTTGACCTCAGTTAGTTCTTTGTT;
ATM基因引物名称：FL-ATM-3-F，序列信息TTTTAGGGAAATCATAAAGTACGTGAGTCT；FL-ATM-3-R，序列信息TTTTATGCTTTCTATTTCTCAGCAAAAACA；ATM gene primer name: FL-ATM-3-F, sequence information TTTTAGGGAAATCATAAAGTACGTGAGTCT; FL-ATM-3-R, sequence information TTTTATGCTTTCTATTTCTCAGCAAAAACA;
tPA基因引物名称：FL-tPA-1-F，序列信息TTTTTAACACTGACAATAACCAAAACCAAG；FL-tPA-1-R，序列信息TTTTCCCAGGTCTGAGTGATCTCATTG；tPA gene primer name: FL-tPA-1-F, sequence information TTTTTAACACTGACAATAACCAAAACCAAG; FL-tPA-1-R, sequence information TTTTCCCAGGTCTGAGTGATCTCATTG;
TGFB1基因引物名称：FL-TGFB1-3-F，序列信息TTTTGTAGCCACAGCAGCGGTAGC；FL-TGFB1-3-R，序列信息TTTTTTCCCTCGAGGCCCTCCTAC；TGFB1 gene primer name: FL-TGFB1-3-F, sequence information TTTTGTAGCCACAGCAGCGGTAGC; FL-TGFB1-3-R, sequence information TTTTTTCCCTCGAGGCCCTCCTAC;
RAD51基因引物名称：FL-RAD51-1-F，序列信息TTTTGCAACTCATCTGGGTTGTGC；FL-RAD51-1-R，序列信息TTTTCTCACACACTCACCTCGGTC；RAD51 gene primer name: FL-RAD51-1-F, sequence information TTTTGCAACTCATCTGGGTTGTGC; FL-RAD51-1-R, sequence information TTTTCTCACACACTCACCTCGGTC;
NFE2L2基因引物名称：FL-NFE2L2-1-F，序列信息TTTTGGGCTAAAGATTTGGACCCAG；FL-NFE2L2-1-R，序列信息TTTTGAATGGAGACACGTGGGAGTT；NFE2L2 gene primer name: FL-NFE2L2-1-F, sequence information TTTTGGGCTAAAGATTTGGACCCAG; FL-NFE2L2-1-R, sequence information TTTTGAATGGAGACACGTGGGAGTT;
GSTA1基因引物名称：FL-GSTA1-1-F，序列信息TTTTATAAGATCAGTACTTACTTTGTTAAA；FL-GSTA1-1-R，序列信息TTTTGAGTGGCTTTTCCCTAACTTGACT；GSTA1 gene primer name: FL-GSTA1-1-F, sequence information TTTTATAAGATCAGTACTTACTTTGTTAAA; FL-GSTA1-1-R, sequence information TTTTGAGTGGCTTTTCCCTAACTTGACT;
MGMT基因引物名称：FL-MGMT-1-F，序列信息TTTTCCCATAGTTCAGTTCTCTGTGTAGG；FL-MGMT-1-R，序列信息TTTTCTAGCACGAGGTGGGCAGAGT；MGMT gene primer name: FL-MGMT-1-F, sequence information TTTTCCCATAGTTCAGTTCTCTGTGTAGG; FL-MGMT-1-R, sequence information TTTTCTAGCACGAGGTGGGCAGAGT;
ATM基因引物名称：FL-ATM-4-F，序列信息TTTTTCTGTAATCTATAGCAGAGTAAAGCG；FL-ATM-4-R，序列信息TTTTAAAAACAAAAAGAGCTGACTACCCA；ATM gene primer name: FL-ATM-4-F, sequence information TTTTTCTGTAATCTATAGCAGAGTAAAGCG; FL-ATM-4-R, sequence information TTTTAAAAAACAAAAAGAGCTGACTACCCA;
TDG基因引物名称：FL-TDG-1-F，序列信息TTTTCCCACATTGTTTCCTTTGAACAGA；FL-TDG-1-R，序列信息TTTTAGTCTATGGCATTCTGAAACAGCAA；FL-TDG-2-F，序列信息TTTTGTCTGAAGACACTTTTGATGATCAAG；FL-TDG-2-R，序列信息TTTTCTCAGGACGTAAAGCAGTTTCTCA；TDG gene primer name: FL-TDG-1-F, sequence information TTTTCCACATTGTTTCCTTTGAACAGA; FL-TDG-1-R, sequence information TTTTAGTCTATGGCATTCTGAAACAGCAA; FL-TDG-2-F, sequence information TTTTGTCTGAAGACACTTTTGATGATCAAG; FL-TDG-2-R, sequence information INFO TTTTCTCAGGACGTAAAGCAGTTTCTCA;
GSK3B基因引物名称：FL-GSK3B-1-F，序列信息TTTTGTTTGCACAACTTCGTGAATCTACTC；FL-GSK3B-1-R，序列信息TTTTTGACCTTAAGTGCATGGACATTGG；GSK3B gene primer name: FL-GSK3B-1-F, sequence information TTTTGTTTGCACAACTTCGTGAATCTACTC; FL-GSK3B-1-R, sequence information TTTTTGACCTTAAGTGCATGGACATTGG;
NOS2基因引物名称：FL-NOS2-2-F，序列信息TTTTCGCCTCTGACAGATACACTCAG；FL-NOS2-2-R，序列信息TTTTCAATCTGCAATAACGCAATAGTGCAA；NOS2 gene primer name: FL-NOS2-2-F, sequence information TTTTCGCCTCTGACAGATACACTCAG; FL-NOS2-2-R, sequence information TTTTCAATCTGCAATAACGCAATAGTGCAA;
XRCC1基因引物名称：FL-XRCC1-5-F，序列信息TTTTCCAGGTCACTTTTGGATTCTGGT；FL-XRCC1-5-R，序列信息TTTTCCTCATGGTGTGTGTATCAGCA；XRCC1 gene primer name: FL-XRCC1-5-F, sequence information TTTTCCAGGTCACTTTTGGATTCTGGT; FL-XRCC1-5-R, sequence information TTTTCCTCATGGTGTGTGTATCAGCA;
FSHR基因引物名称：FL-FSHR-1-F，序列信息TTTTCCGAGAAATCCAGAAATCTAGACTTA；FL-FSHR-1-R，序列信息TTTTTCTACCCAGTGTATCATTCTGCTTACT；FSHR gene primer name: FL-FSHR-1-F, sequence information TTTTCCGAGAAATCCAGAAATCTAGACTTA; FL-FSHR-1-R, sequence information TTTTTTCTACCCAGTGTATCATTCTGCTTACT;
MYO3B基因引物名称：FL-MYO3B-1-F，序列信息TTTTCATGATCCTTTATACTGCCACCAGAA；FL-MYO3B-1-R，序列信息TTTTTGGCAAGTTAATGTCCAAGATGGAATT；MYO3B gene primer name: FL-MYO3B-1-F, sequence information TTTTCATGATCCTTTATACTGCCACCAGAA; FL-MYO3B-1-R, sequence information TTTTTGGCAAGTTAATGTCCAAGATGGAATT;
TGFBR2基因引物名称：FL-TGFBR2-1-F，序列信息TTTTTCTGAATGCCCAGAAGACCTCT；FL-TGFBR2-1-R，序列信息TTTTGGGAAGCATGGAATATGACAAGAGTC；FL-TGFBR2-2-F，序列信息TTTTCCACATCCATTTGGTGCTTTTAATTGA；FL-TGFBR2-2-R，序列信息TTTTCCAGTCGATAACTAAGAAGAAGCTACA；TGFBR2 gene primer name: FL-TGFBR2-1-F, sequence information TTTTTCTGAATGCCCAGAAGACCTCT; FL-TGFBR2-1-R, sequence information TTTTGGGAAGCATGGAATATGACAAGAGTC; FL-TGFBR2-2-F, sequence information TTTTCCACATCCATTTGGTGCTTTTAATTGA; FL-TGFBR2-2-R, sequence information infoTTTTCCAGTCGATAACTAAGAAGAAGCTACA;
5q31基因引物名称：FL-5q31-1-F，序列信息TTTTTTGATAACAGCAGAAGATAGAAGAGTT；FL-5q31-1-R，序列信息TTTTCTGTATTCCCAAGACAAAGCCTCA；5q31 gene primer name: FL-5q31-1-F, sequence information TTTTTTGATAACAGCAGAAGATAGAAGAGTT; FL-5q31-1-R, sequence information TTTTCTGTATTCCCCAAGACAAAGCCTCA;
LIN28B基因引物名称：FL-LIN28B-2-F，序列信息TTTTCAATTGTGTGAAGCCAGAGCAT；FL-LIN28B-2-R，序列信息TTTTACATTCCTACGACATGGAGACAAATG；LIN28B gene primer name: FL-LIN28B-2-F, sequence information TTTTCAATTGTGTGAAGCCAGAGCAT; FL-LIN28B-2-R, sequence information TTTTACATTCCTACGACATGGAGACAAATG;
HSPB1基因引物名称：FL-HSPB1-1-F，序列信息TTTTGCAATGCATGACTGTTACCATCATT；FL-HSPB1-1-R，序列信息TTTTGCAATCTAGTAATCTGGTGTGGTTGTTAG；HSPB1 gene primer name: FL-HSPB1-1-F, sequence information TTTTGCAATGCATGACTGTTACCATCATT; FL-HSPB1-1-R, sequence information TTTTGCAATCTAGTAATCTGGTGTGGTTGTTAG;
NBN基因引物名称：FL-NBN-2-F，序列信息TTTTATTATTTCAGCCAGATGGCAGACT；FL-NBN-2-R，序列信息TTTTTCTGTAAAATAAGAATAGCAAAGGCAGT；NBN gene primer name: FL-NBN-2-F, sequence information TTTTATTATTTCAGCCAGATGGCAGACT; FL-NBN-2-R, sequence information TTTTTCTGTAAAATAAGAATAGCAAAGGCAGT;
TXN基因引物名称：FL-TXN-1-F，序列信息TTTTACATACCCAAGCTTCTTAAATGGAAACT；FL-TXN-1-R，序列信息TTTTCCTTATTTGAAATCCCAGCCTCAGAATT。TXN gene primer name: FL-TXN-1-F, sequence information TTTTACATACCCAAGCTTCTTAAATGGAAACT; FL-TXN-1-R, sequence information TTTTCCTTATTTGAAATCCCAGCCTCAGAATT.
作为本发明的又一种优选方案，在步骤(2)中，多重富集PCR的反应体系为：As another preferred solution of the present invention, in step (2), the reaction system of multiple enrichment PCR is:
1×PCR缓冲液1×PCR buffer
作为本发明的进一步改进方案，该构建方法的试剂盒，包括As a further improvement of the present invention, the kit of the construction method includes
一DNA富集反应组件，由第一扩增引物组组成；A DNA enrichment reaction component, consisting of a first amplification primer set;
一RingCap-Taq酶，由Taq酶、DNA连接酶和DNA末端修饰酶组成；1. RingCap-Taq enzyme, composed of Taq enzyme, DNA ligase and DNA end modification enzyme;
一管阳性质控品；One tube of positive quality control;
和一管阴性质控品。and a tube of negative quality control.
与现有技术相比，本发明具有如下优点：Compared with prior art, the present invention has following advantage:
1、本发明的构建方法针对多个靶序列进行单管，快速完成文库的构建，整个文库构建过程仅需要2～3小时，手动时间仅仅需要45分钟即可，结合高通量测序平台可以十分有效的解决目前对于临床上预测患者接受肿瘤放射治疗是否发生较毒性，而需要对多基因、多靶点检测这一难点，且成本低廉。1. The construction method of the present invention conducts a single tube for multiple target sequences to quickly complete the construction of the library. The entire library construction process only takes 2 to 3 hours, and the manual time only takes 45 minutes. Combined with a high-throughput sequencing platform, it can be very Effectively solve the current problem of multi-gene and multi-target detection for clinically predicting whether patients undergoing tumor radiation therapy will be more toxic, and the cost is low.
2、本发明的构建方法所制备的文库序列可被目前高通量测序系统识别并进行检测，从而实现文库构建进行核酸序列的检测的应用，该核酸检测可应用于目前多种高通量测序平台、基因芯片平台、杂交检测平台，见图1。2. The library sequence prepared by the construction method of the present invention can be recognized and detected by the current high-throughput sequencing system, so as to realize the application of library construction for nucleic acid sequence detection, and the nucleic acid detection can be applied to various current high-throughput sequencing See Figure 1 for the platform, gene chip platform, and hybridization detection platform.
3、本发明的构建方法适用于来自外周血样品所获得的DNA，见图2、图3。3. The construction method of the present invention is applicable to DNA obtained from peripheral blood samples, see Fig. 2 and Fig. 3 .
附图说明Description of drawings
图1为本发明的实施例构建的核酸文库进行高通量测序总数据图；Fig. 1 is the high-throughput sequencing total data diagram of the nucleic acid library constructed in the embodiment of the present invention;
图2为本发明的实施例检测肿瘤放射治疗毒性基因突变的检测均一性结果图；Fig. 2 is the result figure of the detection uniformity of the embodiment of the present invention detecting tumor radiotherapy toxicity gene mutation;
图3为本发明的实施例检测肿瘤放射治疗毒性基因突变的检测突变结果。Fig. 3 is the mutation detection result of the detection of tumor radiotherapy toxicity gene mutation according to the embodiment of the present invention.
具体实施方式detailed description
下面结合附图和具体实施方式对本发明作进一步详细地描述。The present invention will be described in further detail below in conjunction with the accompanying drawings and specific embodiments.
肿瘤治疗心脏毒性基因突变文库的构建方法，覆盖人类基因XRCC1、TNFa、TXNRD2、ERCC2、TGFB1、VEGF、DNMT1、TP53、XRCC3、LIN28B、MTHFR、PON1、GSTP1、ATM、NOS2、APEX1、MLH1、IL8、CD44、LIG4、IL1A、TNF、CYP2C8、IL4、NEIL1、NBN、APC、NFKBIA、DEAD、Tpa、RAD51、NFE2L2、GSTA1、MGMT、TDG、GSK3B、FSHR、MYO3B、TGFBR2、5q31、HSPB1和TXN上的总共61种遗传性变异位点。A method for constructing a cardiotoxic gene mutation library for tumor therapy, covering human genes XRCC1, TNFa, TXNRD2, ERCC2, TGFB1, VEGF, DNMT1, TP53, XRCC3, LIN28B, MTHFR, PON1, GSTP1, ATM, NOS2, APEX1, MLH1, IL8, Total on CD44, LIG4, IL1A, TNF, CYP2C8, IL4, NEIL1, NBN, APC, NFKBIA, DEAD, Tpa, RAD51, NFE2L2, GSTA1, MGMT, TDG, GSK3B, FSHR, MYO3B, TGFBR2, 5q31, HSPB1 and TXN 61 kinds of genetic variation sites.
具体包括如下步骤：Specifically include the following steps:
(1)针对目的基因XRCC1、TNFa、TXNRD2、ERCC2、TGFB1、VEGF、DNMT1、TP53、XRCC3、LIN28B、MTHFR、PON1、GSTP1、ATM、NOS2、APEX1、MLH1、IL8、CD44、LIG4、IL1A、TNF、CYP2C8、IL4、NEIL1、NBN、APC、NFKBIA、DEAD、Tpa、RAD51、NFE2L2、GSTA1、MGMT、TDG、GSK3B、FSHR、MYO3B、TGFBR2、5q31、HSPB1和TXN设计基本扩增引物组，该基本扩增引物组的正向引物和反向引物的5’端设有额外的2～5个T，且该2～5个T的靠近3’端的第一个T具有PNA修饰，同时该基本扩增引物组的Tm值相差不超过1℃；所述扩增引物组包括FL-XRCC1-1-F、FL-XRCC1-1-R、FL-TNFa-1-F、FL-TNFa-1-R、FL-TXNRD2-1-F、FL-TXNRD2-1-R、FL-ERCC2-1-F、FL-ERCC2-1-R、FL-TGFB1-1-F、FL-TGFB1-1-R、FL-VEGF-1-F、FL-VEGF-1-R、FL-DNMT1-1-F、FL-DNMT1-1-R、FL-XRCC1-2-F、FL-XRCC1-2-R、FL-TP53-1-F、FL-TP53-1-R、FL-XRCC3-1-F、FL-XRCC3-1-R、FL-LIN28B-1-F、FL-LIN28B-1-R、FL-XRCC3-2-F、FL-XRCC3-2-R、FL-MTHFR-1-F、FL-MTHFR-1-R、FL-PON1-1-F、FL-PON1-1-R、FL-GSTP1-1-F、FL-GSTP1-1-R、FL-ATM-1-F、FL-ATM-1-R、FL-XRCC1-3-F、FL-XRCC3-1-R、FL-LIN28B-1-F、FL-LIN28B-1-R、FL-XRCC3-2-F、FL-XRCC3-2-R、FL-MTHFR-1-F、FL-MTHFR-1-R、FL-PON1-1-F、FL-PON1-1-R、FL-GSTP1-1-F、FL-GSTP1-1-R、FL-ATM-1-F、FL-ATM-1-R、FL-XRCC1-3-F、FL-XRCC1-3-R、FL-NOS2-1-F、FL-NOS2-1-R、FL-TGFB1-2-F、FL-TGFB1-2-R、FL-APEX1-1-F、FL-APEX1-1-R、FL-VEGF-2-F、FL-VEGF-2-R、FL-XRCC1-4-F、FL-XRCC1-4-R、FL-MLH1-1-F、FL-MLH1-1-R、FL-IL8-1-F、FL-IL8-1-R、FL-CD44-1-F、FL-CD44-1-R、FL-LIG4-1-F、FL-LIG4-1-R、FL-IL1A-1-F、FL-IL1A-1-R、FL-TNF-1-F、FL-TNF-1-R、FL-CYP2C8-1-F、FL-CYP2C8-1-R、FL-IL1A-2-F、FL-IL1A-2-R、FL-IL4-1-F、FL-IL4-1-R、FL-IL4-2-F、FL-IL4-2-R、FL-NEIL1-1-F、FL-NEIL1-1-R、FL-NBN-1-F、FL-NBN-1-R、FL-APC-1-F、FL-APC-1-R、FL-NFKBIA-1-F、FL-NFKBIA-1-R、FL-DEAD-1-F、FL-DEAD-1-R、FL-ATM-2-F、FL-ATM-2-R、FL-IL8-2-F、FL-IL8-2-R、FL-ATM-3-F、FL-ATM-3-R、FL-tPA-1-F、FL-tPA-1-R、FL-TGFB1-3-F、FL-TGFB1-3-R、FL-RAD51-1-F、FL-RAD51-1-R、FL-NFE2L2-1-F、FL-NFE2L2-1-R、FL-GSTA1-1-F、FL-GSTA1-1-R、FL-MGMT-1-F、FL-MGMT-1-R、FL-ATM-4-F、FL-ATM-4-R、FL-TDG-1-F、FL-TDG-1-R、FL-TDG-2-F、FL-TDG-2-R、FL-GSK3B-1-F、FL-GSK3B-1-R、FL-NOS2-2-F、FL-NOS2-2-R、FL-XRCC1-5-F、FL-XRCC1-5-R、FL-FSHR-1-F、FL-FSHR-1-R、FL-MYO3B-1-F、FL-MYO3B-1-R、FL-TGFBR2-1-F、FL-TGFBR2-1-R、FL-TGFBR2-2-F、FL-TGFBR2-2-R、FL-5q31-1-F、FL-5q31-1-R、FL-LIN28B-2-F、FL-LIN28B-2-R、FL-HSPB1-1-F、FL-HSPB1-1-R、FL-NBN-2-F、FL-NBN-2-R、FL-TXN-1-F和FL-TXN-1-R，其序列依次如SEQ ID 01～SEQ ID 122所示；(1) For target genes XRCC1, TNFa, TXNRD2, ERCC2, TGFB1, VEGF, DNMT1, TP53, XRCC3, LIN28B, MTHFR, PON1, GSTP1, ATM, NOS2, APEX1, MLH1, IL8, CD44, LIG4, IL1A, TNF, CYP2C8, IL4, NEIL1, NBN, APC, NFKBIA, DEAD, Tpa, RAD51, NFE2L2, GSTA1, MGMT, TDG, GSK3B, FSHR, MYO3B, TGFBR2, 5q31, HSPB1 and TXN design basic amplification primer sets, the basic amplification The 5' end of the forward primer and reverse primer of the primer set is provided with an additional 2-5 Ts, and the first T near the 3' end of the 2-5 Ts has a PNA modification, and the basic amplification primer The Tm values of the groups differ by no more than 1°C; the amplification primer set includes FL-XRCC1-1-F, FL-XRCC1-1-R, FL-TNFa-1-F, FL-TNFa-1-R, FL -TXNRD2-1-F, FL-TXNRD2-1-R, FL-ERCC2-1-F, FL-ERCC2-1-R, FL-TGFB1-1-F, FL-TGFB1-1-R, FL-VEGF -1-F, FL-VEGF-1-R, FL-DNMT1-1-F, FL-DNMT1-1-R, FL-XRCC1-2-F, FL-XRCC1-2-R, FL-TP53-1 -F, FL-TP53-1-R, FL-XRCC3-1-F, FL-XRCC3-1-R, FL-LIN28B-1-F, FL-LIN28B-1-R, FL-XRCC3-2-F , FL-XRCC3-2-R, FL-MTHFR-1-F, FL-MTHFR-1-R, FL-PON1-1-F, FL-PON1-1-R, FL-GSTP1-1-F, FL -GSTP1-1-R, FL-ATM-1-F, FL-ATM-1-R, FL-XRCC1-3-F, FL-XRCC3-1-R, FL-LIN28B-1-F, FL-LIN28B -1-R, FL-XRCC3-2-F, FL-XRCC3-2-R, FL-MTHFR-1-F, FL-MTHFR-1-R, FL-PON1-1-F, FL-PON1-1 -R, FL-GSTP1-1-F, FL-GSTP1-1-R, FL-ATM-1-F, FL-ATM-1-R, FL-XRCC1-3-F, FL-XRCC1-3-R , FL-NOS2-1-F, FL-NOS2-1-R, FL-TGFB1-2-F, FL-TGFB1-2-R, FL- APEX1-1-F, FL-APEX1-1-R, FL-VEGF-2-F, FL-VEGF-2-R, FL-XRCC1-4-F, FL-XRCC1-4-R, FL-MLH1- 1-F, FL-MLH1-1-R, FL-IL8-1-F, FL-IL8-1-R, FL-CD44-1-F, FL-CD44-1-R, FL-LIG4-1- F, FL-LIG4-1-R, FL-IL1A-1-F, FL-IL1A-1-R, FL-TNF-1-F, FL-TNF-1-R, FL-CYP2C8-1-F, FL-CYP2C8-1-R, FL-IL1A-2-F, FL-IL1A-2-R, FL-IL4-1-F, FL-IL4-1-R, FL-IL4-2-F, FL- IL4-2-R, FL-NEIL1-1-F, FL-NEIL1-1-R, FL-NBN-1-F, FL-NBN-1-R, FL-APC-1-F, FL-APC- 1-R, FL-NFKBIA-1-F, FL-NFKBIA-1-R, FL-DEAD-1-F, FL-DEAD-1-R, FL-ATM-2-F, FL-ATM-2- R, FL-IL8-2-F, FL-IL8-2-R, FL-ATM-3-F, FL-ATM-3-R, FL-tPA-1-F, FL-tPA-1-R, FL-TGFB1-3-F, FL-TGFB1-3-R, FL-RAD51-1-F, FL-RAD51-1-R, FL-NFE2L2-1-F, FL-NFE2L2-1-R, FL- GSTA1-1-F, FL-GSTA1-1-R, FL-MGMT-1-F, FL-MGMT-1-R, FL-ATM-4-F, FL-ATM-4-R, FL-TDG- 1-F, FL-TDG-1-R, FL-TDG-2-F, FL-TDG-2-R, FL-GSK3B-1-F, FL-GSK3B-1-R, FL-NOS2-2- F, FL-NOS2-2-R, FL-XRCC1-5-F, FL-XRCC1-5-R, FL-FSHR-1-F, FL-FSHR-1-R, FL-MYO3B-1-F, FL-MYO3B-1-R, FL-TGFBR2-1-F, FL-TGFBR2-1-R, FL-TGFBR2-2-F, FL-TGFBR2-2-R, FL-5q31-1-F, FL- 5q31-1-R, FL-LIN28B-2-F, FL-LIN28B-2-R, FL-HSPB1-1-F, FL-HSPB1-1-R, FL-NBN-2-F, FL-NBN- 2-R, FL- TXN-1-F and FL-TXN-1-R, the sequences of which are shown in sequence as SEQ ID 01 to SEQ ID 122;
(2)将模板、上述基本扩增引物组置于一含有RingCap-Taq酶的PCR反应体系中进行扩增，纯化后得扩增产物；将扩增产物、多对第一不对称连接探针、通用引物和上述RingCap-Taq酶混合进行PCR，纯化后获得文库产物；(2) Place the template and the above-mentioned basic amplification primer set in a PCR reaction system containing RingCap-Taq enzyme for amplification, and obtain the amplification product after purification; the amplification product, multiple pairs of the first asymmetric connection probe , universal primers and the above-mentioned RingCap-Taq enzyme are mixed for PCR, and the library product is obtained after purification;
(3)将文库产物组成所述用于高通量测序的肿瘤放射治疗毒性基因突变文库；(3) forming the library product into the tumor radiotherapy toxicity gene mutation library for high-throughput sequencing;
(4)通过Ion torrent PGM高通量测序仪进行文库上机检测，获得目标序列信息，通过VC软件进行数据信息比对分析，得到样本突变状态。(4) The Ion torrent PGM high-throughput sequencer was used to detect the library on the computer to obtain the target sequence information, and the data information was compared and analyzed by VC software to obtain the mutation status of the sample.
上述模板所来自的样品适用范围包括外周血标本。The scope of application of the samples from which the above templates come includes peripheral blood samples.
外周血样品，使用Qiagen公司外周血DNA提取试剂盒提取基因组DNA，具体步骤按试剂盒操作说明。每次提取外周血不少于1000μl。所提DNA溶于Tris-HCl(10mmol/L,pH8.0)，经紫外分光光度计检测提取质量，并确定浓度，用Tris-HCl溶液(10mmol/L,pH8.0)调整DNA浓度到2ng/μl作为PCR扩增的模板。For peripheral blood samples, genomic DNA was extracted using the peripheral blood DNA extraction kit from Qiagen, and the specific steps were according to the instructions of the kit. Peripheral blood was extracted not less than 1000μl each time. The extracted DNA was dissolved in Tris-HCl (10mmol/L, pH8.0), the quality of the extraction was detected by a UV spectrophotometer, and the concentration was determined, and the DNA concentration was adjusted to 2ng with Tris-HCl solution (10mmol/L, pH8.0). /μl as a template for PCR amplification.
基于上述构建方法的试剂盒包括：The kit based on the above construction method includes:
一DNA富集反应组件，由扩增引物组组成，每人份的DNA富集反应组件的配方如下表所示：A DNA enrichment reaction component, consisting of an amplification primer set, the recipe of the DNA enrichment reaction component per person is shown in the table below:
一RingCap-Taq酶，由Taq酶、DNA连接酶和DNA末端修饰酶组成，比例为0.8～1.2:0.8～1.2:0.8～1.2，优选比例1:1:1；1. RingCap-Taq enzyme, composed of Taq enzyme, DNA ligase and DNA end modification enzyme, the ratio is 0.8～1.2:0.8～1.2:0.8～1.2, preferably 1:1:1;
一阴性质控品，具体为无核酸水；A negative quality control product, specifically nucleic acid-free water;
和一阳性质控品，具体由10个阳性突变质粒序列野生型基因组DNA混合而成，浓度为2ng/μL。And a positive quality control product, which is specifically prepared by mixing wild-type genomic DNA of 10 positive mutant plasmid sequences, with a concentration of 2ng/μL.
将上述试剂盒用前述的构建方法进行实验，来分析本发明的检测肿瘤放射治疗毒性基因突变的方法。分别用20份临床乳腺癌放射治疗过程无严重毒性的全血样品、20份临床乳腺癌放射治疗过程存在严重毒性的全血样本：The above-mentioned kit was tested with the aforementioned construction method to analyze the method for detecting tumor radiotherapy toxicity gene mutation of the present invention. 20 whole blood samples without serious toxicity during clinical breast cancer radiotherapy and 20 whole blood samples with severe toxicity during clinical breast cancer radiotherapy were used respectively:
所述PCR反应体系的模板量(待测样品、阳性质控品和阴性质控品)为5uL，其余组分如下表所示：The template amount (sample to be tested, positive quality control product and negative quality control product) of described PCR reaction system is 5uL, and all the other components are shown in the following table:
PCR扩增程序设置如下表：The PCR amplification program settings are as follows:
上述PCR反应体系所得的扩增产物的纯化具体如下：The purification of the amplification product obtained by the above-mentioned PCR reaction system is as follows:
取出Agencourt AMPure XP试剂置于室温，同时要打散磁珠，同时配制新鲜的70％的乙醇(230μL无水乙醇+100μL无核酸酶水)，必须是新鲜配制的。Take out the Agencourt AMPure XP reagent and place it at room temperature, and at the same time break up the magnetic beads, and prepare fresh 70% ethanol (230 μL absolute ethanol + 100 μL nuclease-free water), which must be freshly prepared.
第一轮纯化步骤First round of purification steps
(1)向每个样品反应管25μL产物中分别加入12.5μL(0.5x样本体积)的AgencourtAMPure XP试剂，上下吸取吹打5次，完全混匀重悬DNA；(1) Add 12.5 μL (0.5x sample volume) of AgencourtAMPure XP reagent to 25 μL of each sample reaction tube, pipette up and down 5 times, mix completely and resuspend the DNA;
(2)室温孵育5分钟；(2) Incubate at room temperature for 5 minutes;
(3)放置在磁力架上面，孵育5分钟，一直到溶液澄清；(3) Place on the magnetic stand and incubate for 5 minutes until the solution is clear;
(4)小心地吸取上清液置于新的离心管，不要扰动磁珠；注意：上清液中含有扩增产物不要丢弃。(4) Carefully pipette the supernatant into a new centrifuge tube without disturbing the magnetic beads; Note: Do not discard the amplification product contained in the supernatant.
第二轮纯化步骤Second round of purification steps
(1)向上述吸取的上清液25μL中加入30μL(1.2x样本体积)的Agencourt AMPureXP试剂，上下吸取吹打5次，完全混匀重悬DNA；(1) Add 30 μL (1.2x sample volume) of Agencourt AMPureXP reagent to 25 μL of the above-mentioned supernatant, pipette up and down 5 times, mix completely and resuspend DNA;
(2)室温孵育5分钟；(2) Incubate at room temperature for 5 minutes;
(3)放置在磁力架上面，孵育3分钟，一直到溶液澄清，小心地吸走并弃掉上清，不要扰动磁珠；注意：磁珠上含有扩增文库不要丢弃。(3) Place on the magnetic stand and incubate for 3 minutes until the solution is clear, carefully aspirate and discard the supernatant without disturbing the magnetic beads; Note: do not discard the amplified library contained on the magnetic beads.
(4)加入150μl新鲜配制的70％乙醇，没过磁珠样品即可，离心管正反方向移动5次，然后磁力架上孵育2分钟，去除上清液；(4) Add 150 μl of freshly prepared 70% ethanol, just submerge the magnetic bead sample, move the centrifuge tube forward and reverse 5 times, then incubate on the magnetic stand for 2 minutes, and remove the supernatant;
(5)重复上述步骤4，进行第二次洗涤；(5) Repeat above-mentioned step 4, carry out washing for the second time;
(6)确保乙醇液滴已全部从孔中吸走，将板放置于磁力架上，室温空气干燥5分钟，注意不要过度干燥。(6) Make sure that all the ethanol droplets have been absorbed from the wells, place the plate on a magnetic stand, and air dry at room temperature for 5 minutes, taking care not to over dry.
(7)将样品管从磁力架上拿开，在每孔中加入25μL TE(PH8.0)缓冲液充分浸润磁珠。充分振荡混匀，快速离心将液体收集到管底。(也可以选择用枪吸取一半以上的液体上下吹打至少5次来混匀)；注意：上清液含有扩增文库不要丢弃。(7) Remove the sample tube from the magnetic stand, and add 25 μL TE (PH8.0) buffer solution to each well to fully infiltrate the magnetic beads. Shake well to mix, and quickly centrifuge to collect the liquid at the bottom of the tube. (You can also choose to use a gun to absorb more than half of the liquid and pipette up and down at least 5 times to mix); Note: Do not discard the supernatant containing the amplified library.
将样品管置于磁力架上2分钟。上清液中含有扩增产物。取出20μL上清。Place the sample tubes on the magnetic stand for 2 min. The supernatant contains the amplification product. Remove 20 μL of supernatant.
上述纯化所得的扩增产物制备文库产物的PCR反应的模板量为5uL，其余组分如下表所示：The template amount of the PCR reaction for preparing the library product from the above-mentioned purified amplification product is 5uL, and the remaining components are shown in the following table:
PCR扩增程序设置如下表：The PCR amplification program settings are as follows:
PCR产物分别按纯化扩增产物方法进行纯化，制得文库产物。The PCR products were purified according to the method for purifying amplified products to obtain library products.
上述文库产物的检测具体如下：The detection of the above library products is as follows:
采用Ion torrent PGM半导体测序仪(Thermofisher公司)一次可以检测96份样品(包括阴阳性对照)。Using Ion torrent PGM semiconductor sequencer (Thermofisher company) can detect 96 samples (including negative and positive controls) at one time.
前述40份全血样品以及阳性质粒经本发明的体系检测，只有阳性质粒和临床接受肿瘤放射治疗具有严重毒性的有检测到突变序列，而临床接受肿瘤放射治疗无严重毒性样品无突变序列，进一步证明了该方法的特异性，具体结果如图1至图3所示。The aforementioned 40 whole blood samples and positive plasmids are detected by the system of the present invention, only the positive plasmids and those with severe toxicity of clinically accepted tumor radiotherapy have detected mutation sequences, while the clinically accepted samples of tumor radiotherapy without severe toxicity have no mutated sequences, further The specificity of the method is proved, and the specific results are shown in Fig. 1 to Fig. 3 .
重复性试验：每个反应分别加入突变细胞系DNA10ng、1ng和100pg，重复10次进行高通量测序检测，10次结果一致，符合率100％。Repeatability test: Add 10ng, 1ng and 100pg of mutant cell line DNA to each reaction, repeat 10 times for high-throughput sequencing detection, and the results are consistent 10 times, with a coincidence rate of 100%.
以上可知本发明肿瘤放射治疗毒性基因文库构建反应就可以同时检测肿瘤放射治疗毒性基因61个遗传性变异位点，文库构建时间仅需3小时，因此本发明省时省力，准确性高，可满足突变的快速诊断。It can be seen from the above that the construction reaction of the tumor radiation therapy toxicity gene library of the present invention can simultaneously detect 61 genetic mutation sites of tumor radiation therapy toxicity genes, and the library construction time only needs 3 hours. Therefore, the present invention saves time and effort, has high accuracy, and can meet Rapid diagnosis of mutations.
最后说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的宗旨和范围，其均应涵盖在本发明的权利要求范围当中。Finally, it is noted that the above embodiments are only used to illustrate the technical solutions of the present invention without limitation. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be carried out Modifications or equivalent replacements without departing from the spirit and scope of the technical solution of the present invention shall be covered by the claims of the present invention.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012031008A2 (en) * | 2010-08-31 | 2012-03-08 | The General Hospital Corporation | Cancer-related biological materials in microvesicles |
| US20130004481A1 (en) * | 2011-01-12 | 2013-01-03 | Boehringer Ingelheim International Gmbh | Anticancer therapy |
| US20130143747A1 (en) * | 2011-12-05 | 2013-06-06 | Myriad Genetics, Incorporated | Methods of detecting cancer |
| CN103370334A (en) * | 2010-12-23 | 2013-10-23 | 韩诺生物制药株式会社 | Modified human tumor necrosis factor receptor-I polypeptide or fragment thereof and method for preparing same |
| CN106498035A (en) * | 2016-09-30 | 2017-03-15 | 厦门飞朔生物技术有限公司 | A kind of construction method and its application for detecting chemotherapeutics gene SNP variation library for high-flux sequence |
-
2017
- 2017-05-15 CN CN201710339037.0A patent/CN107119331A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012031008A2 (en) * | 2010-08-31 | 2012-03-08 | The General Hospital Corporation | Cancer-related biological materials in microvesicles |
| CN103370334A (en) * | 2010-12-23 | 2013-10-23 | 韩诺生物制药株式会社 | Modified human tumor necrosis factor receptor-I polypeptide or fragment thereof and method for preparing same |
| US20130004481A1 (en) * | 2011-01-12 | 2013-01-03 | Boehringer Ingelheim International Gmbh | Anticancer therapy |
| US20130143747A1 (en) * | 2011-12-05 | 2013-06-06 | Myriad Genetics, Incorporated | Methods of detecting cancer |
| CN106498035A (en) * | 2016-09-30 | 2017-03-15 | 厦门飞朔生物技术有限公司 | A kind of construction method and its application for detecting chemotherapeutics gene SNP variation library for high-flux sequence |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020124472A1 (en) * | 2018-12-20 | 2020-06-25 | 深圳华大智造科技有限公司 | Pcr primer, pcr amplification method and use thereof |
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