DNA測序

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DNA测序DNA sequencing,或譯DNA定序)是指分析特定DNA片段的碱基序列,也就是腺嘌呤(A)、胸腺嘧啶(T)、胞嘧啶(C)與鳥嘌呤的(G)排列方式。快速的DNA测序方法的出现极大地推动了生物学和医学的研究和发现。

在基础生物学研究中,和在众多的应用领域,如诊断,生物技术,法医生物学,生物系统学中,DNA序列知识已成为不可缺少的知识。具有现代的DNA测序技术的快速测序速度已经有助于达到测序完整的DNA序列,或多种类型的基因组测序和生命物种,包括人类基因组和其他许多动物,植物和微生物物种的完整DNA序列。

RNA測序則通常将RNA提取后,反转录为DNA后使用DNA测序的方法进行测序。目前应用最广泛的是由弗雷德里克·桑格发明的Sanger双脱氧链终止法(Chain Termination Method)[1]。新的测序方法,例如454生物科学的方法和焦磷酸测序法

自动化chain-termination DNA测序结果的一个例子.

用途[编辑]

历史[编辑]

基本方法[编辑]

Maxam-Gilbert测序法[编辑]

Sanger测序法[编辑]

Sanger(桑格)双脱氧链终止法是Frederick Sanger于1975年发明的。测序过程需要先做一个聚合酶连锁反应(PCR)。PCR过程中,双脱氧核糖核苷酸可能随机的被加入到正在合成中的DNA片段里。由于双脱氧核糖核苷酸又少了一个原子,一旦它被加入到DNA链上,这个DNA链就不能继续增加长度。最终的结果是获得所有可能获得的、不同长度的DNA片段。目前最普遍最先进的方法,是将双脱氧核糖核苷酸进行不同荧光标记。将PCR反应获得的总DNA通过毛细管电泳分离,跑到最末端的DNA就可以在激光的作用下发出荧光。由于ddATP, ddGTP, ddCTP, ddTTP(4种双脱氧核糖核苷酸)荧光标记不同,计算机可以自动根据颜色判断该位置上碱基究竟是A,T,G,C中的哪一个[2]

高级方法和de novo测序法[编辑]

霰彈槍定序法[编辑]

霰彈槍定序法Shotgun sequencing,又称鸟枪法)是一种广泛使用的为长DNA测序的方法,比傳統的定序法快速,但精確度較差。曾經使用於塞雷拉基因組(Celera Genomics)公司所主持的人類基因組計畫

Bridge PCR[编辑]

新一代测序[编辑]

随着人们对低成本测序的需求与日俱增,推动了高通量测序(或称为新一代测序)的发展,这些技术对测序过程多路复用,同时产生上千或上百万条序列[3][4]。高通量测序技术的目的是降低DNA测序的成本,这个成本比同样可实现测序的染料终止法来得低得多[5]。超高通量测序过程中可同时运行高达500,000次的边合成边测序[6][7][8]

需要根据多个片段序列所重叠的区域将它们全部组装起来。
新一代测序方法的比较 [9][10]
方法 单分子实时测序(Pacific Bio) 离子半导体(Ion Torrent sequencing) 焦磷酸测序(454) 边合成边测序(Illumina) 边连接边测序(SOLiD sequencing) 链终止法(Sanger sequencing)
读长 5,500 bp to 8,500 bp avg (10,000 bp N50); maximum read length >30,000 bases[11][12][13] up to 400 bp 700 bp 50 to 300 bp 50+35 or 50+50 bp 400 to 900 bp
精确度 99.999% consensus accuracy; 87% single-read accuracy[14] 98% 99.9% 98% 99.9% 99.9%
每次运行可获取读段数 50,000 per SMRT cell, or ~400 megabases[15][16] up to 80 million 1 million up to 3 billion 1.2 to 1.4 billion N/A
每次运行耗时 30 minutes to 2 hours [17] 2 hours 24 hours 1 to 10 days, depending upon sequencer and specified read length[18] 1 to 2 weeks 20 minutes to 3 hours
每百万碱基所耗成本(美元) $0.33-$1.00 $1 $10 $0.05 to $0.15 $0.13 $2400
优势 Longest read length. Fast. Detects 4mC, 5mC, 6mA.[19] Less expensive equipment. Fast. Long read size. Fast. Potential for high sequence yield, depending upon sequencer model and desired application. Low cost per base. Long individual reads. Useful for many applications.
劣势 Moderate throughput. Equipment can be very expensive. Homopolymer errors. Runs are expensive. Homopolymer errors. Equipment can be very expensive. Requires high concentrations of DNA. Slower than other methods. Have issue sequencing palindromic sequence.[20] More expensive and impractical for larger sequencing projects.

454生物科学和焦磷酸测序法[编辑]

454测序法由454生物科学发明,是一个类似焦磷酸测序法的新方法。2003年向GenBank提交了一个腺病毒全序列[21],使得他们的技术成为Sanger测序法后第一个被用来测生物基因组全序列的新方法。454使用类似于焦磷酸测序的方法,有着相当高的读取速度,大约为5小时可以测两千万碱基对[22]

正在开发的测序法[编辑]

Nanopore DNA sequencing[编辑]

参考文献[编辑]

  1. ^ http://www.bioon.com/experiment/nua2/89939.shtml
  2. ^ http://en.wikipedia.org/wiki/Chain_termination_method
  3. ^ Hall, Nell. Advanced sequencing technologies and their wider impact in microbiology. J. Exp. Biol. May 2007, 209 (Pt 9): 1518–1525. doi:10.1242/jeb.001370. PMID 17449817. open access publication - free to read
  4. ^ Church, George M.. Genomes for all. Sci. Am. January 2006, 294 (1): 46–54. doi:10.1038/scientificamerican0106-46. PMID 16468433. (需要訂閱才能查看)
  5. ^ 引用错误:无效<ref>标签;未为name属性为pmid18165802的引用提供文字
  6. ^ Kalb, Gilbert; Moxley, Robert. Massively Parallel, Optical, and Neural Computing in the United States. IOS Press. 1992. ISBN 90-5199-097-9. [页码请求]
  7. ^ Ten Bosch, J. R.; Grody, W. W. Keeping Up with the Next Generation. The Journal of Molecular Diagnostics. 2008, 10 (6): 484–492. doi:10.2353/jmoldx.2008.080027. PMC 2570630. PMID 18832462.  编辑open access publication - free to read
  8. ^ Tucker, T.; Marra, M.; Friedman, J. M. Massively Parallel Sequencing: The Next Big Thing in Genetic Medicine. The American Journal of Human Genetics. 2009, 85 (2): 142–154. doi:10.1016/j.ajhg.2009.06.022. PMC 2725244. PMID 19679224.  编辑open access publication - free to read
  9. ^ Quail, Michael; Smith, Miriam E; Coupland, Paul et al. A tale of three next generation sequencing platforms: comparison of Ion torrent, pacific biosciences and illumina MiSeq sequencers. BMC Genomics. 1 January 2012, 13 (1): 341. doi:10.1186/1471-2164-13-341. PMC 3431227. PMID 22827831. open access publication - free to read
  10. ^ Liu, Lin; Li, Yinhu; Li, Siliang et al. Comparison of Next-Generation Sequencing Systems. Journal of Biomedicine and Biotechnology (Hindawi Publishing Corporation). 1 January 2012, 2012: 1–11. doi:10.1155/2012/251364. open access publication - free to read
  11. ^ New Products: PacBio's RS II; Cufflinks | In Sequence | Sequencing | GenomeWeb
  12. ^ After a Year of Testing, Two Early PacBio Customers Expect More Routine Use of RS Sequencer in 2012. GenomeWeb. 10 January 2012. (需註冊)
  13. ^ Pacific Biosciences Introduces New Chemistry With Longer Read Lengths
  14. ^ http://www.nature.com/nmeth/journal/v10/n6/full/nmeth.2474.html
  15. ^ De novo bacterial genome assembly: a solved problem? | In between lines of code
  16. ^ Rasko, David A.; Webster, Dale R.; Sahl, Jason W. et al. Origins of the Strain Causing an Outbreak of Hemolytic–Uremic Syndrome in Germany. N Engl J Med. 25 August 2011, 365 (8): 709–717. doi:10.1056/NEJMoa1106920. open access publication - free to read
  17. ^ Tran, Ben; Brown, Andrew M.K.; Bedard, Philippe L.; Winquist, Eric; Goss, Glenwood D.; Hotte, Sebastien J.; Welch, Stephen A.; Hirte, Hal W.; Zhang, Tong; Stein, Lincoln D.; Ferretti, Vincent; Watt, Stuart; Jiao, Wei; Ng, Karen; Ghai, Sangeet; Shaw, Patricia; Petrocelli, Teresa; Hudson, Thomas J.; Neel, Benjamin G. et al. Feasibility of real time next generation sequencing of cancer genes linked to drug response: Results from a clinical trial. Int. J. Cancer. 1 January 2012: 1547–1555. doi:10.1002/ijc.27817. (需要訂閱才能查看)
  18. ^ van Vliet, Arnoud H.M. Next generation sequencing of microbial transcriptomes: challenges and opportunities. FEMS Microbiology Letters. 1 January 2010, 302 (1): 1–7. doi:10.1111/j.1574-6968.2009.01767.x. open access publication - free to read
  19. ^ Murray, I. A.; Clark, T. A.; Morgan, R. D.; Boitano, M.; Anton, B. P.; Luong, K.; Fomenkov, A.; Turner, S. W.; Korlach, J.; Roberts, R. J. The methylomes of six bacteria. Nucleic Acids Research. 2 October 2012, 40 (22): 11450–62. doi:10.1093/nar/gks891. PMC 3526280. PMID 23034806. 
  20. ^ Yu-Feng Huang, Sheng-Chung Chen, Yih-Shien Chiang, Tzu-Han Chen & Kuo-Ping Chiu. Palindromic sequence impedes sequencing-by-ligation mechanism. BMC systems biology. 2012,. 6 Suppl 2: S10. doi:10.1186/1752-0509-6-S2-S10. PMID 23281822. 
  21. ^ http://www.454.com/about-454/index.asp
  22. ^ http://www.454.com/about-454/index.asp

參見[编辑]