CN111154872A - 用于检测肺癌驱动性基因突变的探针及试剂盒 - Google Patents
用于检测肺癌驱动性基因突变的探针及试剂盒 Download PDFInfo
- Publication number
- CN111154872A CN111154872A CN201911423840.8A CN201911423840A CN111154872A CN 111154872 A CN111154872 A CN 111154872A CN 201911423840 A CN201911423840 A CN 201911423840A CN 111154872 A CN111154872 A CN 111154872A
- Authority
- CN
- China
- Prior art keywords
- seq
- lung cancer
- mutation
- sequences
- probe
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/118—Prognosis of disease development
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Hospice & Palliative Care (AREA)
- Biophysics (AREA)
- Oncology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明公开了一种用于检测肺癌驱动性基因突变的探针及检测试剂盒，包括BRAF、EGFR、KRAS、MET、NRAS和PIK3CA驱动性基因突变的探针及TP53和UGT1A1化疗药物相关基因的探针，共计276条探针。本发明提供的探针特异性高，准确性好，最低检测限可达0.1％。可为寻找靶向治疗药物的肺癌患者提供用药依据；还可以为治疗过程中发生严重毒副作用、耐药等的肺癌患者更换治疗方案。
Description
技术领域
本发明涉及癌症基因检测方法，尤其涉及一种用于检测肺癌驱动性基因突变的探针及试剂盒。
背景技术
根据2018年世界癌症报告数据统计显示，全球肺癌的发病率和致死率在所有癌种中排首位，且逐年呈上升趋势，其中肺癌的全球发病率占癌症总发病人口的11.6％，致死率占癌症总发病人口的18.4％。在中国，肺癌的发病数位居所有癌症的榜首，发病数达78.7万。在男性中，发病比例较高；在女性中，肺癌的发病率仅次于乳腺癌，位居第二。但男女肺癌的死亡率均居癌症死亡榜首，对患者及社会造成了严重的负担。在临床肿瘤治疗过程中发现，人体肿瘤千差万别，即使是同一个部位的肿瘤，治疗效果和方法也应因人而异。此外，肿瘤的异质性高，即使是同一个人的同一部位的肿瘤也有可能包含了多种突变类型的肿瘤细胞，所以给临床治疗带来了很大的挑战。“驱动基因”这一名词的出现是在2012年亚洲肿瘤大会上，广东省人民医院副院长吴一龙教授提出的。他指出与癌症发生发展相关的基因可称为驱动基因，其决定了这个癌症的最主要原因。我们知道了驱动基因，就知道可以采用哪些药物来对抗它，就可以进行个体化治疗。也正是由于这种观念上的巨大改变，基因检测及肿瘤的个体化治疗越来越广泛的引起重视，短短几年之间发展飞速。
基因检测技术主要依赖于高通量测序仪，目前第二代测序(Next GenerationSequencing， NGS)平台主要以Illumina公司的Hiseq，Miseq，Nextseq系列及Life公司的Ion TorrentTM系列测序仪为代表，已被广泛应用于生物学研究、产前诊断、基因诊断和治疗方面。然而肿瘤患者往往携带的是一小片段基因的改变或单个碱基的改变，如果选择全基因组测序会造成成本的浪费，且给患者带来更沉重的经济负担。目标序列捕获测序技术的应用解决了上述问题，该技术可将感兴趣的基因组区域定制成特异性探针与基因组DNA进行杂交，将目标基因组区域的DNA片段进行富集后再利用第二代测序技术进行测序。这种方法与传统聚合酶链式反应(PCR)及基因芯片相比，通量高，准确度高，且可一次性检测已知和未知基因，可以有效检测到样本的高频突变及低频甚至超低频突变。
随着对肺癌基因层面的深入研究，越来越多的与肺癌相关的驱动基因被挖掘，进而开发出多种靶向特异基因突变的药物。目前已知驱动基因如EGFR、KRAS、BRAF、MET、NRAS、PIK3CA等等，还有化疗药的相关基因如TP53和UGT1A1等。常见靶向药物有：EGFR靶药-吉非替尼、厄洛替尼、埃克替尼、阿法替尼、奥希替尼、AZD9291；MET靶药-赛可瑞；BRAF 靶药-达拉非尼。化疗药物如：伊立替康、5-FU、卡培他滨、铂类药物等。
目前，临床上最常用的基因突变检测方法有Sanger测序技术、荧光原位杂交技术(FISH)，扩增阻碍突变系统技术(ARMS-PCR)、荧光PCR技术等。Sanger测序技术作为基因检测的金标准，主要用于寻找与疾病有关的特定的基因突变，难以完成没有明确候选基因或候选基因数量较多的大样本病例的筛查。荧光定量PCR检测目的基因仅需检测样本是否具有扩增信号即可，可检测微小突变，但通量低。ARMS-PCR技术可用于对已知突变基因进行检测，对未知突变基因不可行。FISH技术虽然采用几种不同颜色的荧光素单独或者混合标记的探针进行原位杂交,能同时检测多个基因，但在进行大量位点的检测仍然有限。
本发明提供的一种用于检测肺癌驱动性基因突变的探针及检测试剂盒。可以联合检测肺癌多种基因的突变，大大节省了患者的样本和等待检测的时间，为肺癌患者的个体化治疗提供一种依据和指导，同时也可监测治疗后的疗效及预测预后。
发明内容
本发明的目的在于提供一种用于检测肺癌驱动性基因突变的探针及检测试剂盒，包括 BRAF、EGFR、KRAS、MET、NRAS和PIK3CA驱动性基因突变的探针及TP53和UGT1A1 化疗药物相关基因的探针，通过对这些基因的检测实现肺癌的个体化治疗和预测预后，
本发明提供的一种用于检测肺癌驱动性基因突变的探针，包括下列6组共248条探针：
BRAF驱动突变特异性探针序列：其序列为SEQ ID NO.1至SEQ ID NO.3；
EGFR驱动突变特异性探针序列：其序列为SEQ ID NO.4至SEQ ID NO.98；
KRAS驱动突变特异性探针序列：其序列为SEQ ID NO.99至SEQ ID NO.148；
MET驱动突变特异性探针序列：其序列为SEQ ID NO.149至SEQ ID NO.150；
NRAS驱动突变特异性探针序列：其序列为SEQ ID NO.151至SEQ ID NO.193；
PIK3CA驱动突变特异性探针序列：其序列为SEQ ID NO.194至SEQ ID NO.248；
还包括下列2组共28条探针：
TP53特异性探针核苷酸序列：其序列为SEQ ID NO.249至SEQ ID NO.272；
UGT1A1特异性探针序列：其序列为SEQ ID NO.273至SEQ ID NO.276。
以上特异性探针的序列具体如下：
1.BRAF驱动突变探针序列
SEQ ID NO.1
GATGATCTTCATCTGCTGGTCGGAAGGGCTGTGGAATTGGAATGGATTTTGAAGGAGACGGACTG GTGAGAATTTGGGGCCTGGAAAAATGAAGTCATTGGAAGATAAGATTCAGAGTAA
SEQ ID NO.2
ACTTGGTTTTTTTTTAGTTCTAGCAATGCTGGATACTTACATCAATATTGACAGGTTCTATTGTGTTTATATGCACATTGGGAGCTGATGAGGATCGGTCTCGTTGCCCAAATTGATTTC
SEQ ID NO.3
CATGCCACTTTCCCTTGTAGACTGTTCCAAATGATCCAGATCCAATTCTTTGTCCCACTGTAATCT GCCCATCAGGAATCTCCCAATCATCACTCGAGTCCCGTCTACCAAGTGTTTTCT
2.EGFR驱动突变探针序列
SEQ ID NO.4
GCAGCCTCCGCCCCCCGCACGGTGTGAGCGCCCGACGCGGCCGAGGCGGCCGGAGTCCCGAGC TAGCCCCGGCGGCCGCCGCCGCCCAGACCGGACGACAGGCCACCTCGTCGGCGTCCG
SEQ ID NO.5
CCCGAGTCCCCGCCTCGCCGCCAACGCCACAACCACCGCGCACGGCCCCCTGACTCCGTCCAGT ATTGATCGGGAGAGCCGGAGCGAGCTCTTCGGGGAGCAGCGATGCGACCCTCCGGG
SEQ ID NO.6
ACGGCCGGGGCAGCGCTCCTGGCGCTGCTGGCTGCGCTCTGCCCGGCGAGTCGGGCTCTGGAG GAAAAGAAAGGTAAGGGCGTGTCTCGCCGGCTCCCGCGCCGCCCCCGGATCGCGCCC
SEQ ID NO.7
TGCATTTCTCAGTATTTCATGTGATATCTGTCTTTTTCTTCCAGTTTGCCAAGGCACGAGTAACAA GCTCACGCAGTTGGGCACTTTTGAAGATCATTTTCTCAGCCTCCAGAGGATGTT
SEQ ID NO.8
CAATAACTGTGAGGTGGTCCTTGGGAATTTGGAAATTACCTATGTGCAGAGGAATTATGATCTTTC CTTCTTAAAGGTTGGTGACTTTGATTTTCCTACACAAATAAAATTGGAGAAAAT
SEQ ID NO.9
ATCACGCATTTATGTTTTCTCTTCTTAGACCATCCAGGAGGTGGCTGGTTATGTCCTCATTGCCCTCAACACAGTGGAGCGAATTCCTTTGGAAAACCTGCAGATCATCAGAGGAAATAT
SEQ ID NO.10
GTACTACGAAAATTCCTATGCCTTAGCAGTCTTATCTAACTATGATGCAAATAAAACCGGACTGAA GGAGCTGCCCATGAGAAATTTACAGGGTGAGAGGCTGGGATGCCAAGGCTGGGG
SEQ ID NO.11
GGAATTTAAAGGAGCTGGAAAGAGTGCTCACCGCAGTTCCATTCTCCCGCAGAAATCCTGCATG GCGCCGTGCGGTTCAGCAACAACCCTGCCCTGTGCAACGTGGAGAGCATCCAGTGG
SEQ ID NO.12
CGGGACATAGTCAGCAGTGACTTTCTCAGCAACATGTCGATGGACTTCCAGAACCACCTGGGCA GCTGTAAGTGTCGCATACACACTATCTCTGCCTCCAGCTCCTATGGGGGACAGCTC
SEQ ID NO.13
ATGCATCTATTACTTTTACATTTCAGGCCAAAAGTGTGATCCAAGCTGTCCCAATGGGAGCTGCTG GGGTGCAGGAGAGGAGAACTGCCAGAAACGTAAGTCAGTGAACAGCCTCAGACC
SEQ ID NO.14
TGACCAAAATCATCTGTGCCCAGCAGTGCTCCGGGCGCTGCCGTGGCAAGTCCCCCAGTGACTG CTGCCACAACCAGTGTGCTGCAGGCTGCACAGGCCCCCGGGAGAGCGACTGCCTGG
SEQ ID NO.15
GCTGAGTGTACTTACCTCACTTGCCCAGCGTGTCCTCTCTCCTCCATAGGTCTGCCGCAAATTCCG AGACGAAGCCACGTGCAAGGACACCTGCCCCCCACTCATGCTCTACAACCCCAC
SEQ ID NO.16
CACGTACCAGATGGATGTGAACCCCGAGGGCAAATACAGCTTTGGTGCCACCTGCGTGAAGAAG TGTCCCCGTGAGTCCTCCTCTGTGGGCCCTCTAACTGGTCAGGCATCCTTGTCCCG
SEQ ID NO.17
AGGTAATTATGTGGTGACAGATCACGGCTCGTGCGTCCGAGCCTGTGGGGCCGACAGCTATGAGA TGGAGGAAGACGGCGTCCGCAAGTGTAAGAAGTGCGAAGGGCCTTGCCGCAAAGG
SEQ ID NO.18
AGCTATTCTTAATCCAACAAATGTGAACGGAATACACGTCTCTCTTATCTCTGCAGTGTGTAACGG AATAGGTATTGGTGAATTTAAAGACTCACTCTCCATAAATGCTACGAATATTAA
SEQ ID NO.19
ACACTTCAAAAACTGCACCTCCATCAGTGGCGATCTCCACATCCTGCCGGTGGCATTTAGGGGGT GAGTCACAGGTTCAGTTGCTTGTATAAAGAAAAACAAAATCTGCCTTTTTAACTG
SEQ ID NO.20
AATCACCCTGTTGTTTGTTTCAGTGACTCCTTCACACATACTCCTCCTCTGGATCCACAGGAACTG GATATTCTGAAAACCGTAAAGGAAATCACAGGTTTGAGCTGAATTATCACATGA
SEQ ID NO.21
CATCTGCCTTACAGGGTTTTTGCTGATTCAGGCTTGGCCTGAAAACAGGACGGACCTCCATGCCT TTGAGAACCTAGAAATCATACGCGGCAGGACCAAGCAACAGTAAGTTGACCACAG
SEQ ID NO.22
TTTTCTTCTCTCCAATGTAGTGGTCAGTTTTCTCTTGCAGTCGTCAGCCTGAACATAACATCCTTG GGATTACGCTCCCTCAAGGAGATAAGTGATGGAGATGTGATAATTTCAGGAAAC
SEQ ID NO.23
AAAAATTTGTGCTATGCAAATACAATAAACTGGAAAAAACTGTTTGGGACCTCCGGTCAGAAAA CCAAAATTATAAGCAACAGAGGTGAAAACAGCTGCAGTAAGTCACCGCTTTCTGTT
SEQ ID NO.24
CCCCTCGGGTCCCTGCTCTGTCACTGACTGCTGTGACCCACTCTGTCTCCGCAGAGGCCACAGG CCAGGTCTGCCATGCCTTGTGCTCCCCCGAGGGCTGCTGGGGCCCGGAGCCCAGGG
SEQ ID NO.25
ACTGCGTCTCTTGCCGGAATGTCAGCCGAGGCAGGGAATGCGTGGACAAGTGCAACCTTCTGGA GGGGTAGGAGGTTATTTCTTTAATCCCCTTGCGTTGATCAAAAATAAGGCTCCAGG
SEQ ID NO.26
CTTCCTCCTCTCAGTGAGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATACAGTGCCACCCAG AGTGCCTGCCTCAGGCCATGAACATCACCTGCACAGGACGGGTAAGAGCCCCTTGC
SEQ ID NO.27
GAAAGAGACATGCATGAACATTTTTCTCCACCTTGGTGCAGGGACCAGACAACTGTATCCAGTGT GCCCACTACATTGACGGCCCCCACTGCGTCAAGACCTGCCCGGCAGGAGTCATGG
SEQ ID NO.28
GAGAAAACAACACCCTGGTCTGGAAGTACGCAGACGCCGGCCATGTGTGCCACCTGTGCCATCC AAACTGCACCTACGGGTGAGTGGAAAGTGAAGGAGAACAGAACATTTCCTCTCTTG
SEQ ID NO.29
AGAATGAGAAAAATGTATATTTCTCTTTCACTTCCTACAGATGCACTGGGCCAGGTCTTGAAGGC TGTCCAACGAATGGGTAAGTGTTCACAGCTCTGTGTCACATGGACCTCGTCAAGA
SEQ ID NO.30
GGAATCTGTCAGCAACCTCACCCTTCCTTGTTCCTCCACCTCATTCCAGGCCTAAGATCCCGTCCA TCGCCACTGGGATGGTGGGGGCCCTCCTCTTGCTGCTGGTGGTGGCCCTGGGGA
SEQ ID NO.31
TCGGCCTCTTCATGCGAAGGCGCCACATCGTTCGGAAGCGCACGCTGCGGAGGCTGCTGCAGGA GAGGGAGGTGAGTGCCAGTCCTGGGTGGGCTCAGGAGCCCTCGCACCCCGACAGGA
SEQ ID NO.32
CTTTCCAGCATGGTGAGGGCTGAGGTGACCCTTGTCTCTGTGTTCTTGTCCCCCCCAGCTTGTGG AGCCTCTTACACCCAGTGGAGAAGCTCCCAACCAAGCTCTCTTGAGGATCTTGAA
SEQ ID NO.33
GGAAACTGAATTCAAAAAGATCAAAGTGCTGGGCTCCGGTGCGTTCGGCACGGTGTATAAGGTA AGGTCCCTGGCACAGGCCTCTGGGCTGGGCCGCAGGGCCTCTCATGGTCTGGTGGG
SEQ ID NO.34
TCTGTCATAGGGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGGAAT TAAGAGAAGCAACATCTCCGAAAGCCAACAAGGAAATCCTCGATGTGAGTTTCTG
SEQ ID NO.35
CACTGACGTGCCTCTCCCTCCCTCCAGGAAGCCTACGTGATGGCCAGCGTGGACAACCCCCACG TGTGCCGCCTGCTGGGCATCTGCCTCACCTCCACCGTGCAGCTCATCACGCAGCTC
SEQ ID NO.36
ATGCCCTTCGGCTGCCTCCTGGACTATGTCCGGGAACACAAAGACAATATTGGCTCCCAGTACCT GCTCAACTGGTGTGTGCAGATCGCAAAGGTAATCAGGGAAGGGAGATACGGGGAG
SEQ ID NO.37
CATGATGATCTGTCCCTCACAGCAGGGTCTTCTCTGTTTCAGGGCATGAACTACTTGGAGGACCG TCGCTTGGTGCACCGCGACCTGGCAGCCAGGAACGTACTGGTGAAAACACCGCAG
SEQ ID NO.38
CATGTCAAGATCACAGATTTTGGGCTGGCCAAACTGCTGGGTGCGGAAGAGAAAGAATACCATG CAGAAGGAGGCAAAGTAAGGAGGTGGCTTTAGGTCAGCCAGCATTTTCCTGACACC
SEQ ID NO.39
CCTCATCTCTCACCATCCCAAGGTGCCTATCAAGTGGATGGCATTGGAATCAATTTTACACAGAAT CTATACCCACCAGAGTGATGTCTGGAGCTACGGTGAGTCATAATCCTGATGCTA
SEQ ID NO.40
TTCATTCATGATCCCACTGCCTTCTTTTCTTGCTTCATCCTCTCAGGGGTGACTGTTTGGGAGTTG ATGACCTTTGGATCCAAGCCATATGACGGAATCCCTGCCAGCGAGATCTCCTCC
SEQ ID NO.41
ATCCTGGAGAAAGGAGAACGCCTCCCTCAGCCACCCATATGTACCATCGATGTCTACATGATCAT GGTCAAGTGTGAGTGACTGGTGGGTCTGTCCACACTGCCTAGCTGAGCCTTGGTG
SEQ ID NO.42
TCCTTCCCCAGGCTGGATGATAGACGCAGATAGTCGCCCAAAGTTCCGTGAGTTGATCATCGAAT TCTCCAAAATGGCCCGAGACCCCCAGCGCTACCTTGTCATTCAGGTACAAATTGC
SEQ ID NO.43
CGGGCCATTCTAATAGCCTCAAAATCTCTGCACCAGGGGGATGAAAGAATGCATTTGCCAAGTCC TACAGACTCCAACTTCTACCGTGCCCTGATGGATGAAGAAGACATGGACGACGTG
SEQ ID NO.44
GTGGATGCCGACGAGTACCTCATCCCACAGCAGGGCTTCTTCAGCAGCCCCTCCACGTCACGGA CTCCCCTCCTGAGCTCTCTGGTATGAAATCTCTGTCTCTCTCTCTCTCTCAAGCTG
SEQ ID NO.45
AGCATTCCATGGGCAACTTCTCTGTTTCTTTTTCAGAGTGCAACCAGCAACAATTCCACCGTGGC TTGCATTGATAGAAATGGGGTATGTATGAACACCTTATAAGCCAGAATTTACAGC
SEQ ID NO.46
CTGCAGCTGCAAAGCTGTCCCATCAAGGAAGACAGCTTCTTGCAGCGATACAGCTCAGACCCCA CAGGCGCCTTGACTGAGGACAGCATAGACGACACCTTCCTCCCAGTGCCTGGTGAG
SEQ ID NO.47
TGATTTCTTTCCACTTTCAGAATACATAAACCAGTCCGTTCCCAAAAGGCCCGCTGGCTCTGTGC AGAATCCTGTCTATCACAATCAGCCTCTGAACCCCGCGCCCAGCAGAGACCCACA
SEQ ID NO.48
CTACCAGGACCCCCACAGCACTGCAGTGGGCAACCCCGAGTATCTCAACACTGTCCAGCCCACC TGTGTCAACAGCACATTCGACAGCCCTGCCCACTGGGCCCAGAAAGGCAGCCACCA
SEQ ID NO.49
AATTAGCCTGGACAACCCTGACTACCAGCAGGACTTCTTTCCCAAGGAAGCCAAGCCAAATGGC ATCTTTAAGGGCTCCACAGCTGAAAATGCAGAATACCTAAGGGTCGCGCCACAAAG
SEQ ID NO.50
CAGTGAATTTATTGGAGCATGACCACGGAGGATAGTATGAGCCCTAAAAATCCAGACTCTTTCGA TACCCAGGACCAAGCCACAGCAGGTCCTCCATCCCAACAGCCATGCCCGCATTAG
SEQ ID NO.51
CTCTTAGACCCACAGACTGGTTTTGCAACGTTTACACCGACTAGCCAGGAAGTACTTCCACCTCG GGCACATTTTGGGAAGTTGCATTCCTTTGTCTTCAAACTGTGAAGCATTTACAGA
SEQ ID NO.52
AACGCATCCAGCAAGAATATTGTCCCTTTGAGCAGAAATTTATCTTTCAAAGAGGTATATTTGAAA AAAAAAAAAAGTATATGTGAGGATTTTTATTGATTGGGGATCTTGGAGTTTTTC
SEQ ID NO.53
ATTGTCGCTATTGATTTTTACTTCAATGGGCTCTTCCAACAAGGAAGAAGCTTGCTGGTAGCACTT GCTACCCTGAGTTCATCCAGGCCCAACTGTGAGCAAGGAGCACAAGCCACAAGT
SEQ ID NO.54
CTTCCAGAGGATGCTTGATTCCAGTGGTTCTGCTTCAAGGCTTCCACTGCAAAACACTAAAGATC CAAGAAGGCCTTCATGGCCCCAGCAGGCCGGATCGGTACTGTATCAAGTCATGGC
SEQ ID NO.55
AGGTACAGTAGGATAAGCCACTCTGTCCCTTCCTGGGCAAAGAAGAAACGGAGGGGATGGAATT CTTCCTTAGACTTACTTTTGTAAAAATGTCCCCACGGTACTTACTCCCCACTGATG
SEQ ID NO.56
GACCAGTGGTTTCCAGTCATGAGCGTTAGACTGACTTGTTTGTCTTCCATTCCATTGTTTTGAAAC TCAGTATGCTGCCCCTGTCTTGCTGTCATGAAATCAGCAAGAGAGGATGACACA
SEQ ID NO.57
TCAAATAATAACTCGGATTCCAGCCCACATTGGATTCATCAGCATTTGGACCAATAGCCCACAGCT GAGAATGTGGAATACCTAAGGATAGCACCGCTTTTGTTCTCGCAAAAACGTATC
SEQ ID NO.58
TCCTAATTTGAGGCTCAGATGAAATGCATCAGGTCCTTTGGGGCATAGATCAGAAGACTACAAAA ATGAAGCTGCTCTGAAATCTCCTTTAGCCATCACCCCAACCCCCCAAAATTAGTT
SEQ ID NO.59
TGTGTTACTTATGGAAGATAGTTTTCTCCTTTTACTTCACTTCAAAAGCTTTTTACTCAAAGAGTATATGTTCCCTCCAGGTCAGCTGCCCCCAAACCCCCTCCTTACGCTTTGTCACAC
SEQ ID NO.60
AAAAAGTGTCTCTGCCTTGAGTCATCTATTCAAGCACTTACAGCTCTGGCCACAACAGGGCATTT TACAGGTGCGAATGACAGTAGCATTATGAGTAGTGTGGAATTCAGGTAGTAAATA
SEQ ID NO.61
TGAAACTAGGGTTTGAAATTGATAATGCTTTCACAACATTTGCAGATGTTTTAGAAGGAAAAAAG TTCCTTCCTAAAATAATTTCTCTACAATTGGAAGATTGGAAGATTCAGCTAGTTA
SEQ ID NO.62
GGAGCCCACCTTTTTTCCTAATCTGTGTGTGCCCTGTAACCTGACTGGTTAACAGCAGTCCTTTGT AAACAGTGTTTTAAACTCTCCTAGTCAATATCCACCCCATCCAATTTATCAAGG
SEQ ID NO.63
AAGAAATGGTTCAGAAAATATTTTCAGCCTACAGTTATGTTCAGTCACACACACATACAAAATGT TCCTTTTGCTTTTAAAGTAATTTTTGACTCCCAGATCAGTCAGAGCCCCTACAGC
SEQ ID NO.64
ATTGTTAAGAAAGTATTTGATTTTTGTCTCAATGAAAATAAAACTATATTCATTTCCACTCTATTATGCTCTCAAATACCCCTAAGCATCTATACTAGCCTGGTATGGGTATGAAAGAT
SEQ ID NO.65
AGGCCTCACAGGAGGATGACCAGGTCTCAGTCAGCGGGGAGGTGGAAAGTGCAGGTGCATCAG GGGCACCCTGACCGAGGAAACAGCTGCCAGAGGCCTCCACTGCTAAAGTCCACATAA
SEQ ID NO.66
AAGGCTGAGGTCAGTCACCCTAAACAACCTGCTCCCTCTAAGCCAGGGGATGAGCTTGGAGCAT CCCACAAGTTCCCTAAAAGTTGCAGCCCCCAGGGGGATTTTGAGCTATCATCTCTG
SEQ ID NO.67
TGCACATGCTTAGTGAGAAGACTACACAACATTTCTAAGAATCTGAGATTTTATATTGTCAGTTAA CCACTTTCATTATTCATTCACCTCAGGACATGCAGAAATATTTCAGTCAGAACT
SEQ ID NO.68
CTGGGAAACAGAAGGACCTACATTCTGCTGTCACTTATGTGTCAAGAAGCAGATGATCGATGAG GCAGGTCAGTTGTAAGTGAGTCACATTGTAGCATTAAATTCTAGTATTTTTGTAGT
SEQ ID NO.69
GTTTGAAACAGTAACTTAATAAAAGAGCAAAAGCTATTCTAGCTTTCTTCTTCATATTTTAATTTTCCACCATAAAGTTTAGTTGCTAAATTCTATTAATTTTAAGATTGTGCTTCCCAA
SEQ ID NO.70
AAAATAGTTCTCACTTCATCTGTCCAGGGAGGCACAGTTCTGTCTGGTAGAAGCCGCAAAGCCCT TAGCCTCTTCACGGATCTGGCGACTGTGATGGGCAGGTCAGGAGAGGAGCTGCCC
SEQ ID NO.71
CCAAAGTCCCATGATTTTCACCTAACAGCCCTGATCAGTCAGTACTCAAAGCTTGGACTCCATCC CTGAAGGTCTTCCTGATTGATAGCCTGGCCTTAATACCCTACAGAAAGCCTGTCC
SEQ ID NO.72
CCATTGGCTGTTTCTTCCTCAGTCAGTTCCTGGAAGACCTTACCCCATGACCCCAGCTTCAGATGT GGTCTTTGGAAACAGAGGTCGAAGGAAAGTAAGGAGCTGAGAGCTCACATTCAT
SEQ ID NO.73
ATAGGTGCCGCCAGCCTTCGTGCATCTTCTTGCATCATCTCTAAGGAGCTCCTCTAATTACACCAT GCCCGTCACCCCATGAGGGATCAGAGAAGGGATGAGTCTTCTAAACTCTATATT
SEQ ID NO.74
TTCGCTGTGAGTCCAGGTTGTAAGGGGGAGCACTGTGGATGCATCCTATTGCACTCCAGCTGATG ACACCAAAGCTTAGGTGTTTGCTGAAAGTTCTTGATGTTGTGACTTACCACCCCT
SEQ ID NO.75
CTGCCTCACAACTGCAGACATAAGGGGACTATGGATTGCTTAGCAGGAAAGGCACTGGTTCTCA AGGGCGGCTGCCCTTGGGAATCTTCTGGTCCCAACCAGAAAGACTGTGGCTTGATT
SEQ ID NO.76
TTTTCTCAGGTGCAGCCCAGCCGTAGGGCCTTTTCAGAGCACCCCCTGGTTATTGCAACATTCAT CAAAGTTTCTAGAACCTCTGGCCTAAAGGAAGGGCCTGGTGGGATCTACTTGGCA
SEQ ID NO.77
CACTCGCTGGGGGGCCACCCCCCAGTGCCACTCTCACTAGGCCTCTGATTGCACTTGTGTAGGAT GAAGCTGGTGGGTGATGGGAACTCAGCACCTCCCCTCAGGCAGAAAAGAATCATC
SEQ ID NO.78
TCTGTGGAGCTTCAAAAGAAGGGGCCTGGAGTCTCTGCAGACCAATTCAACCCAAATCTCGGGG GCTCTTTCATGATTCTAATGGGCAACCAGGGTTGAAACCCTTATTTCTAGGGTCTT
SEQ ID NO.79
TTCAGTTGTACAAGACTGTGGGTCTGTACCAGAGCCCCCGTCAGAGTAGAATAAAAGGCTGGGT AGGGTAGAGATTCCCATGTGCAGTGGAGAGAACAATCTGCAGTCACTGATAAGCCT
SEQ ID NO.80
CTGAGACTTGGCTCATTTCAAAAGCGTTCAATTCATCCTCACCAGCAGTTCAGCTGGAAAGGGG CAAATACCCCCACCTGAGCTTTGAAAACGCCCTGGGACCCTCTGCATTCTCTAAGT
SEQ ID NO.81
GTAAGTTATAGAAACCAGTCTCTTCCCTCCTTTGTGAGTGAGCTGCTATTCCACGTAGGCAACAC CTGTTGAAATTGCCCTCAATGTCTACTCTGCATTTCTTTCTTGTGATAAGCACAC
SEQ ID NO.82
ACACTTTTATTGCAACATAATGATCTGCTCACATTTCCTTGCCTGGGGGCTGTAAAACCTTACAGA ACAGAAATCCTTGCCTCTTTCACCAGCCACACCTGCCATACCAGGGGTACAGCT
SEQ ID NO.83
CTTTGTACTATTGAAGACACAGACAGGATTTTTAAATGTAAATCTATTTTTGTAACTTTGTTGCGG GATATAGTTCTCTTTATGTAGCACTGAACTTTGTACAATATATTTTTAGAAACT
SEQ ID NO.84
CTCATTTTTCTACTAAAACAAACACAGTTTACTTTAGAGAGACTGCAATAGAATCAAAATTTGAA ACTGAAATCTTTGTTTAAAAGGGTTAAGTTGAGGCAAGAGGAAAGCCCTTTCTCT
SEQ ID NO.85
CTCTCTTATAAAAAGGCACAACCTCATTGGGGAGCTAAGCTAGGTCATTGTCATGGTGAAGAAGA GAAGCATCGTTTTTATATTTAGGAAATTTTAAAAGATGATGGAAAGCACATTTAG
SEQ ID NO.86
AGCTTGGTCTGAGGCAGGTTCTGTTGGGGCAGTGTTAATGGAAAGGGCTCACTGTTGTTACTACT AGAAAAATCCAGTTGCATGCCATACTCTCATCATCTGCCAGTGTAACCCTGTACA
SEQ ID NO.87
CATGTAAGAAAAGCAATAACATAGCACTTTGTTGGTTTATATATATAATGTGACTTCAATGCAAATTTTATTTTTATATTTACAATTGATATGCATTTACCAGTATAAACTAGACATGTC
SEQ ID NO.88
TCTGGAGAGCCTAATAATGTTCAGCACACTTTGGTTAGTTCACCAACAGTCTTACCAAGCCTGGG CCCAGCCACCCTAGAGAAGTTATTCAGCCCTGGCTGCAGTGACAT
SEQ ID NO.89
GCCAAAGTTGAGAACTACTGGCCTAGGGATTAGCCACAAGGACATGGACTTGGAGGCAAATTCT GCAGGTGTATGTGATTCTCAGGCCTAGAGAGCTAAGACACAAAGACCTCCACATCT
SEQ ID NO.90
TGTCGCTGAGAGTCAAGAACCTGAACAGAGTTTCCATGAAGGTTCTCCAAGCACTAGAAGGGA GAGTGTCTAAACAATGGTTGAAAAGCAAAGGAAATATAAAACAGACACCTCTTTCCA
SEQ ID NO.91
ATTTCCTAAGGTTTCTCTCTTTATTAAGGGTGGACTAGTAATAAAATATAATATTCTTGCTGCTTATGCAGCTGACATTGTTGCCCTCCCTAAAGCAACCAAGTAGCCTTTATTTCCCAC
SEQ ID NO.92
CAGTGAAAGAAAACGCTGGCCTATCAGTTACATTACAAAAGGCAGATTTCAAGAGGATTGAGTA AGTAGTTGGATGGCTTTCATAAAAACAAGAATTCAAGAAGAGGATTCATGCTTTAA
SEQ ID NO.93
AGAAACATTTGTTATACATTCCTCACAAATTATACCTGGGATAAAAACTATGTAGCAGGCAGTGTG TTTTCCTTCCATGTCTCTCTGCACTACCTGCAGTGTGTCCTCTGAGGCTGCAAG
SEQ ID NO.94
GTCTGTCCTATCTGAATTCCCAGCAGAAGCACTAAGAAGCTCCACCCTATCACCTAGCAGATAAA ACTATGGGGAAAACTTAAATCTGTGCATACATTTCTGGATGCATTTACTTATCTT
SEQ ID NO.95
TTAAAAAAAAAGGAATCCTATGACCTGATTTGGCCACAAAAATAATCTTGCTGTACAATACAATCT CTTGGAAATTAAGAGATCCTATGGATTTGATGACTGGTATTAGAGGTGACAATG
SEQ ID NO.96
GTAACCGATTAACAACAGACAGCAATAACTTCGTTTTAGAAACATTCAAGCAATAGCTTTATAGCT TCAACATATGGTACGTTTTAACCTTGAAAGTTTTGCAATGATGAAAGCAGTATT
SEQ ID NO.97
TTGTACAAATGAAAAGCAGAATTCTCTTTTATATGGTTTATACTGTTGATCAGAAATGTTGATTGTGCATTGAGTATTAAAAAATTAGATGTATATTATTCATTGTTCTTTACTCCTGAG
SEQ ID NO.98
GTACCTTATAATAATAATAATGTATTCTTTGTTAACAATGCCATGTTGGTACTAGTTATTAATCATATCTAACCAACTGTAGGTGTTCTTTCCTGATAACTTTTTTAAAAAATCAATAAA
3.KRAS驱动突变探针序列
SEQ ID NO.99
AGAGACCAAACCCCTTCTTTGCAAAACTAAAATACGCATCGTGTTATCTCTGGGTCGTATACCAA AGGCCTTAGTAAGATATTACAGACCACACTAGCACTACCTAAGGACCGGGATTAT
SEQ ID NO.100
GCCAAATAAAAAAACAATATAATCAAGTTTATTCCTTTAAAACAATGAAGTGATTTACATAAAGTA GCTTGATCGAAGAGTTTCAGTGGAATCGTAGCAAAACAATTATAGAGCTGGCAC
SEQ ID NO.101
TTTAAGTATATTTTAATTACTTATGCAGAGAAAACTGGAATATTACACATTTGGGTCAATATGAATATCTGACATACACCTTAATGTGTACAGTAATTGTCCTAAAAGAATCACAGTTAT
SEQ ID NO.102
CCAAAGATCTGTAGTTTCACATAGCAATTCAGAAATCATAGTGATTTTTACATAGAAGTTTCCTTG TCTGTGAACTAGTTCAGGCACCTGTTTATTTGTACCCAGATAAAACTATTAATT
SEQ ID NO.103
AAAATTCAAGAGGCCTAAATATCCCCTCATAAGCACTGCAGTTCCTGAAGTATGGCCATTTCTTTC TCTGTGTAGAAACGAGGTACTGTGTAAGTCTTAACACCCTACCTAAACAGTGTT
SEQ ID NO.104
CATTTATTTCTAGAATTCTCCCCCTTTAAAATCTCTACAAAAACAAATCTTTTGTTAAACCATTCAAAGTTCACATAAAGGTAATTAACCACTACCTTAAAAAAATGCCCATCTACATCA
SEQ ID NO.105
ACAATATACGTCTGCTATATTCTTCCACAAACATGTTAATGCCTAAGTCTATGTAATTTAGCTTTTTTTAAAAAAACTTCAACAAGGATTTTTGTCTTTAAGGCTGTAATAATTAGGTAA
SEQ ID NO.106
ATTGTGCAATGGTGACAACAGTTTTGATAACCTATAAAAGTTAGGTTCTAAATTCCTATGCAGTGT GACTCAGTTAAATAGAGCCTAGAATGCCTACTTGGGAACATTCACTCAAATGAT
SEQ ID NO.107
ACTGTTTGAAGAAAAAATGTTTAGAAGAAAAAAAAAATCAATGGAATACAAATGAGATGAACTT GTGCAAACTGTAACTTAACATGCCCCACAAAGTTTCTATGTATATATTAGGACAAA
SEQ ID NO.108
AACTGCTGGGTTCTAAAAAACATTACTACACAATTATCAAGAAATCATTACTTTTTGACAAATGGA AATCTTCAGATAGTTTTTGCTGTCTAAAAAAAAATCCCCTAAAAAAAGTTATAT
SEQ ID NO.109
TGAGTATCTTTCTTTAGAAAGAAAGTTTCATTTTATGACAGCTATTCAGTTTCTCAATGCAGAATTCATGCTATCCAGTATTAACACAGAAGTTACTAAATATAAATTCAGCTTTAAGGT
SEQ ID NO.110
TAACTTTTTTTTCCCCTAAATTCATCTAAATTACCTATCATTATCCCAAACAGGCACTTCAAACTATTAAACTAAAACACAGATCTTAATCTAGTTATGACTATTCTTCAAGAACTCATG
SEQ ID NO.111
AAGTATTTTCAACATGAAAACACAAGACAGTGGAATTGGAAACTTTCGGATAAAACACTGTAAC CCAGTTAGCTCTGTGGGGGTGTGGGGGGAGAGATGGGCCCTCAACATATCTGCAGA
SEQ ID NO.112
GTACAAAATGTGCATGTTTCAGTTTACACTATACAAAAATAGTTAAAATACATTCCAGGTAAACAT GTTACATTAAGAAATAGTACTAGTAAGAAATTGGCACTCAAAGGAAAAATGCAA
SEQ ID NO.113
TTCAATTAAAAGAGTGGTCATTTTTAATGTTTGATATGACCAACATTCCTAGGTCAGCGCAACCAA ATGATGGAAAACAACTGGATCACACTGCATATGTCCCACAAAAGAAAGCACAAT
SEQ ID NO.114
AGTCACTGTAACTATTTTTATTACATTACAATAATTAGGAGTAGTACAGTTCATGACAAAAATATTACAAATTTTAGATCACTTCACAGCACATACTCCTATAAACATTTAAAAGTTAAT
SEQ ID NO.115
ATACTTCTAACAAGCTGCATAAAAGATTCACTGGTATACTATAGTGTCTATGGCTTATAAAGTATTCCATATCCATTTCGGGGCAAACAGTTCAAATTTCATGAATAAATACACACTCAT
SEQ ID NO.116
ATGTTATCCTATTTTTTTGTCATAAAGGCAGATTTGTTTTGCATCTACTTCTAAATATGGTTTTAAGTTTAAGCAAACACGCCTTTACATAATATCCACAGCACCTTTTAGAAATAAAGG
SEQ ID NO.117
TACATAACTAACTATTCAATTATTTCCATTATTAATGTTGCTGCTGCTACCTTTGGGCCTAAAACCAAGTCACCTGTTGTGTATCAATTACCTTCTTTGATAAAAGGAAATAAATAAATA
SEQ ID NO.118
AGGTTCACTACAAAACAAACAGTTCCTGGTAATGATTTAAATGTAGTTATAGAAATAAATAATATG TATGGAGTCATTACTTCTGACCTTGAAATAGCCTGCTGGTGACTGGCATTAACA
SEQ ID NO.119
CAACACCCTGAAATACATAAAAAGTATTAAAATGTGAATATATACGATGGCTTCATGTGTACAGGT AACAAATTTCATTAATGGAAAAAATATTAAGAAAGGATTCTTTATGTTTCTCTT
SEQ ID NO.120
CAAATTGTATTTACATAATTACACACTTTGTCTTTGACTTCTTTTTCTTCTTTTTACCATCTTTGCTCATCTTTTCTTTATGTTTTCGAATTTCTCGAACTAATGTATAGAAGGCATCAT
SEQ ID NO.121
TAAAAGCTAACAGTCTGCATGGAGCAGGAAAAAAATTAGGTAATGCTAAAACAAATGCTAATAAT TTAGTGTAATGTACAAAAATTACCACTTGTACTAGTATGCCTTAAGAAAAAAGTA
SEQ ID NO.122
ATCTTAGGTATTCAGTTTCTTTTTCACAGGCATTGCTAGTTCAAAAACCAAAACTCTGGGAATACT GGCACTTAGAGGAAAAAAAAACTTCCACTGTCATTTTAAAATAAGCATTTAAGG
SEQ ID NO.123
GATAAAACACAGAATAGGGATGATTCAAAAGCTTCATTAATTTGTTTCACACCAACATTCACAATT GGTAAGAAAAATAAGAAGTAATCAACTGCATGCACCAAAAGCCCCAAGACAGAA
SEQ ID NO.124
ACATGATGCCTAGAAGAATCATCATCAGGAAGCCCATAAATTTGTGTTCCCTCAATGTTTCAGTAA AAACCAATTAGAAGGTCTCAACTGAAATTAGTAATTAATCCATTTATGTGACTA
SEQ ID NO.125
CTTTTTATAGAAAAAATATAATATTTTGGGGAGAGTGACCATGACTAATAGCAGTGGAAAGGAGA CAAAACCTTTGTGAACAGTGTAACTTTACATTCATCAGGGATGACAAACTATAGG
SEQ ID NO.126
CATACTGGGTCTGCCTTAACAGGAAAAGCTATTAGGAGTCTTTATAGTAATTTATCTAATGTGAAA AGGAAATGGCCTTATAATAGTTTCCATTGCCTTGTAATTTTTTTCCATTTTTTT
SEQ ID NO.127
GGAGACTACATTTAATTCCTATGAGAATTTTTTATACTTGTTAAAATCTTTTCAATTATTATAAAAATTTAGTAGCATGTAAATATAGCCCCAAAATGGTTGCTATAATAATCCCCATTT
SEQ ID NO.128
AATAAGCTATAACTGGCCCAAATAATCTTTTAATGTCACAAGCAGAATTAAAACTATCTTCAAAGA CTCAAGTTGAAGAAAAGATTTAAAGTTATACTATGAAAGAGCAGTCTGACACAG
SEQ ID NO.129
TAACCAATGCATGACAACACTGGATGACCGTGGGGACACAGTCCATGCTGTGAAACTCTCTATGA AAGCTCAAAGGTTCACACAGGGCCTGGCCTTGCAACCTTGGTCTCTTCAACACCT
SEQ ID NO.130
AAAGAGTTTGTTTTCTTAAGAAACAAAAGCAATGCTCTTGATTTGTCAGCAGGACCACCACAGA GTGAGATTGTATCTTGTTGAGCTATCCAAACTGCCCTAGTCCCTCCCCATTTTGAC
SEQ ID NO.131
GCCAAACCTAGAGATTGTAAAACTTTTTCACTTCATTGTTTAAAAAAAAAATTAATGTCTTGGCAC ACCACCACCCCAAAATCTCAACTTTTGAGTTAATATTTAAAAGTAATTTTTAAA
SEQ ID NO.132
AGTAACATTTTAAATTTATCAAAAGGATTGTTTTTATTTTTATTTTAAAGCATTATTAAATATGGATCAGACTTGAAAAGTGTTTATGCAATGTTAATTTAACCAGTGTTAAGAGAACTA
SEQ ID NO.133
AAGTGATGTCCTCAAAATCAGAGTCCTAAAAGACACCTATCTAGAACCTAAGTCACCTTCTTCCT AGTCCAGTGATACTTTCACCTCACCATGCCATCTCACTTCATTTATTTTAAAATA
SEQ ID NO.134
ATTGTGCTGAGCTTGACAAATAAGTGTATCCTTATGTAAATGGAATATAAATTACATAGTTGTAAAAAAAAAAAACTAAGAGTTTGAGATGACTTCTTTTAACATGAAGAAATGGATAGT
SEQ ID NO.135
ACTAATATGGAAGAAGAGTCCTAAAACGAGAATGGATATTCAAATATAAACTTCACCTCTTGCAC AATTTTGCCCAAGACTGGCACTGAAGATGGTGTAACATAGGTTAAAAATTTACAG
SEQ ID NO.136
TCAGTGGGAAAACTTCATGGAGATATCCACAGCAGCAGTAAATCTTATGGTTAGGGGAATTACAA GTATTAAAACTGCATCAAGTCATGGGGCATGTGGAAGGTAGGGAGGCAAGATGAC
SEQ ID NO.137
GAGATGTTCAAAGCATCAGCCACCACCTGAAAATTAGTGATTAGGTCAAATCCCTTTATGGTATCT GTCAGATTCTCTTGAGCCCTGAGGAAATAAGATGTAGGGCATTTCTGATGTGAC
SEQ ID NO.138
GATTACCTAAGGAATTCTTTCAGCACTATCTTTATTAAATTCTCCTTCCACTGGATAGGGTTCTGTCTATTCATACCAGGTTTGAAAAATCCTACTGTCGCTAATGGATTGGGCAGCAAA
SEQ ID NO.139
CTCTGACACCAAAAAAAAAAAGTAAGCTTCATGAATTAAAGTATTTTTCATTGCATGAAGATTTC TGGTTACTAAAACAATGGAATGTATTACTGTTACCAGGAGTAGTCCTAGTTATA
SEQ ID NO.140
TTCTAGAAGGCAAATCACATTTATTTCCTACTAGGACCATAGGTACATCTTCAGAGTCCTTAACTC TTTTAATTTGTTCTCTGGGAAAGAAAAAAAAGTTATAGCACAGTCATTAGTAAC
SEQ ID NO.141
TTGCAGAAAACAGATCTGTATTTATTTCAGTGTTACTTACCTGTCTTGTCTTTGCTGATGTTTCAATAAAAGGAATTCCATAACTTCTTGCTAAGTCCTGAGCCTGTTTTGTGTCTACTG
SEQ ID NO.142
ATTGCACTGTACTCCTCTTGACCTGCTGTGTCGAGAATATCCAAGAGACAGGTTTCTCCATCAATT ACTACTTGCTTCCTGTAGGAATCCTGAGAAGGGAGAAACACAGTCTGGATTATT
SEQ ID NO.143
GTCAGCTTATTATATTCAATTTAAACCCACCTATAATGGTGAATATCTTCAAATGATTTAGTATTATTTATGGCAAATACACAAAGAAAGCCCTCCCCAGTCCTCATGTACTGGTCCCTC
SEQ ID NO.144
CTCTTGCCTACGCCACCAGCTCCAACTACCACAAGTTTATATTCAGTCATTTTCAGCAGGCCTTAT AATAAAAATAATGAAAATGTGACTATATTAGAACATGTCACACATAAGGTTAAT
SEQ ID NO.145
TTTATCTGTATCAAAGAATGGTCCTGCACCAGTAATATGCATATTAAAACAAGATTTACCTCTATTGTTGGATCATATTCGTCCACAAAATGATTCTGAATTAGCTGTATCGTCAAGGCA
SEQ ID NO.146
ACCTGGGAGCCGCTGAGCCTCTGGCCCCGCCGCCGCCTTCAGTGCCTGCGCCGCGCTCGCTCCC AGTCCGAAATGGCGGGGGCCGGGAGTACTGGCCGAGCCGCCGCCACCTTCGCCGCC
SEQ ID NO.147
CGCCCCTCCGGGGACCCCTAATTCATTCACTCGCCGCCGGCCCCGCCCGGCGCCGGCAAAGAGG GTCGGGACCCGGGCAGGGGCCCAGGAGGGGTGGTCCGCTCCGTACCTCTCTCCCGC
SEQ ID NO.148
TCCGCCGCCGCGGCCGCCGCCTAGGAAAATCGAGCTCCGAGCACACCGATGAGTTCGGGGCCGG GCGGCCGCAGAGGGCAGAGCTATCGATGCGTTCCGCGCTCGATTCTTCTTCAGAC
4.MET驱动突变探针序列
SEQ ID NO.149
GGCCCATGATAGCCGTCTTTAACAAGCTCTTTCTTTCTCTCTGTTTTAAGATCTGGGCAGTGAATT AGTTCGCTACGATGCAAGAGTACACACTCCTCATTTGGATAGGCTTGTAAGTGC
SEQ ID NO.150
CCGAAGTGTAAGCCCAACTACAGAAATGGTTTCAAATGAATCTGTAGACTACCGAGCTACTTTTC CAGAAGGTATATTTCAGTTTATTGTTCTGAGAAATACCTATACATATACCTCAGT
5.NRAS驱动突变探针序列
SEQ ID NO.151
CATAGCCTAAAAACCACGTTTTATACCACACTGAAGTTATTCAGCTTTTAACTATAAGTTTGTCAC TCCTTTGTGCCAAAATTCACACCTAGGTGTAACTTTTTTTTTGGAGACAAGTCT
SEQ ID NO.152
AACATGTAACCTTTAAAAAAACCAAAAGGAATATGTAATTTATGACAGCTGGCCAGGCACTTAAT TTGGGGAAAGAGAAGGATTTTGAGGTAAACTAGAATTCTTTTCAGCAATCACAAA
SEQ ID NO.153
TATGATATTGTATTAAGTAACATTCTATAGTAAGCCGTTGGAGCAAATACAGCATTTAACAGGTTCATTTACAGATCCGGCATGGTAGCCTTCAGACAGAACATCTTATTTTCACTCTTG
SEQ ID NO.154
TTTCAAGTCATGACAAAAAAATTCACAGCAGTTTAAAGGATGGTGAAAGACTAAACATGGGCCC ACTAAAATAGAGATTAATTTTACCTATTACACTCCTATAGTAAAAATTGTAATAAG
SEQ ID NO.155
GGTTGTAGCTGCCAACTTAGGAGTTCATGACTATAAATATAAAGCAAATATGATTATGCTCACAAG CTCTGGAATGGTAAAAAAACCCCACATATCTCTAAAGTGTTTCTCTATCCTTGC
SEQ ID NO.156
TATAGTCCCTTGTTTATAGAAACCATCCAAATAAAATATTTTCCCTAATTTTCCTTGCTTCTTATAATTTATTAGCAGAGATGAATTACAAGAAGCAGAACGCACCATTTTTTGGTTCTT
SEQ ID NO.157
TACTCTGGTAACAAGAATACTTTTAAAACAATTCTTATAGAAAACAAATACCTTAGATAATTGTGT GGCACATTAGACTGCTCAAAGAAACAGCAAAGATGAAAAATAATGTACAAGAAT
SEQ ID NO.158
AAAGTCTTACAACTTCCACGGACATCCTCCCCTCCCTCCAGAGCAGAGTTCATACAATTCCATCTT AGGTCAGTACTCAGGCTTTTAAAAATCATCTTACGTTTTGAAATATAATACAAC
SEQ ID NO.159
ATAAAATAATCAACTTAAAATATCGGCCCTTCCATTTAGGGCCAAGGAGGCCAATAGTTCCTGTTT AAACAGCAGAATTGCACAATTATTTTTACCTATATTTGATGGCACAAAAAAATA
SEQ ID NO.160
CTTCAGTGAATATAAGAATTATGACTAAGCCAAGAACTTCCAGTTTTTATTTTTTAAACATCATTTAACAAGAAAAAACATTCAACCAAATTAAAAAGAACTAGGTTGGATTAATTTACA
SEQ ID NO.161
CTTCTTTCCTTGCAGGGCTAACTGCAGCTACCTAGCAAAATTACTAGCTGGAGTTACTGGTGCAAT GAGCAAATGCAGAAGAATTTCTTTCTCTGTGATGCTAACTGGATTGTTCAAGTT
SEQ ID NO.162
CAAATGAGAATATTGGCTTAATTTAGAGTTTCTACATGGCTAAAAGAAAAAAAAATGTTTAAATAC GTGATACTGAGTTTTCCAGTCATGGTAAAGGGTTTGTGCTAACTGACCTCTTCT
SEQ ID NO.163
CTGTTTTGAGCAAACTCATCAGCTTAGATTTATCAACATCTTGTTTTAAACCCTTCAAAGAACATT ATGAATAAAGAGAATTTAAAACGTTTCTCAGCTGAGACATCCTCATTCTCAAAT
SEQ ID NO.164
TATTTTTCATGAGAATTACCAGGGTATTATCTGCAAATTAAAATGCCAAGCCTCCAAATCCTCATAGGTATCAATTAATTATATTTTCCATTCCTGTCTCAACAATTTCAACTTCCTGTT
SEQ ID NO.165
ATAAATGAAAATTCATAATATAGAAGATGGAGGACCTCAGTAAGTATTTAGATATTAAAGACAGGA ACAAAGTGGCCATTTTGGAATTAGTCTTTTATCAAAAGTTATCCAAGTCTATTT
SEQ ID NO.166
CTGTGAAAGCTCCCAGAATTAGCAGTAAGAAGCACAACTCTGCAGGTTCATTATACTCTGGACAC AGTATATGTTTCTTCTCTTCTGAATTTCAAGGTAATATGACATTTGCTTAATAAA
SEQ ID NO.167
ACATTTAAAAAAGAAATGACTGAGGTGATAAGCCTCAGGAAAATTACAGAGTAGCACTGCTCCA GAAGGAGCAGAAACAGAAAAGCAGAATTTCCATTTACAAATGATGACAGTACTGTG
SEQ ID NO.168
GTACATACATACAAGGGCTATTGGCACAAGAGCCCAAGATGAGTAACTATCTTTCTCCCTAATTTG ACATCAAATTTAATAGATTTTTAAGAAAAAGAATCACTGCCAGTCACAGACATT
SEQ ID NO.169
GGCCACCATGCCCGGCTAATTGTAACTTCTTAAGGTATTCAAAGAACATACTCAAGTCTGAATTTT GGCCCCATTTAGAAACTTCTGTGCTTAGAACATACTTGGAAAACTCTAAGTACA
SEQ ID NO.170
GTCAGTGCAGCTGAAATAGAAACAGAATTTGTATAAGTGACACATTCAGAGTATAAGAAAAACA AATATACAAAATTAACAGGAATGAGAATACATTTCCAAACTTGTCCTACCTTGAAA
SEQ ID NO.171
CTTCTCAAATAGAGATTATTGTACTAAACTACTGAGAGCTGGGGAAGTAGCAGGAGCTTCTCTGT GAGACTGAAGACAGCAACAGGAATACTTCTCCTCCAGGGAAGTCAGGACCAGGGT
SEQ ID NO.172
CCTGGGGTGGCAGAGGTGTGTTTGTGCTGTGGAAGAACCCAGGGCAGAAAAATAACAGGGAGT AACAAGAGGTGCATTCTCTGGTCAGACAGCCAAGTGAGGAGGTAGTTATTCTGAGAA
SEQ ID NO.173
AAAGAAAAAAAAAATCACCAGCAGTTGCTACTTTAGAGCTCAAGACACTGTTTTCAATAGAATT TCACGTTTGCGGTTTGGTTCTCTGTTTCAGACATGAACTGCTTTTCAGATGAAAAA
SEQ ID NO.174
TAGAGAATAGAGCCGATAACATTATAAACAAACCAAACAGCAATAATAACTATATTCTTGGCAAA ACAGTAATTTATGACATTTCTCCAAAAATTAGCATAGTTCTAAGTTCAACAGTAA
SEQ ID NO.175
TAGGAAAAAGGCACTTCACTGTGAAAGTTGGATTTAATTACGTCAAAGTTGTGAAGACTAGGATA GACTCAAAATTCAGTATTCATTTTTGTATTTATGAATCTAGAGCAGATGCCAGTT
SEQ ID NO.176
ATAGCTTTCCTTCAATGGTAGATAAGTCACAGACGTATCTCAGACATTCATTTCATGCATAAGGATTTTTGAGACATCTATTCCACTGAAATATTATAAAGGAAGTCTACAAACCACTTC
SEQ ID NO.177
TTATTAAGTGAAATTATTCATGTGCTTTCAGTTTCATCTTTCTCCTGGGAAAGCCCCAAAACAGGC CTCTGGAAAACCAATTTCATACATGTACAAAATGGCATCTGCTCTCAAATAGAT
SEQ ID NO.178
CACAAATTTGAATACAAATGGAAGTTCATTGAATATTAGGTTTATCAGTAAGCTAGGGGAAACAA ATTACATACATTGTATAGGTAGTAACCTATGAAAAAAGTGGAGATTAGGTAAAAA
SEQ ID NO.179
TGTAATTTGTTAATATACTATATTTGAGGTTTGAAAATCACTGATCACAGCTATTAAATAACAATGCACCAAAGTTTTACAATATTTGAACATCTGCAAATGTAGAGCTTTCTGGTATGA
SEQ ID NO.180
TTTACTCAACTGAAATAATCAAGCCCCTATTGCTGTGGGGGAAGCTGTAGAGGGTGGGGTGAGA AGGGGATTGAAGGCAATTATATACATCATTAAGATACCATGATATACAGTGAAAA
SEQ ID NO.181
AAATTAACAGGAATGAGAATACATTTCCAAACTTGTCCTACCTTGAAAGTGGCTCTTTTCTGACA AAACTTTAAAAGTATCTGTAAAAAAAGAACAGAAACCATGTCCTTATTAACTAGT
SEQ ID NO.182
AGTTTTTTCATTCGGTACTGGCGTATTTCTCTTACCAGTGTGTAAAAAGCATCTTCAACACCCTATAAAAGGAAAAAATGAAAAAAAATGAGAGAGCTAGCTCAACGGACACAATCCAAA
SEQ ID NO.183
AAATTGCCCAATACTATATACTAAGATTTGTAATTATGCCAAGAAACCATATGCTCACCTTGTTACATCACCACACATGGCAATCCCATACAACCCTGAGTCCCATCATCACTGCTGTTG
SEQ ID NO.184
TCCTTGTTGGCAAATCACACTTGTTTCCCACTAGCACCATAGGTACATCATCCGAGTCTTTTACTC GCTTAATCTGCTCCCTAAAAACGGGAATATATTATCAGAACATAAGAAAAACAA
SEQ ID NO.185
GATGCAAACTCTTGCACAAATGCTGAAAGCTGTACCATACCTGTCTGGTCTTGGCTGAGGTTTCA ATGAATGGAATCCCGTAACTCTTGGCCAGTTCGTGGGCTTGTTTTGTATCAACTG
SEQ ID NO.186
CCTTGTTGGCAAATCACACTTGTTTCCCACTAGCACCATAGGTACATCATCCGAGTCTTTTACTCG CTTAATCTGCTCCCTAAAAACGGGAATATATTATCAGAACATAAGAAAAACAAG
SEQ ID NO.187
ATGCAAACTCTTGCACAAATGCTGAAAGCTGTACCATACCTGTCTGGTCTTGGCTGAGGTTTCAA TGAATGGAATCCCGTAACTCTTGGCCAGTTCGTGGGCTTGTTTTGTATCAACTGT
SEQ ID NO.188
ATGGCACTGTACTCTTCTTGTCCAGCTGTATCCAGTATGTCCAACAAACAGGTTTCACCATCTATA ACCACTTGTTTTCTGTAAGAATCCTGGGGGTGTGGAGGGTAAGGGGGCAGGGAG
SEQ ID NO.189
TCCTTTCAGAGAAAATAATGCTCCTAGTACCTGTAGAGGTTAATATCCGCAAATGACTTGCTATTATTGATGGCAAATACACAGAGGAAGCCTTCGCCTGTCCTCATGTATTGGTCTCTC
SEQ ID NO.190
TTCCCAACACCACCTGCTCCAACCACCACCAGTTTGTACTCAGTCATTTCACACCAGCAAGAACC TGTTGGAAACCAGTAATCAGGGTTAATTGGCGAGCCACATCTACAGTACTTTAAA
SEQ ID NO.191
GATGATCCGACAAGTGAGAGACAGGATCAGGTCAGCGGGCTACCACTGGGCCTCACCTCTATGG TGGGATCATATTCATCTACAAAGTGGTTCTGGATTAGCTGGATTGTCAGTGCGCTT
SEQ ID NO.192
CTACGTAATCAGTCGGCGCCCCAGGCGCCTGAGTCCCCGCCCCGGCCACGTGGGCCTCCGAACC ACGAGTCATGCGGCAGGCCGCACCCAGACCCGCCCCTCCCACACGGGACGTTTCAA
SEQ ID NO.193
CCTCAAGCTCCACTGCCTCTGCTTTGGACAGATTTAGGACCACAGCCGGGAAAAATGTTGGAGA CCCCGGAACCGCCATGAACAGCCCCCACCAAGGAGCGGCACTTCCGGCCCCGCCCG
6.PIK3CA驱动突变探针序列
SEQ ID NO.194
GTTTTATAATTTAGACTAGTGAATATTTTTCTTTGTTTTTTAAGGAACACTGTCCATTGGCATGGGGAAATATAAACTTGTTTGATTACACAGACACTCTAGTATCTGGAAAAATGGCTT
SEQ ID NO.195
TGAATCTTTGGCCAGTACCTCATGGATTAGAAGATTTGCTGAACCCTATTGGTGTTACTGGATCAA ATCCAAATAAAGTAAGGTTTTTATTGTCATAAATTAGATATTTTTTATGGCAGT
SEQ ID NO.196
ATCCAGAGGGGAAAAATATGACAAAGAAAGCTATATAAGATATTATTTTATTTTACAGAGTAACAG ACTAGCTAGAGACAATGAATTAAGGGAAAATGACAAAGAACAGCTCAAAGCAAT
SEQ ID NO.197
TTCTACACGAGATCCTCTCTCTGAAATCACTGAGCAGGAGAAAGATTTTCTATGGAGTCACAGGT AAGTGCTAAAATGGAGATTCTCTGTTTCTTTTTCTTTATTACAGAAAAAATAACT
SEQ ID NO.198
TTTGAGCAAAGACCTGAAGGTATTAACATCATTTGCTCCAAACTGACCAAACTGTTCTTATTACTT ATAGGTTTCAGGAGATGTGTTACAAGGCTTATCTAGCTATTCGACAGCATGCCA
SEQ ID NO.199
AATCTCTTCATAAATCTTTTCTCAATGATGCTTGGCTCTGGAATGCCAGAACTACAATCTTTTGATGACATTGCATACATTCGAAAGACCCTAGCCTTAGATAAAACTGAGCAAGAGGCT
SEQ ID NO.200
TTTGGAGTATTTCATGAAACAAATGAATGATGCACATCATGGTGGCTGGACAACAAAAATGGATT GGATCTTCCACACAATTAAACAGCATGCATTGAACTGAAAAGATAACTGAGAAAA
SEQ ID NO.201
ATGAAAGCTCACTCTGGATTCCACACTGCACTGTTAATAACTCTCAGCAGGCAAAGACCGATTGC ATAGGAATTGCACAATCCATGAACAGCATTAGAATTTACAGCAAGAACAGAAATA
SEQ ID NO.202
AAAATACTATATAATTTAAATAATGTAAACGCAAACAGGGTTTGATAGCACTTAAACTAGTTCATTTCAAAATTAAGCTTTAGAATAATGCGCAATTTCATGTTATGCCTTAAGTCCAAA
SEQ ID NO.203
AAAGGTAAACTTTGAAGATTGTTTGTATCTTTTTTTAAAAAACAAAACAAAACAAAAATCCCCAA AATATATAGAAATGATGGAGAAGGAAAAAGTGATGGTTTTTTTTGTCTTGCAAAT
SEQ ID NO.204
TGTTCTATGTTTTGAAATGTGGACACAACAAAGGCTGTTATTGCATTAGGTGTAAGTAAACTGGA GTTTATGTTAAATTACATTGATTGGAAAAGAATGAAAATTTCTTATTTTTCCATT
SEQ ID NO.205
TGCTGTTCAATTTATAGTTTGAAGTGGGTTTTTGACTGCTTGTTTAATGAAGAAAAATGCTTGGGG TGGAAGGGACTCTTGAGATTTCACCAGAGACTTTTTCTTTTTAATAAATCAAAC
SEQ ID NO.206
CCTTTTGATGATTTGAGGTTTTATCTGCAGTTTTGGAAGCAGTCACAAATGAGACCTGTTATAAGG TGGTATTTTTTTTTTTCTTCTGGACAGTATTTAAAGGATCTTATTCTTATTTCC
SEQ ID NO.207
CCAGGGAAATTCTGGGCTCCCACAAAGTAAAAAAAAAAAAAAATCATAGAAAAAGAATGAGCA GGAATAGTTCTTATTCCAGAATTGTACAGTATTCACCTTAAGTTGATTTTTTTTCTC
SEQ ID NO.208
CCTTCTGCAATTGAACTGAATACATTTTTCATGCATGTTTTCCAGAAAATAGAAGTATTAATGTTATTAAAAAGATTATTTTTTTTATTAAAGGCTATTTATATTATAGAAACTATCATT
SEQ ID NO.209
TAATATATATTCTTTATTTACATGATCTGTCCCATAGTCATGCATTGTTTTGCACCCCAAATTTTTTATTGTTCATAGCAGCATGGTCAGCTTTCTTCTTGATCTATAGATGAGGCTCAG
SEQ ID NO.210
GGCACTATCCCATTTATACCAATAACCAGTGTATAACTACTTAAGGAAAACATAAAAACTTCATCT TCTTTCCTTTTATTTCTTATGTGAATCTCCCGTCTTCCATTCTCTTTTATAATT
SEQ ID NO.211
TGAGAATGTCTCAATCATATGAAATTAGTTACCAGAATTAACACAATTTAGACTATCTTCCTGATTCCTTAAACCCCTTTACTGAAGTATACTCATGAATAATACTTTAAAATATGGGGG
SEQ ID NO.212
GAATAGAAACCATGAACTTTTTACCTTTTTAAACTATTTATCCATATCTCCAAAGTAGAACATTAAACCATTTTAAGATATGTCTCATTCCCAAGTAGTCAGAGCTCACTCTCCAACTTT
SEQ ID NO.213
TATTAAATACTATTTGAGCACAGGACACATTCTTAAACATTTTGAAAAACATTAACCCAAGATGTA GAGGCTACTGCTAGTCGTCATTCTAGAATCTGATATTTTACTCTGTATTTGAAA
SEQ ID NO.214
ATGAATGATTAATGTCCTAGGAAATTAGCTTTAGCAGATGTCCAGGTGCCACATCAAAAAAGTGC AATAATTATTGACAGTTTTTTAGATTAGGCATATTATTGGAAAACAACTTTATAA
SEQ ID NO.215
AAGAGTGAACATTGTATACTCTAGTAAAACAGCATCACTTTAAAAATATTCATTTATGAAATCTGTTACCTATAGTTGAAGTCTTGAGTAGTGAACAAGGGACTCTAATACCAATACTCT
SEQ ID NO.216
TTAATATCTGGCTATTTTAGATCCCTTAAAGGGCATAATTATTGGAAATTTAGGTATTTCACTAAAGCATGTATATAATATTGCCAACAAGAAAAGTAAATTTGAAGATTAAGGGAACTT
SEQ ID NO.217
TACTTCTGCAAACTGTCTTGCGATAGTTAAGCAGAATTTAAACTCTGTTTTAAGCAGGAAACCAG AAAGATTATTTTGCAGTTGTAGAAGATTTCATAACTTATTAAAACTTATTAACAT
SEQ ID NO.218
TTTTGTGTTGTTTAGATATAGGCAGTTGATACATACTAACATCCCAGCCTTTTCAATATCAGGGTTAAATTATAGGAAAACTCAGTAAAATGGTACAAATCTGAAAGTTTGATGGTAGAA
SEQ ID NO.219
AACTGAAGATTTAACAGAGAACTGTGTTTTACCCGAGTGCCAAAAATGCTGTGAGCCTCCTTGC ACAAAATTTATACCACTTTTGCATTTTTATCTATCAGTCCAGATAGTTGTCTCCCC
SEQ ID NO.220
CTCCTTCTCCCAGGACCTCTCCACCATTAAAATGCACAAACCACATGGCCGATTTCACCATTTACA TTTATTTTCAAAAGTTACTACAACCAAATTAATTCTATTAGAAGAAATGTAGAC
SEQ ID NO.221
CAAATTCTATAAAGACTATAGATTGTGACCTAAGAAAGAAATGAGGCAAAGAACCAAACATTGAA TTAAATGCTACATGGGTGACTAAGATCTGTTTCAAGTCAGTGATAATATAGCCAC
SEQ ID NO.222
CTTCTGGGTACTTCAGTATCAGAGATCAGTTCTCGTGGTTTAGACAGTTCCTATCTATAGCTGACTATCCTTGTCCTTGAATATGGTGTAACTGACTATTGGCTCTACAGTTTTATTGGG
SEQ ID NO.223
GCCACTTAAGAAATATTTCCTTGAATAATTATTTTGAGAAAAAGTCTAAAAGTAATAAAAATAATT TTAAACACACTGTAGTAAGAAATGACTGTTGGAAAATTATGCTTTCACTTTCTA
SEQ ID NO.224
ACCATATTCTCAGCTATACAAAACCATTTATTTTGAAGATTTTTAGACTACTGTTAATTTGAAATCTGTTACTCTTATTGTGGAATTTGTTTTTTTAAAAAAGATGTTTCTAATTGGATT
SEQ ID NO.225
TTTTAAAAGAAGAATGGAATTTGGTTGCTATTTTACAATAGAACCTAAGCTTTTTGTGGTTCTTAG TGTCCTATGTAAAACTTAGTGTCAAAGTAATCAACTTTGAGATTTTCCCTTCTA
SEQ ID NO.226
ATTCTGCTTTATATTAAAAGCCCATTAGAAAATGGGAACCTGGTGAATATATAATGAATTGTAAAATATTTTAATGTGTAACTTTTTCAACTGTGAAACTGACTTGATTTTTTGATGAAA
SEQ ID NO.227
AACAGCTGCTGATAAAGTATTTTGTGTAAAGTGTAGTTCTTATTAATCAGGAAAATGATGACTTGA TTAGACTGTATATGCCCTCTTGGATTTTATTTTAAATGGATTGGTGACTTTCAC
SEQ ID NO.228
CATAGGTAAAACACAGTCCATCTGTATTCTTTTTTCCATCAAAAATCGAGTGATTTGGAATTATAA AAAAATTGTGAGCAGCCTATTTGAAAGGCATCATGGAAATTTCACAGCACAATA
SEQ ID NO.229
AACACGGATTTGTTTTTTCTTAATGATGTAAATCCGTTTAATTCATACTTTGATCAATAGCCCATGCTTGCCAACTCTGAAGAAATTTAATTTCCAGCAGTATTTTAAAGCTAGCCTGTT
SEQ ID NO.230
TAACTTTTTCTGAATATTTAAAGTTCCTCTTTTTTCTATGTCTGCACAAACTGCAGACCTGGGCTG GACCCACATACTCAAGAGTCCACCTTAAGAAATTATTTTGATGTCCAAGACATC
SEQ ID NO.231
CACTAAAATATTTAAGTTTAAAGATAATATGTGGTGTTAATAGATTGTGGTGCTTTTACTATTTAAAGACAACTTTCATACTTCAGATGTTTTTGAGAAGAGGGGAATGTGAGGGGAGGG
SEQ ID NO.232
GGGCAGAACAGGGAGGAGTTGTTTGAATGAATTACATTCTTTATATCCATCCTGCTCATTTGGGGC ATGTCTTTAAGAGAAGGCTGAAAGTTGTGAGAGTATATTGTATACCGTAAGAGA
SEQ ID NO.233
AATCAACTCTTCATCATGGATGGGATTGTGAAGGCTGAACTATAAAATTCAGCATTGACAGCATCC TCAATTAATAATTCTTGGTGACAGAATAATACAGCTGGGCTGTTTTTTAAAATA
SEQ ID NO.234
CATTAAAAATTGTAAATATATCTATGTGCCATGGCCTGGGAAGCCTGCTTTCTTTTTTCATAAAAATTATTTTTACTGTATGAAAAGATCATGGGGTTTAGCTCAAAATATCTGTGGTCC
SEQ ID NO.235
TGATAAAATTGGATTGGTAACTCTACCTCAGAAGGAAAATGGGAAAAAAAAATAGATGAGTCAC AATTCAATACTTCAAGCTCAGAAACTGTGCAGATCACTGAATTTTAGATTTATAAA
SEQ ID NO.236
GTCAGAGTTGGCATGCCTTGTTTTTAATGATATGGAAGACCTTAAGAAAAAAACTTGGCTGAAGT TTAATCGTTGGTCCAGCCATTTGAAAAAGGCAATAGTTTGAGGAGGTTCCCGAAT
SEQ ID NO.237
TCGGCATTTGAAATTCATTTTGTTCTCTCTTCTTCATTATTAGTGCATTTGGTGTGTGTATACTTGCACACAATTCTGTTTGTGTACACACTGCTTGCTTAGCCCTAGTCAAGAGGCATC
SEQ ID NO.238
TTTTATAAAAGGTGTAAAGAAATATCAAGGTTCTAAAATTCGGAAGAGTTTAGAATTTATTAGGAG TTTCCCAAGTTGGGATGTTAGTCTTTAAATAAACTTCATGCACCTATTCCACTT
SEQ ID NO.239
AAGGTTTTGCACCTCCTTTTTATTAGTGCAGTGCCATTTCTTCTGCTTGATTTTAGGTATGTTAATATTCCAGCCTTGCTAGTTAGCATAAAGTGACAGGTGTGAGCCATGAGGAAATTT
SEQ ID NO.240
TCTGACTTAATTTGTACACAACTACATATAAGAGTTTTAGTGGAGGAAAAAAATTAGTCCCTTGTG CGTATACAGTAGTTAGGTAAATGATTTTTCTACCAACAGTATACTCCATTCCTC
SEQ ID NO.241
ATGTAGGTAAGTACAGAAAAGGTTTTTAAATGTATTTTTTTAGCCAGTTAAAGTCTATGAATCTATCTGCAACCTTATTTAATCTGTCACTATAATAATTTTGTGGTTATGCTAAGAACC
SEQ ID NO.242
ATGTATACTTTTAGGTATTCTTATTTTTGTCAATTTTTCTAGGTTGGCAAGGAGGCAGAAAACCTTCATTGTTTCATATTAAAATATAATTAGACTAAACTTAATTCTAGTATGAATTTC
SEQ ID NO.243
CAAAATCATTATCTATTTATTTCATTTTTATTTAATTTTGTTTTTATTTCATTTTTAAAAGTCCCTTGTTCAATTTAACTTATGTTCCTAAGAGAGGTTGGAGAACTTGGCCTTCATCTG
SEQ ID NO.244
ATTTCAAAAATGTTTTGAGTTTCAAATGAAGTTAATGGTTTCAGTGTGATTCAGTCCTCAGACCTA ATTGGGTTGAATAAAATCTAAAAGAATATACCCTTTTGGAGCATAACATTTTAA
SEQ ID NO.245
TACCTTGGGGAATGTGGCACTACCAAAAGAAGACTACTAACACGTCAGATGTTCACCTGGAAGC TTTATCAAGAAATTCGAACCACCCTTTTGGCCCCATTAATTGTAGCAAGTTTATTT
SEQ ID NO.246
CTCTATATTTTGTCATTCAGTGAATTGAAGTCCTGTGGTATACTGCATTCATTAGAAGAAAAACGT TTTTAATGTCCTTTTAATGATGGCCCAGAAAGCATTTGACACAGCAAGATGCAT
SEQ ID NO.247
GTGTTACTATATTGAGAATATAGAATAATAACAGTATCACTAAATTTAAGACCTCTTCCCAGTCTTGCTGTTCCTAGCAAGAAGTTTGGCCTGTGACTGCACTTACTGTTTATGCTCATC
SEQ ID NO.248
AGAAACTGTCAATGTCTGCTTTTCTTTAACTCTGCAGTCTGTAACATCACGCTGTTTATTAAAAAA AAAAAGAAAAATTACTTTTTGTGTCCAAACAATCCTTAGTGTACTACATAAGCA
7.TP53化疗药物基因探针
SEQ ID NO.249
GATCGATATAAAAATGGGATATAAAAAGGGAGAAGGAGGGGAAGGGTGGGGTGAAAATGCAGAT GTGCTTGCAGAATGTAAAAGATGTTGACCCTTCCAGCTGGACGTGGTGGCTCACAA
SEQ ID NO.250
TCAGGTGGCTGCTTCCCAGCACCTCCTCACTCACCCCTGCACCTGCTGACCCCAGTAGCCTGCAC TGGCGTTCACCCCTCAGACACACAGGTGGCAGCAAAGTTTTATTGTAAAATAAGA
SEQ ID NO.251
GCTGCCCAACTGTAGAAACTACCAACCCACCGACCAACAGGGAGAGGGAACAAGCACCCTCAA GGGGGTCAAGTTCTAGACCCCATGTAATAAAAGGTGGTTTCAAGGCCAGATGTACAT
SEQ ID NO.252
GGGATGGGGTGAGATTTCCTTTTAGGTACTAAGGTTCACCAAGAGGTTGTCAGACAGGGTTTGG CTGGGCCAGCAGAGACTTGACAACTCCCTCTACCTAACCAGCTGCCCAACTGTAGA
SEQ ID NO.253
GAAAAAAAAAAAAAAAAAAGAAAAAAGAAATTGACCCTGAGCATAAAACAAGTCTTGGTGGAT CCAGATCATCATATACAAGAGATGAAATCCTCCAGGGTGTGGGATGGGGTGAGATT
SEQ ID NO.254
CAGTGGGGAACAAGAAGTGGAGAATGTCAGTCTGAGTCAGGCCCTTCTGTCTTGAACATGAGTT TTTTATGGCGGGAGGTAGACTGACCCTTTTTGGACTTCAGGTGGCTGTAGGAGACA
SEQ ID NO.255
GCCCCGGGACAAAGCAAATGGAAGTCCTGGGTGCTTCTGACGCACACCTATTGCAAGCAAGGGT TCAAAGACCCAAAACCCAAAATGGCAGGGGAGGGAGAGATGGGGGTGGGAGGCTGT
SEQ ID NO.256
ATTTCTTACATCTCCCAAACATCCCTCACAGTAAAAACCTTAAAATCTAAGCTGGTATGTCCTACT CCCCATCCTCCTCCCCACAACAAAACACCAGTGCAGGCCAACTTGTTCAGTGGA
SEQ ID NO.257
TATGGGCCTTGAAGTTAGAGAAAATTCAACAGTGAGGGACAGCTTCCCTGGTTAGTACGGTGAA GTGGGCCCCTACCTAGAATGTGGCTGATTGTAAACTAACCCTTAACTGCAAGAAC
SEQ ID NO.258
CACTCACCTGGAGTGAGCCCTGCTCCCCCCTGGCTCCTTCCCAGCCTGGGCATCCTTGAGTTCCA AGGCCTCATTCAGCTCTCGGAACATCTCGAAGCGCTCACGCCCACGGATCTGCAG
SEQ ID NO.259
TAAGAGGTCCCAAGACTTAGTACCTGAAGGGTGAAATATTCTCCATCCAGTGGTTTCTTCTTTGG CTGGGGAGAGGAGCTGGTGTTGTTGGGCAGTGCTAGGAAAGAGGCAAGGAAAGGT
SEQ ID NO.260
TGCGCCGGTCTCTCCCAGGACAGGCACAAACACGCACCTCAAAGCTGTTCCGTCCCAGTAGATT ACCACTACTCAGGATAGGAAAAGAGAAGCAAGAGGCAGTAAGGAAATCAGGTCCTA
SEQ ID NO.261
GCATAACTGCACCCTTGGTCTCCTCCACCGCTTCTTGTCCTGCTTGCTTACCTCGCTTAGTGCTCC CTGGGGGCAGCTCGTGGTGAGGCTCCCCTTTCTTGCGGAGATTCTCTTCCTCTG
SEQ ID NO.262
CTGACCTGGAGTCTTCCAGTGTGATGATGGTGAGGATGGGCCTCCGGTTCATGCCGCCCATGCAG GAACTGTTACACATGTAGTTGTAGTGGATGGTGGTACAGTCAGAGCCAACCTAGG
SEQ ID NO.263
GACCTCAGGCGGCTCATAGGGCACCACCACACTATGTCGAAAAGTGTTTCTGTCATCCAAATACT CCACACGCAAATTTCCTTCCACTCGGATAAGATGCTGAGGAGGGGCCAGACCTAA
SEQ ID NO.264
CGGGTGCCGGGCGGGGGTGTGGAATCAACCCACAGCTGCACAGGGCAGGTCTTGGCCAGTTGG CAAAACATCTTGTTGAGGGCAGGGGAGTACTGTAGGAAGAGGAAGGAGACAGAGTTG
SEQ ID NO.265
TGTCGTCTCTCCAGCCCCAGCTGCTCACCATCGCTATCTGAGCAGCGCTCATGGTGGGGGCAGCG CCTCACAACCTCCGTCATGTGCTGTGACTGCTTGTAGATGGCCATGGCGCGGACG
SEQ ID NO.266
CCTGGGTCTTCAGTGAACCATTGTTCAATATCGTCCGGGGACAGCATCAAATCATCCATTGCTTGG GACGGCAAGGGGGACTGTAGATGGGTGAAAAGAGCAGTCAGAGGACCAGGTCCT
SEQ ID NO.267
GAAGGGACAGAAGATGACAGGGGCCAGGAGGGGGCTGGTGCAGGGGCCGCCGGTGTAGGAGC TGCTGGTGCAGGGGCCACGGGGGGAGCAGCCTCTGGCATTCTGGGAGCTTCATCTGGA
SEQ ID NO.268
CATTGAAGTCTCATGGAAGCCAGCCCCTCAGGGCAACTGACCGTGCAAGTCACAGACTTGGCTG TCCCAGAATGCAAGAAGCCCAGACGGAAACCGTAGCTGCCCTGGTAGGTTTTCTGG
SEQ ID NO.269
CCTCCAGGTCCCCAGCCCTCCAGGTCCCCAGCCCAACCCTTGTCCTTACCAGAACGTTGTTTTCA GGAAGTCTGAAAGACAAGAGCAGAAAGTCAGTCCCATGGAATTTTCGCTTCCCAC
SEQ ID NO.270
TCCACTCACAGTTTCCATAGGTCTGAAAATGTTTCCTGACTCAGAGGGGGCTCGACGCTAGGATC TGACTGCGGCTCCTCCATGGCAGTGACCCGGAAGGCAGTCTGGCTGCTGCAAGAG
SEQ ID NO.271
CTGCTCCCTGGACGGTGGCTCTAGACTTTTGAGAAGCTCAAAACTTTTAGCGCCAGTCTTGAGCA CATGGGAGGGGAAAACCCCAATCCCATCAACCCCTGCGAGGCTCCTGGCACAAAG
SEQ ID NO.272
GACTCAGAGAGGACTCATCAAGTTCAGTCAGGAGCTTACCCAATCCAGGGAAGCGTGTCACCGT CGTGGAAAGCACGCTCCCAGCCCGAACGCAAAGTGTCCCCGGAGCCCAGCAGCTAC
8.UGT1A1化疗药物基因探针
SEQ ID NO.273
TGGCACTTGGTAAGCACGCAATGAACAGTCATAGTAAGCTGGCCAAGGGTAGAGTTCAGTTTGA ACAAAGCAATTTGAGAACATCAAAGGAAGTTTGGGGAACAGCAAGGGATCCAGAAT
SEQ ID NO.274
GGGGTACAGTTGTGTTCTTTTCTTTCTAAAAGGCTTTCTAAAAAGCCTTCTGTTTAATTTCTGGAA AAGAAGCCTAACTTGTTCACTACATAGTCGTCCTTCTTCCTCTCTGGTAACACT
SEQ ID NO.275
TAGTCACGTGACACAGTCAAACATTAACTTGGTGTATCGATTGGTTTTTGCCATATATATATATATAAGTAGGAGAGGGCGAACCTCTGGCAGGAGCAAAGGCGCCATGGCTGTGGAGTC
SEQ ID NO.276
CAGCAGAGGGGACATGAAATAGTTGTCCTAGCACCTGACGCCTCGTTGTACATCAGAGACGGAG CATTTTACACCTTGAAGACGTACCCTGTGCCATTCCAAAGGGAGGATGTGAAAGAG
该检测试剂盒的用药指导方案，包括：BRAF、EGFR、KRAS、MET、NRAS、PIK3CA 驱动性基因突变及TP53和UGT1A1化疗药物相关基因突变的用药指导方案。(1)携带BRAF V600E突变的非小细胞肺癌对BRAF抑制剂达拉非尼联合MEK抑制剂曲美替尼敏感。但携带 BRAFG466V、G469A、Y472C等非V600E突变的非小细胞肺癌可能对当前选择性BRAF抑制剂不敏感。(2)携带EGFR活化突变(如外显子18、19、20和21)的非小细胞肺癌对第一代和第二代EGFR-TKI敏感。携带EGFR T790M突变的非小细胞肺癌可能对第一代和第二代 EGFR-TKI耐药，但对第三代EGFR-TKI敏感。携带EGFR20号外显子插入突变的非小细胞肺癌可能对现有EGFR-TKI都不敏感。携带EGFR扩增的肺鳞癌可能对抗EGFR抗体联合化疗VS 单纯化疗更加敏感。(3)携带KRAS突变的非小细胞肺癌可能对EGFR-TKI等目前靶向治疗药物耐药。(4)携带MET14号外显子可变剪切突变的晚期非小细胞肺癌对MET抑制剂敏感。携带MET拷贝数高水平扩增的非小细胞肺癌可能对MET抑制剂敏感。(5)携带NRAS突变的可能对MEK抑制剂敏感。(6)携带PIK3CA基因突变(如外显子9和20)，主要靶向药是brparlisib (BKM120)。如果PIK3CA发生激活突变，则可能对EGFR靶点、HER2靶点耐药，赫赛汀和易瑞沙、特罗凯效果欠佳。(7)TP53的rs1042522位点可预测对顺铂、卡培他滨、紫杉醇和奥沙利铂的敏感性。(8)UGT1A1的rs8175347和rs4148323可预测对伊立替康的毒副作用风险的高低。
本发明还提供一种检测肺癌驱动性基因突变的检测试剂盒，包括：上述的6组共248条探针和2组共28条探针。
本发明的有益效果是：本发明提供的探针特异性高，准确性好，最低检测限可达0.1％。可为寻找靶向治疗药物的肺癌患者提供用药依据；还可以为治疗过程中发生严重毒副作用、耐药等的肺癌患者更换治疗方案。
具体实施方式
下面通过具体实施例对本发明进行描述，应当指出的是下面实施例只用于对本发明的进一步说明，但并不能用作对本发明保护范围的限制，本领域的技术人员应当理解，基于本发明的技术前提下，做出的改进和变型也视为本发明的保护范围。
本实施例中检测2例肺癌患者的新鲜全血样本及3种不同突变频率的标准品(0.1％、1％和5％)。本发明中样本的形式是可以提取核酸的任意形式的肿瘤样本，其中全血样本为优，包括但不限于组织样本，穿刺样本、血清和血浆；且组织样本包括但不限于石蜡包埋组织、新鲜组织和冰冻切片组织。
本实施例中，与肺癌相关的基因筛选自NCCN指南、COSMIC数据库及药物相关数据库。具体基因如下：BRAF、EGFR、KRAS、MET、NRAS、PIK3CA、TP53和UGT1A1。探针序列参考SEQID NO.1-SEQ ID NO.276。
本发明采用建库杂交捕获的方法对本发明所涉及的探针进行验证，具体步骤如下：
1样本的准备
1.1分离血浆和血细胞
将2例肺癌患者的新鲜全血样本分别离心3000rpm，5min，取上清至新的离心管中，再次将上清离心3000rpm,5min，取上清(血浆)保存于新的离心管中备用，做好标记(如不立即使用可放-20℃冻存)。
1.2cfDNA的提取
严格按照磁珠法大体积游离核酸提取试剂盒说明书(天根，货号：DP710-01)从血浆中提取 cfDNA，Qubit3.0检测浓度，无需进一步片段化。
1.3gDNA的提取和片段化
1.3.1gDNA的提取
严格按照QIAamp DNABlood Mini Kit(QIAGEN，货号：163026054)从血细胞中提取gDNA， Qubit3.0检测浓度，琼脂糖凝胶电泳检测DNA完整性。
1.3.2gDNA的片段化
1.3.2.1提前打开打断仪，使其温度降至4℃。
1.3.2.2取300ng的gDNA加入到打断管中，使用1x IDTE Buffer补齐至80μL，涡旋混匀，瞬时离心1-3s。
1.3.2.3设置5(run 30s,stop 30s)×3次的程序进行打断。
1.3.2.4Agilent 2200检测打断后DNA的片段大小(一般为200-300bp)。
1.3.2.5将室温平衡30min后的
AMPure XP Beads(磁珠)涡旋混匀后取144μL至新的1.5mL离心管中，做好标记。
1.3.2.6将片段化后的产物转移至步骤1.3.2.5中的1.5mL离心管中，涡旋混匀，室温孵育5min。 1.3.2.7将步骤1.3.2.6的1.5mL离心管置于磁力架，静置直至溶液完全澄清，吸弃上清，注意避免吸到磁珠。
1.3.2.8加入200μL新鲜配制的80％乙醇，室温孵育30sec，吸弃上清。
1.3.2.9重复1.3.2.8。
1.3.2.10将步骤1.3.2.9的1.5mL离心管微离心后静置于磁力架，吸弃残留溶液，开盖室温晾干直至乙醇完全挥发。
1.3.2.11加入52μL NFW，涡旋混匀，室温孵育2min，置于磁力架，待溶液完全澄清后转移 50μL上清至新的已标记好的0.2mL PCR管进入下一步反应。
1.3.2.12取1μL用Qubit3.0检测浓度。
2文库构建
2.1末端修复和3'端加“A”
2.1.1取30ng cfDNA/40ng标准品，用NFW补齐至50μL，涡旋混匀并离心。
2.1.2在0.2mL PCR Tube中配制以下反应体系:
| 组分 | 体积(μL) |
| cfDNA/gDNA | 50 |
| End Repair&A-Tailing Buffer | 7 |
| End Repair&A-Tailing Enzyme Mix | 3 |
| 总体积 | 60 |
2.1.3涡旋混匀，离心后进行以下反应:
2.2加Adapter 2.2.1将上一步反应产物先离心，然后配制以下反应体系：
| 组分 | 体积(μL) |
| End Repair and A-Tailing Reaction Product | 60 |
| NFW | 5 |
| Ligation Buffer | 30 |
| Duplex Seq Adapters(3μM) | 5 |
| DNA Ligase | 10 |
| 总体积 | 110 |
2.2.2涡旋混匀，离心后进行以下反应：
| 步骤 | 反应温度 | 反应时间 |
| Adapter Ligation | 20℃ | 15min |
| Hold | 4℃ | ∞ |
2.3连接后纯化
2.3.1将室温平衡30min后的
AMPure XP Beads(磁珠)涡旋混匀后取88μL至新的1.5mL离心管中，做好标记。
2.3.2将接头连接后的产物转移至步骤2.3.1中的1.5mL离心管中，涡旋混匀，室温孵育5min。 2.3.3将步骤2.3.2的1.5mL离心管置于磁力架，静置直至溶液完全澄清，吸弃上清，注意避免吸到磁珠。
2.3.4加入200μL新鲜配制的80％乙醇，室温孵育30sec，吸弃上清。
2.3.5重复2.3.4。
2.3.6将步骤2.3.5的1.5mL离心管微离心后静置于磁力架，吸弃残留溶液，开盖室温晾干直至乙醇完全挥发。
2.3.7加入22μL NFW，涡旋混匀，室温孵育2min，置于磁力架，待溶液完全澄清后转移20μL 上清至新的已标记好的0.2mL PCR管。
2.4PCR扩增
2.4.1在0.2mL PCR Tube中，配制以下反应体系：
| 组分 | 体积(μL) |
| 2X KAPA HiFi HotStart ReadyMix | 25 |
| UDI Primer Mix(5μM/Primer) | 5 |
| Adapter-ligated Library | 20 |
| 总体积 | 50 |
2.4.2涡旋离心，离心后进行以下反应
2.5PCR产物纯化
2.5.1将室温平衡30min后的
AMPure XP Beads(磁珠)涡旋混匀后取50μL至新的1.5mL离心管中，做好标记。
2.5.2将PCR产物转移至步骤2.5.1中的1.5mL离心管中，涡旋混匀，室温孵育5min。
2.5.3将步骤2.5.2的1.5mL离心管置于磁力架，静置直至溶液完全澄清吸弃上清，注意避免吸到磁珠。
2.5.4加入200μL新鲜配制的80％乙醇，室温孵育30sec，吸弃上清。
2.5.5重复2.5.4。
2.5.6将步骤2.5.5中的1.5mL离心管微离心后静置于磁力架，吸弃残留溶液，开盖室温晾干直至乙醇完全挥发。
2.5.7加入22μL NFW，涡旋混匀，室温孵育2min，置于磁力架，待溶液完全澄清后转移 20μL上清至新的已标记好的1.5mL离心管中。
2.5.8质检
取1μL DNA文库溶液用Qubit荧光定量仪检测浓度。
3杂交
3.1在1.5ml EP管中配置如下反应：
| Blocker component | Volume per reaction(μL) |
| Human Cot DNA | 5 |
| xGen Blocking Oligos based on your library adapters | 2 |
3.2混合文库2ug，加至步骤3.1的混合液中。
3.3真空抽干。
3.4配置下列体系于已真空干燥的样本EP管中
| Hybridization Master Mix component | Volume per reaction(μL) |
| xGen 2X Hybridization Buffer | 8.5 |
| xGen Hybridization Buffer Enhancer | 2.7 |
| xGen Lockdown Panel or custom probes | 4 |
| Nuclease-Free Water | 1.8 |
| Total | 17 |
3.5振荡,离心，重复两次。
3.6进行以下PCR反应程序:
4捕获。
按照下表配置清洗buffer：
按照下表配置Bead Resuspension Mix
| Bead Resuspension Mix component | Volume per reaction(μL) |
| xGen 2X Hybridization Buffer | 8.5 |
| xGen Hybridization Buffer Enhancer | 2.7 |
| Nuclease-Free Water | 5.8 |
| Total | 17 |
4.1清洗链霉亲和素磁珠
4.1.1振荡混匀磁珠，按每个捕获50μL吸取磁珠至同一1.5mL EP管中。
4.1.2按每个捕获加入100μL Bead Wash Buffer，用枪吹打10次以上，放于磁力架静置，直至完全澄清，弃去上清。
4.1.3重复4.1.2操作两次。
4.1.4加入已配置的Bead Resuspension Mix，按照每个捕获17μL，吹打混匀，移至0.2mL PCR 管中。
4.2捕获
4.2.1探针杂交结束后，迅速将梯度PCR仪程序调至下表
4.2.2迅速将杂交后的样本全部转入步骤4.1.4中的含有Bead Resuspension Mix的0.2mL PCR 管中，吹打混匀并离心。
4.2.3将样本放入步骤4.2.1已设置好程序的PCR仪中，放置45min，每隔10-12min，取出快速振荡混匀，确保磁珠处于重悬状态。
4.3热洗
4.3.1恒温仪65℃预热1X Wash Buffer 1和1X Stringent Wash Buffer至少15min。
4.3.2加入100μL预热的1X Wash Buffer 1至4.2.3的样本中，用枪快速吹打混匀，避免产生气泡。
4.3.3置于磁力架至澄清，弃上清，移除磁力架。
4.3.4加入150μL预热的1X Stringent Wash Buffer至样本，用枪快速吹打混匀，避免产生气泡。 65℃孵育5min，置于磁力架至澄清，弃上清。
4.3.5重复步骤4.3.4。
4.4室温洗
4.4.1向热洗步骤4.3.5中的样本加入150μL Wash Buffer 1，振荡离心。室温孵育2min，每30s 振荡一次(不要太剧烈)，确保磁珠处于重悬状态。
4.4.2孵育结束后，微离心。置于磁力架1min，至澄清，弃上清。
4.4.3加入150μL Wash Buffer 2，振荡混匀。室温孵育2min，每30s振荡一次(不要太剧烈)，确保磁珠处于重悬状态。
4.4.4孵育结束后，微离心。置于磁力架1min，至澄清，弃上清。
4.4.5加入150μL Wash Buffer 3，振荡混匀。室温孵育2min，30s振荡一次(不要太剧烈)，确保磁珠处于重悬状态。
4.4.6孵育结束后，微离心。置于磁力架1min，至澄清，弃上清。
4.4.7用枪吸弃残余的Wash Buffer 3，移除磁力架。
4.4.8每个捕获加入20μL NFW，用枪吹打混匀，并转移至0.2mL PCR管中。
5PCR扩增和纯化
5.1PCR扩增
5.1.1在各个捕获中加入如下体系
5.1.2进行如下的PCR扩增
5.2PCR产物纯化
5.2.1提前配置好80％乙醇，每个捕获需要250μL。
5.2.2每个捕获加入75μL 
AMPure XP beads(AMP)，用枪吹打混匀，室温孵育 5-10min。置磁力架2-5min，至澄清，弃上清。
5.2.3加入125μL 80％乙醇，室温静置1min，弃上清。
5.2.4重复步骤5.2.3。
5.2.5室温干燥磁珠，大约1-3min。
5.2.6待磁珠干燥完毕，移除磁力架，加入22μL TE，吹打混匀。
5.2.7室温孵育5min。置磁力架至澄清，转移20μL至新的1.5mL EP管中保存。
5.2.8文库质检
使用Qubit 3.0和Agilent 2200进行检测。
6.Nextseq上机测序。
7.数据分析结果
两位患者样本的主要质量控制如表1所示：DNA质量评估和测序质量评估均合格，可进行下一步数据分析。肺癌患者和标准品的数据分析结果如下：
(1)一位肺癌患者的测序数据分析结果显示：(1)检测到EGFR基因Exon 20存在c.2294 T>G突变，导致所编码第765位上的缬氨酸(V)变为甘氨酸(G)。目前尚无专门针对该变异位点的药物，有被FDA或cFDA批准的该基因靶向药物。(2)UGT1A1基因的rs8175347 位点基因型为杂合型，提示使用伊立替康的毒副作用风险较大(中性粒细胞减少症，腹泻，无力)。
另外一位肺癌患者的测序数据分析结果显示：UGT1A1基因rs8175347位点(*28)基因型为杂合型，提示使用伊立替康的毒副作用风险较大；TP53基因的rs1042522位点基因型为杂合型，提示使用顺铂、卡培他滨、紫杉醇、奥沙利铂的敏感性可能较低。
(2)三种标准品的检测结果如表2所示：等位基因频率分别为0.1％、1％和5％的已知突变位点均被检测出来。说明该检测试剂盒可以检测超低频突变的基因位点，最低检测限可达0.1％。
表1：
表2：
| 基因 | 已知突变位点 | 预期的等位基因频率(％) | 检测的等位基因频率(％) |
| EGFR | T790M | 5.0 | 4.7 |
| EGFR | delE746-A750 | 5.0 | 1.8 |
| EGFR | L858R | 5.0 | 3.9 |
| EGFR | V769-D770insASV | 5.0 | 3.0 |
| PIK3CA | E545K | 6.3 | 5.9 |
| KRAS | G12D | 6.3 | 5.0 |
| EGFR | T790M | 1.0 | 0.52 |
| EGFR | delE746-A750 | 1.0 | 0.19 |
| EGFR | L858R | 1.0 | 0.58 |
| EGFR | V769-D770insASV | 1.0 | 0.39 |
| PIK3CA | E545K | 1.3 | 0.25 |
| KRAS | G12D | 1.3 | 0.25 |
| EGFR | T790M | 0.1 | 0.0021 |
Claims (2)
1.一种用于检测肺癌驱动性基因突变的探针，其特征在于，包括下列6组共248条探针：
BRAF驱动突变特异性探针序列：其序列为SEQ ID NO.1至SEQ ID NO.3；
EGFR驱动突变特异性探针序列：其序列为SEQ ID NO.4至SEQ ID NO.98；
KRAS驱动突变特异性探针序列：其序列为SEQ ID NO.99至SEQ ID NO.148；
MET驱动突变特异性探针序列：其序列为SEQ ID NO.149至SEQ ID NO.150；
NRAS驱动突变特异性探针序列：其序列为SEQ ID NO.151至SEQ ID NO.193；
PIK3CA驱动突变特异性探针序列：其序列为SEQ ID NO.194至SEQ ID NO.248；
还包括下列2组共28条探针：
TP53特异性探针核苷酸序列：其序列为SEQ ID NO.249至SEQ ID NO.272；
UGT1A1特异性探针序列：其序列为SEQ ID NO.273至SEQ ID NO.276。
2.一种检测肺癌驱动性基因突变的检测试剂盒，包括：权利要求1所述的6组共248条探针和2组共28条探针。
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911423840.8A CN111154872A (zh) | 2019-12-31 | 2019-12-31 | 用于检测肺癌驱动性基因突变的探针及试剂盒 |
| PCT/CN2020/094277 WO2021135072A1 (zh) | 2019-12-31 | 2020-06-04 | 用于检测肺癌驱动性基因突变的探针及检测试剂盒 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911423840.8A CN111154872A (zh) | 2019-12-31 | 2019-12-31 | 用于检测肺癌驱动性基因突变的探针及试剂盒 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111154872A true CN111154872A (zh) | 2020-05-15 |
Family
ID=70560607
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911423840.8A Pending CN111154872A (zh) | 2019-12-31 | 2019-12-31 | 用于检测肺癌驱动性基因突变的探针及试剂盒 |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN111154872A (zh) |
| WO (1) | WO2021135072A1 (zh) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021135072A1 (zh) * | 2019-12-31 | 2021-07-08 | 浙江大学 | 用于检测肺癌驱动性基因突变的探针及检测试剂盒 |
| CN113699242A (zh) * | 2021-10-18 | 2021-11-26 | 浙江省人民医院 | 检测kras基因突变、adamts1与bnc1甲基化的引物探针，试剂盒与方法 |
| CN115851927A (zh) * | 2022-08-09 | 2023-03-28 | 上海善准医疗科技有限公司 | 肺鳞癌分子分型及生存风险基因群及诊断产品和应用 |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104294371A (zh) * | 2014-09-30 | 2015-01-21 | 天津华大基因科技有限公司 | 构建测序文库的方法及其应用 |
| CN105339507A (zh) * | 2013-02-21 | 2016-02-17 | 托马生物科学公司 | 用于核酸分析的方法、组合物和试剂盒 |
| CN107475370A (zh) * | 2017-07-13 | 2017-12-15 | 天津脉络医学检验有限公司 | 用于肺癌诊断的基因群和试剂盒及诊断方法 |
| CN108424955A (zh) * | 2018-05-09 | 2018-08-21 | 合肥中科金臻生物医学有限公司 | 一种检测多种变异类型基因的高通量测序方法及其应用 |
| KR20190078715A (ko) * | 2017-12-26 | 2019-07-05 | (주)에스피메드 | 암 유전자 및 항암제 대사 관련 유전자 변이 분석을 위한 차세대 염기 서열 분석(ngs) 기반 혼합 진단 패널 |
| CN110452907A (zh) * | 2019-08-07 | 2019-11-15 | 浙江大学 | 靶向长链非编码rna ddx11-as1的aso、试剂盒及在肝癌治疗中的应用 |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2740742B1 (en) * | 2011-08-04 | 2018-03-14 | National Cancer Center | Fusion gene of kif5b gene and ret gene, and method for determining effectiveness of cancer treatment targeting fusion gene |
| CN102443626B (zh) * | 2011-09-22 | 2013-10-16 | 厦门艾德生物医药科技有限公司 | 肺癌驱动性基因突变的检测试剂盒 |
| WO2015148904A1 (en) * | 2014-03-28 | 2015-10-01 | Driver Group | Methods for predicting egfr tyrosine kinase inhibitor efficacy |
| CN107723351A (zh) * | 2016-08-12 | 2018-02-23 | 嘉兴允英医学检验有限公司 | 一种循环肿瘤dna肺癌驱动基因的高通量检测方法 |
| CN110387400B (zh) * | 2018-04-19 | 2023-03-21 | 上海迪赢生物科技有限公司 | 一种同时捕获基因组目标区域正反义双链的平行液相杂交捕获方法 |
| CN111154872A (zh) * | 2019-12-31 | 2020-05-15 | 浙江大学 | 用于检测肺癌驱动性基因突变的探针及试剂盒 |
-
2019
- 2019-12-31 CN CN201911423840.8A patent/CN111154872A/zh active Pending
-
2020
- 2020-06-04 WO PCT/CN2020/094277 patent/WO2021135072A1/zh active Application Filing
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105339507A (zh) * | 2013-02-21 | 2016-02-17 | 托马生物科学公司 | 用于核酸分析的方法、组合物和试剂盒 |
| CN104294371A (zh) * | 2014-09-30 | 2015-01-21 | 天津华大基因科技有限公司 | 构建测序文库的方法及其应用 |
| CN107475370A (zh) * | 2017-07-13 | 2017-12-15 | 天津脉络医学检验有限公司 | 用于肺癌诊断的基因群和试剂盒及诊断方法 |
| KR20190078715A (ko) * | 2017-12-26 | 2019-07-05 | (주)에스피메드 | 암 유전자 및 항암제 대사 관련 유전자 변이 분석을 위한 차세대 염기 서열 분석(ngs) 기반 혼합 진단 패널 |
| CN108424955A (zh) * | 2018-05-09 | 2018-08-21 | 合肥中科金臻生物医学有限公司 | 一种检测多种变异类型基因的高通量测序方法及其应用 |
| CN110452907A (zh) * | 2019-08-07 | 2019-11-15 | 浙江大学 | 靶向长链非编码rna ddx11-as1的aso、试剂盒及在肝癌治疗中的应用 |
Non-Patent Citations (2)
| Title |
|---|
| XIAOWEI MAO等: "Capture-Based Targeted Ultradeep Sequencing in Paired Tissue and Plasma Samples Demonstrates Differential Subclonal ctDNA-Releasing Capability in Advanced Lung Cancer", 《JOURNAL OF THORACIC ONCOLOGY》 * |
| 林晓等: "浙江地区晚期非小细胞肺癌患者循环肿瘤DNA突变特征的检测", 《中国现代医生》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021135072A1 (zh) * | 2019-12-31 | 2021-07-08 | 浙江大学 | 用于检测肺癌驱动性基因突变的探针及检测试剂盒 |
| CN113699242A (zh) * | 2021-10-18 | 2021-11-26 | 浙江省人民医院 | 检测kras基因突变、adamts1与bnc1甲基化的引物探针，试剂盒与方法 |
| CN115851927A (zh) * | 2022-08-09 | 2023-03-28 | 上海善准医疗科技有限公司 | 肺鳞癌分子分型及生存风险基因群及诊断产品和应用 |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2021135072A1 (zh) | 2021-07-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107475375B (zh) | 一种用于与微卫星不稳定性相关微卫星位点进行杂交的dna探针库、检测方法和试剂盒 | |
| CN109609647B (zh) | 基于二代测序的用于泛癌种靶向、化疗及免疫用药的检测Panel、检测试剂盒及其应用 | |
| CN108374046B (zh) | 肿瘤细胞微卫星不稳定状态检测体系 | |
| CN108753967A (zh) | 一种用于肝癌检测的基因集及其panel检测设计方法 | |
| CN111154872A (zh) | 用于检测肺癌驱动性基因突变的探针及试剂盒 | |
| CN111575380B (zh) | 多基因检测用的探针库、杂交试剂盒和多基因检测的方法 | |
| CN113278611B (zh) | 捕获测序探针及其用途 | |
| CN111690748B (zh) | 使用高通量测序检测微卫星不稳定的探针组、试剂盒及微卫星不稳定的检测方法 | |
| CN111424087A (zh) | 基于二代测序的用于泛癌种检测或靶向用药的检测Panel、试剂盒及应用 | |
| CN114480660A (zh) | 一种用于检测泛癌种的基因Panel、探针及应用 | |
| CN110791500B (zh) | 一种基于ngs方法检测肺癌突变基因的探针组合物及试剂盒 | |
| CN113403381A (zh) | 一种用于他汀类药物疗效预测的检测试剂盒及其检测方法和应用 | |
| CN112259165A (zh) | 用于检测微卫星不稳定性状态的方法及系统 | |
| CN110438206B (zh) | 一组检测egfr基因19外显子缺失突变的引物、探针和试剂盒 | |
| CN116445621A (zh) | 同时检测肺癌和结直肠癌的dna和rna流程引物组和试剂盒 | |
| WO2023041077A1 (zh) | eccDNA相关标志物、用途、系统及方法 | |
| CN111235251A (zh) | 降高血压药物尼群地平个体化用药指导基因检测试剂盒 | |
| CN114525344A (zh) | 一种用于检测或辅助检测肿瘤相关基因变异的试剂盒及其应用 | |
| CN103667267A (zh) | 用于与kras基因杂交的dna探针库及采用其富集kras基因片段的方法 | |
| CN111748621A (zh) | 一种检测肺癌相关41基因的探针库、试剂盒及其应用 | |
| CN111172288A (zh) | 一种大肠癌多基因筛选探针及其应用 | |
| CN111304305A (zh) | 一种用于检测egfr基因甲基化的试剂盒及方法 | |
| CN110564851A (zh) | 一组用于非超突变型直肠癌分子分型的基因及其应用 | |
| CN107227368B (zh) | 肠癌临床用药突变基因检测试剂盒 | |
| CN111235255A (zh) | 通过引物组合物进行质谱法判别尼群地平个体化用药的方法 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200515 |
|
| RJ01 | Rejection of invention patent application after publication |