CN111763729A - 用于braf基因全外显子二代测序的探针组合物、试剂及控制系统 - Google Patents
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Abstract
本发明提供一种用于BRAF基因全外显子二代测序的探针组合物、试剂及控制系统。所述用于BRAF基因全外显子二代测序的探针组合物包括SEQ ID NO:01至SEQ ID NO:94所示的序列。本发明提供的所述用于BRAF基因全外显子二代测序的探针组合物、试剂及控制系统,解决了以下现有的技术问题:1.现有技术主要检测BRAF基因的点突变,检测范围有限;2.实时荧光定量PCR检测、基因芯片只能检测已知位点;3.Sanger测序检验长度小于800bp的范围;4.不能检测低频突变;5.检测多个位点时操作繁琐、费用高。
Description
技术领域
本发明涉及基因测序领域,具体涉及一种用于BRAF基因全外显子二代测序的探针组合物、试剂及控制系统。
背景技术
BRAF(B-raf)基因编码一种属于丝氨酸/苏氨酸蛋白激酶的蛋白质,是RAS/RAF/MEK/ ERK/ MAP信号传导通路中重要的转导因子,参与调控MAPKinse/ERK信号传导途径,从而影响诸如细胞生长、分化和凋亡等。研究表明,BRAF基因也是重要致癌基因之—,在多种人类恶性肿瘤中,如恶性黑色素瘤、结直肠癌(CRC)、非小细胞肺癌(NSCLC)、甲状腺癌、肝癌、胰腺癌以及毛细胞性白血病等,均检出不同比例的BRAF基因突变。
BRAF基因的转录本NM_001354609.1全长2342bp,有18个外显子,已知最常见的突变形式主要是外显子15上第1860位(编码序列的第1799位)及附近的不同形式的点突变,导致第600位缬氨酸V突变为其他种氨基酸(谷氨酸E、天冬氨酸D、赖氨酸K、甲硫氨酸M、丙氨酸A),对于其他位点的研究较少。
针对BRAF基因突变,目前常见的是运用Sanger测序、PCR检测、基因芯片等方法,检测BRAF基因的外显子15上第1860位附近的点突变,检测范围小,且当涉及多个位点时操作极度繁琐、费用高昂、可行性低。
全外显子测序的难点在于探针的设计和优化,要求探针序列组合成型后,性能稳定、操作简便,且其片段长度更加适合illumina测序平台。
专利“CN103667268B-用于与BRAF基因杂交的DNA探针库及采用其富集BRAF基因片段的方法”,其中涉及到的探针一共只有6条,每条探针可捕获的序列至多600bp,由于BRAF基因有多达17个内含子,该方法至多可以检测18条外显子中的6条,仍有大量的位点没有覆盖到。
发明内容
为解决现有技术的的BRAF基因突变检测多为针对BRAF基因的外显子15上第1860位点进行检测,存在多种局限性的技术问题,本发明提供一种解决上述问题的用于BRAF基因全外显子二代测序的探针组合物、试剂及控制系统。
用于BRAF基因全外显子二代测序的探针组合物,包括SEQ ID NO:01至 SEQ IDNO:94所示的序列。
用于BRAF基因全外显子二代测序的试剂,包括上文所述的探针组合物及二代测序所需试剂。
用于BRAF基因全外显子二代测序的控制系统包括:
检测模块,采用上文所述的用于BRAF基因全外显子二代测序的试剂进行二代测序;
显示模块,将检测结果转换为可视化数据,并将可视化的数据于显示设备进行展示;
分析模块:基于预设的质控程序对检测结果进行验证,并将验证结果于同一显示设备进行展示。
其中,运行所述检测模块包括以下步骤:
步骤一:文库构建,包括gDNA浓度检测及稀释、gDNA片段化、末端修复和A碱基的添加、接头的连接及连接产物的纯化、文库片段筛选、PCR扩增及扩增产物的纯化、终文库QC;
步骤二:杂交捕获,包括文库Pooling、变性与杂交、捕获洗脱、PCR扩增与扩增产物的纯化;
步骤三:上机测序,经过稀释、变性等步骤,调整至符合测序仪的标准,并按测序仪的标准操作进行测序、输出检测结果。
所述显示模块将步骤三中的检测结果转化为包括“7号染色体概图”、“测序结果”、“参考序列”三项数据的图表。
所述分析模块直接以所述显示模块转化得到的图表为原始数据,与预设的数据进行对比,并向所述显示模块输出检测结果。
相较于现有技术,本发明提供的所述用于BRAF基因全外显子二代测序的探针组合物,经设计优化后得到的探针一共94条,具有以下优点:1.能够100%覆盖BRAF基因的全外显子区域,能够全面、准确的检测待测样本BRAF基因的突变情况;2.能够检测出Sanger测序、实时荧光定量PCR、基因芯片所不能检测出的低频突变,具有极高的科研、临床价值,且操作简单、成本较低。
附图说明
图1是探针法 BRAF 二代测序结果概图;
图2是探针法 BRAF 二代测序结果细节图。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述,所描述的实施例仅是本发明的一部分实施例,而不是全部的实施例。
实施例1:
用于BRAF基因全外显子二代测序的探针组合物包括SEQ ID NO:01~ SEQ ID NO:94所示的序列。
将94条探针按照每管100p的标准稀释,取一个新的1.5ml离心管,将每种探针分别加入2ul,补水至总体积为200ul。维持终浓度是1pmol,震荡混匀,-20℃保存备用。
用于BRAF基因全外显子二代测序的试剂包括上述探针组合物及二代测序所需的试剂,具体的内容在以下用于BRAF基因全外显子二代测序的方法中说明。
用于BRAF基因全外显子二代测序的方法为采用上述的试剂对BRAF基因全外显子进行二代测序,包括以下步骤:
1、gDNA浓度检测及稀释:Qubit3.0测定gDNA样品浓度,将样品稀释至10 ng/ul,样品体积大于35ul,总DNA质量大于350ng。
2、gDNA片段化、末端修复和A碱基的添加,包括:
2.1、将FX Buffer和FX Enzyme Mix从-20℃取出冰上解冻,混匀,微离心;
2.2、配制反应体系:
2.3、吹打混匀,微离心。
2.4、将PCR仪的热盖温度调至70℃并使加热板预先冷却至4℃,按下表设置反应程序进行反应:
3、接头的连接及连接产物的纯化,包括:
3.1、将Ligation Buffer、DNA ligase和Aapter从-20℃取出冰上解冻,混匀,微离心;
并预先取出 Agencourt AMPure XP beads放置室温;
3.2、配制接头连接反应体系:
3.3、吹打混匀,微离心;
3.4、20℃孵育30分钟(PCR仪的热盖调为:Lif off);
3.5、取一新的EP管,将连接产物与80ul(0.8x)Agencourt AMPure XP beads混匀,室温孵育5分钟;
3.6、磁力架上放置2分钟,弃上清;
3.7、加入200 ul新鲜配制的80%乙醇,转动EP管几次,静置30 s后吸去上清;
3.8、重复(3.7)步骤一次;
3.9、室温放置5-10分钟干燥磁珠;
3.10、将EP管从磁力架上取下,加入52.5 ul Resuspension Buffer重悬磁珠,室温静置2分钟,磁力架上放置2分钟,转移50 ul上清至新的EP管中。
4、文库片段筛选,包括:
4.1、在50ul文库中加入35ul Agencourt AMPure XP beads,吹打混匀,室温放置5分钟;
4.2、微离心后放置磁力架上2分钟,移取上清至新的96孔板中,并加入10ul AgencourtAMPure XP beads,吹打混匀,室温静置5分钟;
4.3、微离心,放置磁力架上2分钟,弃上清;
4.4、加入100 ul新鲜配制的80%乙醇,静置30s,吸去上清;
4.5、重复上一步骤一次;
4.6、室温放置5-10分钟干燥磁珠;
4.7、将96孔板从磁力架上取下,加入26 ul Resuspension Buffer重悬磁珠,室温静置2分钟,微离心后放置磁力架上2分钟,转移23.5 ul上清至新的96孔板中进行下一步PCR反应。
5、PCR扩增及扩增产物的纯化,包括:
5.1、将HiFi PCR Master Mix和Primer Mix从-20℃取出冰上解冻,混匀,微离心;
5.2、配制PCR反应体系:
试剂 | 体积(ul) |
HiFi PCR Master Mix,2x | 25 |
Primer Mix (10 μM each) | 1.5 |
Library DNA | 23.5 |
总量 | 50 |
5.3、吹打混匀,微离心;
5.4、PCR扩增反应程序:
温度 | 时间 | 循环数 |
98℃ | 1min | 1 |
98℃ | 20s | 8 |
60℃ | 30s | |
72℃ | 30s | |
72℃ | 1min | 1 |
10℃ | ∞ | 1 |
5.5、取一新的EP管,将PCR产物与50 ul(1.0x)Agencourt AMPure XP beads混匀,室温孵育5分钟;
5.6、磁力架上放置2分钟,弃上清;
5.7、加入200 ul新鲜配制的80%乙醇,转动EP管几次,静置30 s后吸去上清;
5.8、重复(5.7)步骤一次;
5.9、室温放置5-10分钟干燥磁珠;
5.10、将EP管从磁力架上取下,加入20 ul Resuspension Buffer重悬磁珠,室温静置2分钟,磁力架上放置2分钟,转移17 ul上清至新的EP管中,-20℃保存。
6、终文库QC,包括:
6.1、用Qubit3.0对终文库进行浓度测定(文库浓度处于30—50ng/ ul);
6.2、采用琼脂糖凝胶电泳检测文库片段大小,片段大小处于200-500bp左右。
7、文库Pooling:取出构号的文库,按照200 ng/sample;每个pool不超过6000 ng的量混合文库于0.2ml PCR管中,建好的文库于-20℃保存。
8、变性与杂交,包括:
8.1、-20℃冰箱取出COT Human DNA和xGen® Universal Blockers,溶解后震荡离心;
8.2、向pooling文库中加入以下试剂:
试剂 | 体积(ul) | 耗材类型 |
混合文库 | <6000ng | 0.2ml PCR管 |
Human Cot-1 DNA® | 5 | |
xGen® Universal Blockers-TS Mix | 2 | |
总量 | 7 |
8.3、将PCR管放至真空抽滤系统(60℃)干燥成干粉,约20min;(预热15min,选择V-AQ模式,Brake-off);
8.4、根据下表向干粉中依次加入相应试剂,吹打混匀后室温放置5min,置于PCR仪上进行变性反应:
试剂 | 体积(ul) |
xGen 2X Hybridization Buffer | 8.5 |
xGen Hybridization Buffer Enhancer | 2.7 |
Nuclease-Free Water | 1.8 |
总量 | 13 |
反应程序为:
温度 | 时间 | 循环数 |
95℃ | 10min | |
4℃ | ∞ |
8.5、反应结束后将样本放置冰板上,加入4 ul探针混合液;
8.6、混匀,置于PCR仪上反应16-18h;
反应程序为:
温度 | 时间 | 循环数 |
65℃ | ∞ |
(热盖温度:75℃)。
9、捕获洗脱,包括:
9.1、取出Dynabeads® M-270 Streptavidin beads捕获磁珠平衡至室温。按下表配制xGen 2X Bead Wash Buffer;
试剂 | 原体积(ul) | ddH2O体积(ul) | 1X Buffer(ul) |
xGen 2X Bead Wash Buffer | 250 | 250 | 500 |
9.2、吸取100 ul /Pool M270捕获磁珠,放置磁力架上,弃上清,加入200 ul 1X BeadWash Buffer重悬磁珠,离心后放置磁力架上,弃上清;重复清洗磁珠一次;
9.3、捕获磁珠重悬:加入100 ul 1X Bead Wash Buffer重悬磁珠,并转移至0.2mL PCR管中,离心后放置磁力架上,弃上清;
9.4、磁珠与杂交产物结合:将杂交产物17 ul转移至捕获磁珠中,吹打重悬磁珠后放置PCR仪(65℃,热盖75℃)孵育45min,中间间隔15min振荡混匀一次;
9.5、buffer配制:根据下表配制1X Wash Buffer I、1X Wash Buffer II、1X WashBuffer III和1X Stringent Wash Buffer,将100ul 1X Wash Buffer I/Pool和400 ul 1XStringent Wash Buffer放置于65℃中温育;按下表配制1X Bead Wash Buffer;
试剂 | 原体积(ul) | ddH2O体积(ul) | 1X Buffer(ul) | 备注 |
xGen 10X Wash Buffer I | 30 | 270 | 300 | 100ul 65℃ 预热 |
xGen 10X Wash Buffer II | 20 | 180 | 200 | |
xGen 10X Wash Buffer III | 20 | 180 | 200 | |
xGen 10X Stringent Wash Buffer | 40 | 360 | 400 | 65℃ 预热 |
xGen 2X Bead Wash Buffer | 250 | 250 | 500 |
10、PCR扩增与扩增产物的纯化,包括:
10.1、试剂解冻后震荡离心,按下表分装:
试剂 | 体积(ul) |
KAPA HiFi HotStart ReadyMix | 25 |
Post-LM-PCR Oligos | 5 |
总体积 | 30 |
带磁珠产物 | 20 |
反应体系总体积 | 50 |
10.2、按上表加入Mix,混匀,微离心后置于PCR仪上反应;
反应程序为:
10.3、扩增完成后,将PCR管置于磁力架上,吸取50ul上清到新的1.5mL的EP管中,并加入75ul Agencourt AMPure XP beads,混匀,室温放置5min;
10.4、微离心后,置于磁力架上2min,弃上清;
10.5、加入200.0 ul 80%乙醇,静置30 s,弃上清;
10.6、加入200.0 ul 80%乙醇,静置30 s,弃上清;
10.7、室温放置10 min,待磁珠干燥;
10.8、加入22.0 ul Resuspension Buffer (RSB),重悬磁珠;
10.9、室温静置2 min,离心后磁力架上放置2min;
10.10、转移20.0 ul上清至新0.2 mL PCR管中,放置4℃或-20℃中保存;
10.11、用Qubit®3.0 Fluorometer测定浓度并记录。
11、上机测序,包括:
11.1、将文库用Resuspension Buffer (RSB)稀释至1.0nM;
11.2、制备20.0ul 0.2N的NaOH;
11.3、将20.0ul 1nM的混合文库与20.0ul 0.2N的NaOH混匀,室温变性5min;
11.4、与960.0ul Hybridization Buffer混匀;
11.5、移取100.0ul与1200.0ul Hybridization Buffer混匀,加入至ReagentCartridge 10号孔。
12、按illumina NextSeq550二代测序仪的标准操作流程测序。
13、分析结果:
请同时参阅图1、图2,分别是探针法BRAF二代测序结果概图和细节图。测序结果经过去除接头、重复序列、低质量序列等生物信息学处理之后,利用igv 软件打开,结果如下图1和图2 所示。
图中由上至下表示的信息均依次为:“7号染色体概图”、“测序结果”、“参考序列”,由图示可以直观的看出本款探针有效。
实施例2:
通过用于BRAF基因全外显子二代测序控制系统,控制专用设备来实施实施例1中所述的用于BRAF基因全外显子二代测序方法。
以上所述仅为本发明的实施例,并非因此限制本发明的专利范围,凡是利用本发明说明书内容所作的等效结构或等效流程变换,或直接或间接运用在其它相关的技术领域,均同理包括在本发明的专利保护范围之内。
序列表
<110> 长沙金域医学检验所有限公司
<120> 用于BRAF基因全外显子二代测序的探针组合物、试剂及控制系统
<130> 0005
<160> 94
<170> SIPOSequenceListing 1.0
<210> 1
<211> 120
<212> DNA
<213> Artificial sequence
<400> 1
ccggcgccgg cgccggcgcc gcggcctctt cggctgcgga ccctgccatt ccggaggagg 60
tgagtgctgg cgccaccctg ccgccctccc gactccgggc tcggcggctg gctggtgttt 120
<210> 2
<211> 120
<212> DNA
<213> Artificial sequence
<400> 2
attttttagg tgtggaatat caaacaaatg attaagttga cacaggaaca tatagaggcc 60
ctattggaca aatttggtgg ggagcataat ccaccatcaa tatatctgga ggtaagcttt 120
<210> 3
<211> 120
<212> DNA
<213> Artificial sequence
<400> 3
atgtcaggac aaagtccgga ttgaatataa gtctgcttta ttttataggc ctatgaagaa 60
tacaccagca agctagatgc actccaacaa agagaacaac agttattgga atctctgggg 120
<210> 4
<211> 120
<212> DNA
<213> Artificial sequence
<400> 4
aacggaactg atttttctgt ttctagctct gcatcaatgg ataccgttac atcttcttcc 60
tcttctagcc tttcagtgct accttcatct ctttcagttt ttcaaaatcc cacagatgtg 120
<210> 5
<211> 120
<212> DNA
<213> Artificial sequence
<400> 5
gcacggagca accccaagtc accacaaaaa cctatcgtta gagtcttcct gcccaacaaa 60
cagaggacag tggtgagtca gttttaatat caccattttg ctgtttcttt gtatttttca 120
<210> 6
<211> 120
<212> DNA
<213> Artificial sequence
<400> 6
taccataggt acctgcaagg tgtggagtta cagtccgaga cagtctaaag aaagcactga 60
tgatgagagg tctaatccca gagtgctgtg ctgtttacag aattcaggat gggtatggtt 120
<210> 7
<211> 120
<212> DNA
<213> Artificial sequence
<400> 7
tttttcagag agaagaaacc aattggttgg gacactgata tttcctggct tactggagaa 60
gaattgcatg tggaagtgtt ggagaatgtt ccacttacaa cacacaactt tgtatgtatc 120
<210> 8
<211> 120
<212> DNA
<213> Artificial sequence
<400> 8
gttttcttgt gagtttttga aatctctgtg attttttact ttgcaggtac gaaaaacgtt 60
tttcacctta gcattttgtg acttttgtcg aaagctgctt ttccagggtt tccgctgtca 120
<210> 9
<211> 120
<212> DNA
<213> Artificial sequence
<400> 9
aacatgtggt tataaatttc accagcgttg tagtacagaa gttccactga tgtgtgttaa 60
ttatgaccaa cttgagtaag taatccaaaa atatctcttt tctacctacc attttacact 120
<210> 10
<211> 120
<212> DNA
<213> Artificial sequence
<400> 10
tttggtatat gaagcttctg ggttttgcac aagttaggtt tgttttgttt tgcctcacag 60
tttgctgttt gtctccaagt tctttgaaca ccacccaata ccacaggaag aggcgtcctt 120
<210> 11
<211> 120
<212> DNA
<213> Artificial sequence
<400> 11
agcagagact gccctaacat ctggatcatc cccttccgca cccgcctcgg actctattgg 60
gtatggtttg acttctgctc ttgggcgaca tgctacttga accgctttct tttggatctc 120
<210> 12
<211> 120
<212> DNA
<213> Artificial sequence
<400> 12
ttactctgaa tcttatcttc caatgacttc atttttccag gccccaaatt ctcaccagtc 60
cgtctccttc aaaatccatt ccaattccac agcccttccg accagcagat gaagatcatc 120
<210> 13
<211> 120
<212> DNA
<213> Artificial sequence
<400> 13
gaaatcaatt tgggcaacga gaccgatcct catcagctcc caatgtgcat ataaacacaa 60
tagaacctgt caatattgat gtaagtatcc agcattgcta gaactaaaaa aaaaccaagt 120
<210> 14
<211> 120
<212> DNA
<213> Artificial sequence
<400> 14
tatgcttgct tggaataaat atacattact atttatttgt aggacttgat tagagaccaa 60
ggatttcgtg gtgatggagg taagtagtga tttcaggttt ttttaaaaac tcaaggaaac 120
<210> 15
<211> 120
<212> DNA
<213> Artificial sequence
<400> 15
ttaacatact tgctcctcct taatgtatac atttttcttt tcactaattt aggatcaacc 60
acaggtttgt ctgctacccc ccctgcctca ttacctggct cactaactaa cgtgaaagcc 120
<210> 16
<211> 120
<212> DNA
<213> Artificial sequence
<400> 16
ttacagaaat ctccaggacc tcagcgagaa aggaagtcat cttcatcctc agaagacagg 60
aatcgaatgg taagagtata tgatatcttt ttttctctga attctttctt cttagaagtc 120
<210> 17
<211> 120
<212> DNA
<213> Artificial sequence
<400> 17
gaaaacactt ggtagacggg actcgagtga tgattgggag attcctgatg ggcagattac 60
agtgggacaa agaattggat ctggatcatt tggaacagtc tacaagggaa agtggcatgg 120
<210> 18
<211> 120
<212> DNA
<213> Artificial sequence
<400> 18
gtaacaattg ccttttaggt gatgtggcag tgaaaatgtt gaatgtgaca gcacctacac 60
ctcagcagtt acaagccttc aaaaatgaag taggagtact caggtgagct tgtgtgaatt 120
<210> 19
<211> 120
<212> DNA
<213> Artificial sequence
<400> 19
tttaatgttt actgtcacat ttctttgtac aggaaaacac gacatgtgaa tatcctactc 60
ttcatgggct attccacaaa gccacaactg gctattgtta cccagtggtg tgagggctcc 120
<210> 20
<211> 120
<212> DNA
<213> Artificial sequence
<400> 20
agcttgtatc accatctcca tatcattgag accaaatttg agatgatcaa acttatagat 60
attgcacgac agactgcaca gggcatggag taagttccat tcgttaaatg tcttgtaaat 120
<210> 21
<211> 120
<212> DNA
<213> Artificial sequence
<400> 21
gccctactca ggttaaaatg atgttttgtt tttcagttac ttacacgcca agtcaatcat 60
ccacagagac ctcaagagta atagtatcct tcctgaaatt tgtctgcgaa gtttgaaaac 120
<210> 22
<211> 120
<212> DNA
<213> Artificial sequence
<400> 22
atatatttct tcatgaagac ctcacagtaa aaataggtga ttttggtcta gctacagtga 60
aatctcgatg gagtgggtcc catcagtttg aacagttgtc tggatccatt ttgtggatgg 120
<210> 23
<211> 120
<212> DNA
<213> Artificial sequence
<400> 23
ttcagtcttt attcaattga agtgaagaat atttttcttt gtatgttcta acaggcacca 60
gaagtcatca gaatgcaaga taaaaatcca tacagctttc agtcagatgt atatgcattt 120
<210> 24
<211> 120
<212> DNA
<213> Artificial sequence
<400> 24
ggaattgttc tgtatgaatt gatgactgga cagttacctt attcaaacat caacaacagg 60
gaccaggtaa atatttacca cgtcttggtg tttattttac cgtctatata caaggctcca 120
<210> 25
<211> 120
<212> DNA
<213> Artificial sequence
<400> 25
ccacttcttg gcattttagg tgctttgtcc tccatgggag tataataaat gatgtggcaa 60
gggcttactc tccatgagag gaatgtgtga ccaacagaag ggtaaggcct ttactagtta 120
<210> 26
<211> 120
<212> DNA
<213> Artificial sequence
<400> 26
ttttcaactt atctacaagg ttgaattatt tccctttttt ttctctctcc agataatttt 60
tatggtggga cgaggatacc tgtctccaga tctcagtaag gtacggagta actgtccaaa 120
<210> 27
<211> 120
<212> DNA
<213> Artificial sequence
<400> 27
agccatgaag agattaatgg cagagtgcct caaaaagaaa agagatgaga gaccactctt 60
tccccaagta agtaaaagct tcatgctatc caaaagaaca gactaacatt catagacaga 120
<210> 28
<211> 120
<212> DNA
<213> Artificial sequence
<400> 28
tttttttttc tttttttttt tgtagattct cgcctctatt gagctgctgg cccgctcatt 60
gccaaaaatt caccgcagtg catcagaacc ctccttgaat cgggctggtt tccaaacaga 120
<210> 29
<211> 120
<212> DNA
<213> Artificial sequence
<400> 29
ggattttagt ctatatgctt gtgcttctcc aaaaacaccc atccaggcag ggggatatgg 60
tgcgtttcct gtccactgaa acaaatgagt gagagagttc aggagagtag caacaaaagg 120
<210> 30
<211> 120
<212> DNA
<213> Artificial sequence
<400> 30
aaaataaatg aacatatgtt tgcttatatg ttaaattgaa taaaatactc tctttttttt 60
taaggtgaac caaagaacac ttgtgtggtt aaagactaga tataattttt ccccaaacta 120
<210> 31
<211> 120
<212> DNA
<213> Artificial sequence
<400> 31
aaatttatac ttaacattgg atttttaaca tccaagggtt aaaatacata gacattgcta 60
aaaattggca gagcctcttc tagaggcttt actttctgtt ccgggtttgt atcattcact 120
<210> 32
<211> 120
<212> DNA
<213> Artificial sequence
<400> 32
tggttatttt aagtagtaaa cttcagtttc tcatgcaact tttgttgcca gctatcacat 60
gtccactagg gactccagaa gaagacccta cctatgcctg tgtttgcagg tgagaagttg 120
<210> 33
<211> 120
<212> DNA
<213> Artificial sequence
<400> 33
gcagtcggtt agcctgggtt agataaggca aactgaacag atctaattta ggaagtcagt 60
agaatttaat aattctatta ttattcttaa taatttttct ataactattt ctttttataa 120
<210> 34
<211> 120
<212> DNA
<213> Artificial sequence
<400> 34
caatttggaa aatgtggatg tcttttattt ccttgaagca ataaactaag tttcttttta 60
taaattttga gtgcaggtga ccaaaaatat tgctgaggag tggcacgttt gacatgagta 120
<210> 35
<211> 120
<212> DNA
<213> Artificial sequence
<400> 35
ctctacagga gaatttgcag ccttcaagta gccaccatca tggcagcatc tgctcttatt 60
tcttaagtct tgtgttcgta caatttgtta acatcaaaac acagttctgt tcctcaaatc 120
<210> 36
<211> 120
<212> DNA
<213> Artificial sequence
<400> 36
aattgaattg gaaaaaaaca atacttccca ccagaattat atatcctgaa aattgtattt 60
ttgttatata aacaactttt aagaaagatc attatccttt tctctaccta aatatgagga 120
<210> 37
<211> 120
<212> DNA
<213> Artificial sequence
<400> 37
gtcttagcat aatgacaaat atttataatt tttcaattaa tggtacttgc tggatccaca 60
ctaacatctt tgctaataat ctcattgttt cttccaactg attcctaaca ctatatccca 120
<210> 38
<211> 120
<212> DNA
<213> Artificial sequence
<400> 38
catcttcttt ctagtctttt atctagaata tgcaacctaa aataaaaatg gtggcgtctc 60
cattcattct ccttcttcct tttttcccaa gcctggtctt caaaaggttg ggcaatttgg 120
<210> 39
<211> 120
<212> DNA
<213> Artificial sequence
<400> 39
cagctgaatt cccagacaga gaatagagca attttaggga tattaggact gagggagggt 60
gtgggaaagc tgtcatcagt tgtttttata gaaagaactg gcattcatta agaacctaaa 120
<210> 40
<211> 120
<212> DNA
<213> Artificial sequence
<400> 40
tcttatcttt gcacaaatgg aaaatataac ctagttatag cttcctttgg cctttattaa 60
agggtaatat caatcacagt catagcaaag aaagcggatg tattaatggc aaattaatgg 120
<210> 41
<211> 120
<212> DNA
<213> Artificial sequence
<400> 41
aaaacctccc ttatcaggaa tctagactca gaatttagga acacaaatca aatcagacca 60
accaagctat agccaaggac ttgaaagaaa ttaaacaaga cccagaataa atcaaggaat 120
<210> 42
<211> 120
<212> DNA
<213> Artificial sequence
<400> 42
tagaaattgt tatttaaaaa tttcagattg taactccagg ccctgctgtc tatattgcag 60
ccactaaaag ctcactacca ttagattttt gctaacatac atgtattcag aagaaagcct 120
<210> 43
<211> 120
<212> DNA
<213> Artificial sequence
<400> 43
attgaaattt tcattgtctt gtaaaaggtt gtcctagtaa aatggaaaag atccttaagt 60
tattaatcag tttgaaaagc aaatttgttt ttaagtttta catcagcagg gcagtgtctt 120
<210> 44
<211> 120
<212> DNA
<213> Artificial sequence
<400> 44
acaaaattca gaaattgcaa aggtggaaat aattcacgct gatttgaaga acatcttctg 60
tgcaataata ctgcctctct tgaaaagcat tggctgtttt ttctttttaa atatatctct 120
<210> 45
<211> 120
<212> DNA
<213> Artificial sequence
<400> 45
agatgctttt aaatgtggct gtgttccctt taccaagatt ggcttcaagt ttccgcaggt 60
agagagacct gggcttgaac aagaggatgt gtttcatgtc ctgctgagga ggtagaacat 120
<210> 46
<211> 120
<212> DNA
<213> Artificial sequence
<400> 46
tttttttaaa gatacaaaat ttccaatgca taagctgatg tggaacagaa tggaatttcc 60
catccaacaa aagaggaaag aatgttttag gaaccagaat tctctgctgc cagtgtttct 120
<210> 47
<211> 120
<212> DNA
<213> Artificial sequence
<400> 47
gtgcagcctg ggtccgggac tgcctccgtg gggcaggggc aggggcggta ccattaggga 60
ggaagcttag catttcagtt tcttaaacaa tattcagggt gatacacttt ttcttccctt 120
<210> 48
<211> 120
<212> DNA
<213> Artificial sequence
<400> 48
gcattttaga ataggctggt atctcatttg aacgggggag cagacttgat ctcaaatgaa 60
gctgtgccca ggagccaggc ttagcatatt gagattttta tagatacctt aaaaaataaa 120
<210> 49
<211> 120
<212> DNA
<213> Artificial sequence
<400> 49
atatttaaac ctctcttttc ttcctttttc tatgaaatag gttttttctc tagtttacaa 60
atgacatgaa aataggtttt atttgtgttt tatctgcttt attttttgat gcttagacaa 120
<210> 50
<211> 120
<212> DNA
<213> Artificial sequence
<400> 50
cagttagact tactgagctc ctaaaaaaac gaggaagaag tccttatttg tgaaaagcac 60
tttatgagta attgtataga cagtatgtgg ctgcgtcact gatcatcttg taagggtgta 120
<210> 51
<211> 120
<212> DNA
<213> Artificial sequence
<400> 51
acagtcttgt ctgtaaagtg gctgcagtgc cttctgtagt gtgttttatt tttggtaggg 60
agaggtgaag ccttctgaaa aatttgagag caactacaga ggattgtttg taactgtgta 120
<210> 52
<211> 120
<212> DNA
<213> Artificial sequence
<400> 52
gtattcctga tggacttttt tcatcgttag agtcaaggac ctagactttt gccactgaaa 60
taatattgac caaaaaaata gtttataaaa gggatttgtg aatagaaaat tcagtgtgat 120
<210> 53
<211> 120
<212> DNA
<213> Artificial sequence
<400> 53
catttgttgt taatgtgcac cttaaaagaa gattctgtct agctgtcaaa ttctggttcc 60
cgaatatctc acccctgatt gtatttgaga tctagtaggg catactgggg cattttagaa 120
<210> 54
<211> 120
<212> DNA
<213> Artificial sequence
<400> 54
gataaaatcc catacaaatg atatatgcta tatttatgtt ggtgttggag aagaaagagc 60
agtatataaa gaaataattc aagactgcag cactgtcaac ctgaaacttt gtaaatattt 120
<210> 55
<211> 120
<212> DNA
<213> Artificial sequence
<400> 55
cctagcttct ggtttggtgc ggtgacagca ctttcatcac aggatgttac cttgtattca 60
ccaggcggag tgcgagctgc tgcacatcct cctcagatct cacctgtccc cactgtacat 120
<210> 56
<211> 120
<212> DNA
<213> Artificial sequence
<400> 56
ccacccgcca gctgcttgca aacctcatct ctagctttag ttcgaaacca cattgcaggg 60
ttcaggtgac ctctacaaaa aactacctct tcagaatgag gtaatgaata gttatttatt 120
<210> 57
<211> 120
<212> DNA
<213> Artificial sequence
<400> 57
tcaacaaaaa taccacgagc atacaagtct gcccagtccc aggaagaaag aggagagacc 60
ctgaattctg accttttgat ggtcaggcat gatggaaaga aactgctgct acagcttggg 120
<210> 58
<211> 120
<212> DNA
<213> Artificial sequence
<400> 58
ttaaaatatg aaaagtcagg agctctagaa catgacgatg atttaagatt ttaacttttt 60
tgtgtacttg tatttgagca ctctcatttt gtcctaaagg gcattataca tttaagcagt 120
<210> 59
<211> 120
<212> DNA
<213> Artificial sequence
<400> 59
aatactgtaa aaaaatgtgt tgctcggaat atctgaatgt tgttgaaagt ggtgccagaa 60
ccggtttagg ggtacgtttc agaatcttaa ccttgagtca attgcatgaa attaaatagc 120
<210> 60
<211> 120
<212> DNA
<213> Artificial sequence
<400> 60
tgtggtatca cttcactaac agtgatgtaa ttttaatttt cagtaggctt ggcatgacag 60
tacatcctca taatgagttt gctgcagctt tgtcacatgc acaggcattc atagaaagac 120
<210> 61
<211> 120
<212> DNA
<213> Artificial sequence
<400> 61
cacccagcta agagggtaga atgattactc tttttgcaag attctcttct ttgtccaagt 60
tggcattgtt agtgctagga ataccagcac cttgagacga gcagattcca accattaggc 120
<210> 62
<211> 120
<212> DNA
<213> Artificial sequence
<400> 62
tataaacacc atagccagag atggaaggtt tactgtgagt atgaacagca aatagcttac 60
aggtcatgag ttgaaatggt gtaggtgagg ctctagaaaa ataccttgac aatttgccaa 120
<210> 63
<211> 120
<212> DNA
<213> Artificial sequence
<400> 63
atgatcttac tgtgccttca tgatgcaata aaaaagctaa cattttagca gaaatcagtg 60
atttgtgaag agagcagcca ctctggttta actcagctgt gttaataatt tttagagtgc 120
<210> 64
<211> 120
<212> DNA
<213> Artificial sequence
<400> 64
aatttagact gcataggtaa atgcactaaa gagtttatag ccaaaatcac atttaacaat 60
gagaaaacac acaggtaaat tttcagtgaa caaaattatt tttttaaagc acataatccc 120
<210> 65
<211> 120
<212> DNA
<213> Artificial sequence
<400> 65
tagtatagtc agatatattt atcacataga gcaactaggt tgcaaatata gttcagtgac 60
atttctagag aaactttttc tactcccata ggctcttcaa agcatggaac ttttatacaa 120
<210> 66
<211> 120
<212> DNA
<213> Artificial sequence
<400> 66
cagaaatgtt gacagaaatt gctgtagttt agggttgaag tactgtatga tgggcagcaa 60
tcatgtatta acttagaagg ggaaattgaa atataggacc gaatttggtt ttatcagttt 120
<210> 67
<211> 120
<212> DNA
<213> Artificial sequence
<400> 67
ccagagtact gctgccaacc tagacactga tttttcagag tttgaaatgt aaatttcttc 60
ccgggacttg attgcacatg aagctggact gcgttagtca tcctgtccca aagcgctgtg 120
<210> 68
<211> 120
<212> DNA
<213> Artificial sequence
<400> 68
agatttgcta tggaaagtct gccagtcaac tttgcccttc taaccaccag atcaatttgt 60
ggctgatcat ctgatggggc agtttcaatc accaagcatc gttctctttc ctgttctgga 120
<210> 69
<211> 120
<212> DNA
<213> Artificial sequence
<400> 69
ggggccaggg tggaggtctc aaggcatcct ttatgacctg gccattggat gtaaaagaaa 60
acatattcca tgctgtggtt cttgtatctt gtttcattcc tcaccattga aagagaaagt 120
<210> 70
<211> 120
<212> DNA
<213> Artificial sequence
<400> 70
ccatgtattg tctccagcac atccttgaaa tgttatactg ggatggatta ctgatgccca 60
tcggtagttg agccccagaa gagggtagta gcatctctgc ctcaggtgat gatttgtagc 120
<210> 71
<211> 120
<212> DNA
<213> Artificial sequence
<400> 71
ttggccagag gagagcggag tcaccagtat atctgtggtc catgttgcta gctctggtaa 60
aattaaaaat actggtaaga tgtttgtttt attagtacac tagacagtaa gctctgtttt 120
<210> 72
<211> 120
<212> DNA
<213> Artificial sequence
<400> 72
gttgttttca aataacctat tttcactttt gtttgggcaa agacatttaa attgaaattc 60
aattctaatt tttgttaatt gtggaaaggg taattaacag ttcctatcag gtatttttaa 120
<210> 73
<211> 120
<212> DNA
<213> Artificial sequence
<400> 73
tgtggaaaag gacagaaacc caactcctaa aatcttaaat taaggtaaca gtgctttaaa 60
aaaaaaaaat gcatggggca attagtcggc aactcaatga gtgactaaag tacttttatt 120
<210> 74
<211> 120
<212> DNA
<213> Artificial sequence
<400> 74
taacatccac aacttcaact gttaagtttt attaattact aaatcagctt tattaaaatg 60
ttgacattta tttagctatt ttgaataatt atagtgactt gacgagtgtg tatgaggaca 120
<210> 75
<211> 120
<212> DNA
<213> Artificial sequence
<400> 75
cagccaatgt aagccagtgt atccattttt tagaggtgca ttttttttta aagaattctg 60
tagatagaag tgctctgaaa acaactaaaa tatgtttatt catggtagta tcaaaaaatg 120
<210> 76
<211> 120
<212> DNA
<213> Artificial sequence
<400> 76
tttgtacaaa ccatctgctt ctcccggcca gccgagttca ttctccagca ccgtgaccgc 60
tggttctcat gtacagcaca tatgcgggag agttggcaga aaatttgtga agagatgccg 120
<210> 77
<211> 120
<212> DNA
<213> Artificial sequence
<400> 77
caaaggaagg gtctgttgac gggtgggatt gggggttttg atgaagttgc ttagtcctgg 60
ttttgttttg aaaattactg cgttgcattt ttgtgttaag tttttgaacc cacgtgtgtt 120
<210> 78
<211> 120
<212> DNA
<213> Artificial sequence
<400> 78
ttggtggagt atgagttgga agtcactgca aactagcata aacaacaaag ctcacagagt 60
aggcacagat gtagagaaca gagaccaaaa tggggtgagg tggcagtaaa tctaggatag 120
<210> 79
<211> 120
<212> DNA
<213> Artificial sequence
<400> 79
attttgtttt ggagctcttt cccctagtga ccaccagtta gtttctgagg gatggaacaa 60
aaatgcagct tgccctttct atgtggtgcg tgttcaggcc ttgacagatt ttatcaaaag 120
<210> 80
<211> 120
<212> DNA
<213> Artificial sequence
<400> 80
ggaaaaatta atgtgagggt gggaaataaa ctgtaattac ctgaaatcaa atgtaagagt 60
gcaataagta tgctttttat tctaagctgt gaacggtttt tttaagaatc attccttcct 120
<210> 81
<211> 120
<212> DNA
<213> Artificial sequence
<400> 81
aatacatttg tgtatgttcc atagctgatt aaaaccagct atatcaacat ataatgcctt 60
tttattcatg ttaatgacca acgtaagtgg ctagccttta tgtcttattt atcttcatgt 120
<210> 82
<211> 120
<212> DNA
<213> Artificial sequence
<400> 82
tatgttagtt tacatacagg ggtgtatgtc tctgtgctgt ccccttctcc tgccttcatt 60
ttaaaatgca tccatgggtc ctccgtgttt cctttggcca tgccacatat atagactcag 120
<210> 83
<211> 120
<212> DNA
<213> Artificial sequence
<400> 83
tttggccttc atgatatcgc ctgatttttg aggactgtat cacagtgata tgtatttgtg 60
gtaatctcat ttgttggttg tacatctgat cctttcctca acatggcaat tgctgccttt 120
<210> 84
<211> 120
<212> DNA
<213> Artificial sequence
<400> 84
cctaagatag gatcatacaa ctgatcaggg gattgaattt gatcattcat caacatgtgt 60
ctctgaattt tattcagtag ttgtcattgc tctttggttt agaccaagaa aaaggaaatc 120
<210> 85
<211> 120
<212> DNA
<213> Artificial sequence
<400> 85
cccccttttc atgtattcct tggtttgagg acatgactcc tgtaagggag aggaaaggga 60
gatgcttcct gtttgaactg cagtgaattc acggttcctg tttcaccact ccaaacctta 120
<210> 86
<211> 120
<212> DNA
<213> Artificial sequence
<400> 86
tggcgactca cacacacatt cctcttttct gttactgcca aaggttcggg tttagtacac 60
ttcagttcca ctcaagcatt gaaaaggttc tcgtggagtc tggggcgtgc ccagtgaaaa 120
<210> 87
<211> 120
<212> DNA
<213> Artificial sequence
<400> 87
gatggggact ttttaattgt ccacagacct ctctatacct gctttgcaaa aattacaatg 60
gagtaactat ttttaaagct tatttttcaa ttcataaaaa agacatttat tttcagtcaa 120
<210> 88
<211> 120
<212> DNA
<213> Artificial sequence
<400> 88
atggatgatg tctccctctt ttcccctatt ctcaatgttt gcttgaatct tttattattt 60
tttttaattc tcccccatac ccacttcctg atactttggt tctctttcct gctcaggtcc 120
<210> 89
<211> 120
<212> DNA
<213> Artificial sequence
<400> 89
cttcatttgt actttggagt ttttctcatg taaatttgta taacagaaaa tattgttcag 60
tttggataga aagcatggag aataaaaaaa gatagctgaa attcagattg aagaaattta 120
<210> 90
<211> 120
<212> DNA
<213> Artificial sequence
<400> 90
gaaactattt tatttaaatg gaggctgagt ggtgagtaga tgtgtcttgg tatggaggaa 60
aagggcatgc tgcatcttct tcctgacctc cggggtctct ggccttttgt ttccttgctc 120
<210> 91
<211> 120
<212> DNA
<213> Artificial sequence
<400> 91
tttctgtgta aagttattta aaaactgtat tatataaaag gcaaaaaaag ttctatgtac 60
ttgatgtgaa tatgcgaata ctgctataat aaagattgac tgcatggaga agtcttcatc 120
<210> 92
<211> 120
<212> DNA
<213> Artificial sequence
<400> 92
actgaggggt ctgtctaacc aagcaggcta gatagtgctg gcacacattg ccttctttct 60
cattgggtcc agcaatgaag ataagtgttt gggttttttt tttttcctcc acaatgtagc 120
<210> 93
<211> 120
<212> DNA
<213> Artificial sequence
<400> 93
aaattctcag gaaatacagt ttatatcttc ctcctatgct cttccagtca ccaactactt 60
atgcggctac tttgtccagg gcacaaaatg ccgtggcagt atctaactaa acccccacaa 120
<210> 94
<211> 120
<212> DNA
<213> Artificial sequence
<400> 94
aactgcttaa taacagtttt gaatgtgaga aatttagata atttaaatat aaggtacagg 60
ttttaatttc tgagtttctt cttttctatt tttattaaaa agaaaataat tttcagattt 120
Claims (6)
1.一种用于BRAF基因全外显子二代测序的探针组合物,其特征在于:包括SEQ ID NO:01至 SEQ ID NO:94所示的序列。
2.一种用于BRAF基因全外显子二代测序的试剂,其特征在于:包括权利要求1所述的用于BRAF基因全外显子二代测序的探针组合物及二代测序所需试剂。
3.一种用于BRAF基因全外显子二代测序的控制系统,其特征在于,包括:
检测模块,采用权利要求2所述的用于BRAF基因全外显子二代测序的试剂进行二代测序;
显示模块,将检测结果转换为可视化数据,并将可视化的数据于显示设备进行展示;
分析模块:基于预设的质控程序对检测结果进行验证,并将验证结果于同一显示设备进行展示。
4.根据权利要求3所述的用于BRAF基因全外显子二代测序的控制系统,其特征在于:
运行所述检测模块,包括以下步骤:
步骤一:文库构建,包括gDNA浓度检测及稀释、gDNA片段化、末端修复和A碱基的添加、接头的连接及连接产物的纯化、文库片段筛选、PCR扩增及扩增产物的纯化、终文库QC;
步骤二:杂交捕获,包括文库Pooling、变性与杂交、捕获洗脱、PCR扩增与扩增产物的纯化;
步骤三:上机测序,经过稀释、变性等步骤,调整至符合测序仪的标准,并按测序仪的标准操作进行测序、输出检测结果。
5.根据权利要求4所述的用于BRAF基因全外显子二代测序的控制系统,其特征在于:
所述显示模块将步骤三中的检测结果转化为包括“7号染色体概图”、“测序结果”、“参考序列”三项数据的图表。
6.根据权利要求5所述的用于BRAF基因全外显子二代测序的控制系统,其特征在于:
所述分析模块直接以所述显示模块转化得到的图表为原始数据,与预设的数据进行对比,并向所述显示模块输出检测结果。
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CN107723352A (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肝癌驱动基因高通量检测方法 |
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