基于基因芯片的荧光分析方法在甲状腺乳头状癌相关BRAFV600E突变高灵敏及特异性检测中的应用
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摘要
研发了一种基于DNA芯片(DNA microarray)的荧光分析方法,并将其用于检测甲状腺乳头癌相关的BRAFV600突变。本方法采用三链杂交体系,首先将单链DNA(ssDNA)捕获探针固定在微阵列芯片基底上,制备DNA芯片;以其为反应平台,加入ssDNA目标物与ssDNA捕获探针进行杂交;最后加入荧光分子Cy5的修饰ssDNA标记ssDNA目标物,检测荧光信号。在优化的实验条件下,本方法对BRAFV600E突变型ssDNA目标物的检测灵敏度达到亚纳摩尔级(0.25 nmol/L),且具有3个数量级的线性范围,并能够从突变型ssDNA目标物和野生型ssDNA目标物的混合物中区分出低至0.1%的BRAFV600E突变型ssDNA目标物。利用本方法对15例临床甲状腺组织样本的BRAFV600E突变水平进行了分析,实验结果与常规Sanger测序法检测结果一致,表明本方法具有良好的实用性。
A DNA microarray-based fluorescence assay was developed for profiling BRAFV600 mutations through formation of three single-stranded DNA(ssDNA) sequences hybridization systems on microarray. In this assay, the target ssDNAs were recognized by correspondent capturing oligonucleotide probes on microarray, followed by hybridization with a Cy5 modified ssDNA for fluorescence detection. Under the optimal experimental conditions, the DNA microarray-based fluorescence assay achieved a limit of detection(S/N) at sub-nanomolar level for the target ssDNA with BRAFV600 E mutation, and could easily differentiate as low as 0.1% BRAFV600 E mutation sequence in the mixture of target ssDNAs. The profile results of BRAFV600 E mutation level in 15 clinical thyroid tissue samples demonstrated that the DNA microarray-based fluorescence assay had good practicability.
A DNA microarray-based fluorescence assay was developed for profiling BRAFV600 mutations through formation of three single-stranded DNA(ssDNA) sequences hybridization systems on microarray. In this assay, the target ssDNAs were recognized by correspondent capturing oligonucleotide probes on microarray, followed by hybridization with a Cy5 modified ssDNA for fluorescence detection. Under the optimal experimental conditions, the DNA microarray-based fluorescence assay achieved a limit of detection(S/N) at sub-nanomolar level for the target ssDNA with BRAFV600 E mutation, and could easily differentiate as low as 0.1% BRAFV600 E mutation sequence in the mixture of target ssDNAs. The profile results of BRAFV600 E mutation level in 15 clinical thyroid tissue samples demonstrated that the DNA microarray-based fluorescence assay had good practicability.
引文
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2 Agianian B,Gavathiotis E.J.Med.Chem.,2018,61(14):5775-5793
3 Karoulia Z,Gavathiotis E,Poulikakos P I.Nat.Rev.Cancer,2017,17(11):676-691
4 Davies H,Bignell G R,Cox C,Stephens P,Edkins S,Clegg S,Teague J,Woffendin H,Garnett M J,Bottomley W,Davis N,Dicks E,Ewing R,Floyd Y,Gray K,Hall S,Hawes R,Hughes J,Kosmidou V,Menzies A,Mould C,Parker A,Stevens C,Watt S,Hooper S,Wilson R,Jayatilake H,Gusterson B A,Cooper C,Shipley J,Hargrave D,Pritchard-Jones K,Maitland N,Chenevix-Trench G,Riggins G J,Bigner D D,Palmieri G,Cossu A,Flanagan A,Nicholson A,Ho J W,Leung S Y,Yuen S T,Weber B L,Seigler H F,Darrow T L,Paterson H,Marais R,Marshall C J,Wooster R,Stratton M R,Futreal P A.Nature,2002,417(6892):949-954
5 Chen H C,Sierra J,Yu L J,Cerchio R,Wall B A,Goydos J,Chen S.Oncotarget,2018,9(5):5861-5875
6 Lang A H,Drexel H,Geller-Rhomberg S,Stark N,Winder T,Geiger K,Muendlein A.J.Mol.Diagn.,2011,13(1):23-28
7 Weyant G W,Wisotzkey J D,Benko F A,Donaldson K J.J.Mol.Diagn.,2014,16(5):481-485
8 Miotke L,Lau B T,Rumma R T,Ji H P.Anal.Chem.,2014,86(5):2618-2624
9 Hatae R,Hata N,Yoshimoto K,Kuga D,Akagi Y,Murata H,Suzuki S O,Mizoguchi M,Iihara K.PloS One,2016,11(8):e0160489
10 Tu M,Chia D,Wei F,Wong D.Analyst,2016,141(2):393-402
11 Chen D,Qi W,Zhang P,Zhang Y,Liu Y,Guan H,Wang L.Pathology,2018,214(2):303-307
12 Huber F,Lang H P,Glatz K,Rimoldi D,Meyer E,Gerber C.Nano Lett.,2016,16(9):5373-5377
13 Li P,He H,Wang Z,Feng M,Jin H,Wu Y,Zhang L,Zhang L,Tang X.Anal.Chem.,2016,88(1):883-889
14 Du Y,Pothukuchy A,Gollihar J D,Nourani A,Li B,Ellington A D.Angew.Chem.,2017,56(4):992-996
15 Wang Y,Tian K,Shi R,Gu A,Pennella M,Alberts L,Gates K S,Li G,Fan H,Wang M X,Gu L Q.ACS Sens.,2017,2(7):975-981
16 Wu T,Chen W,Yang Z,Tan H,Wang J,Xiao X,Li M,Zhao M.Nucleic Acids Res.,2018,46(4):e24
17 Mukherjee S,Sathanoori M,Ma Z,Andreatta M,Lennon P A,Wheeler S R,Prescott J L,Coldren C,Casey T,Rietz H,Fasig K,Woodford R,Hartley T,Spence D,Donnelan W,Berdeja J,Flinn I,Kozyr N,Bouzyk M,Correll M,Ho H,Kravtsov V,Tunnel D,Chandra P.Cancer Genet.,2017,216-217:1281-1241
18 Kim I J,Kang H C,Jang S G,Ahn S A,Yoon H J,Park J G.J.Mol.Diagn.,2007,9(1):55-63
19 DONG Fen,ZHANG Biao,SHAN Guang-Liang.China Oncol.,2016,26(1):47-52董芬,张彪,单广良.中国癌症杂志,2016,26(01):47-52
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23 Wang Z,Zhang J,Yin Z,Wu S,Mandler D,Zhang H.Nanoscale,2012,4(8):2728-2733
24 Liu X,Wang J Y,Mao X B,Ning Y,Zhang G J.Anal.Chem.,2015,87(18):9352-9359
25 Koirala D,Yu Z,Dhakal S,Mao H.J.Am.Chem.Soc.,2011,133(26):9988-9991
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