DNA測序

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DNA測序DNA sequencing,或譯DNA定序)是指分析特定DNA片段的鹼基序列,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測序方法的出現極大地推動了生物學和醫學的研究和發現。

在基礎生物學研究中,和在眾多的應用領域,如診斷,生物技術,法醫生物學,生物系統學中,DNA序列知識已成為不可缺少的知識。具有現代的DNA測序技術的快速測序速度已經有助於達到測序完整的DNA序列,或多種類型的基因組測序和生命物種,包括人類基因組和其他許多動物,植物和微生物物種的完整DNA序列。

RNA測序則通常將RNA提取後,反轉錄為DNA後使用DNA測序的方法進行測序。目前應用最廣泛的是由弗雷德里克·桑格發明的Sanger雙脫氧鏈終止法(Chain Termination Method)[1]。新的測序方法,例如454生物科學的方法和焦磷酸測序法

自動化chain-termination DNA測序結果的一個例子.

用途[編輯]

歷史[編輯]

基本方法[編輯]

Maxam-Gilbert測序法[編輯]

Sanger測序法[編輯]

Sanger(桑格)雙脫氧鏈終止法是Frederick Sanger於1975年發明的。測序過程需要先做一個聚合酶連鎖反應(PCR)。PCR過程中,雙脫氧核糖核苷酸可能隨機的被加入到正在合成中的DNA片段里。由於雙脫氧核糖核苷酸多脫了一個氧原子,一旦它被加入到DNA鏈上,這個DNA鏈就不能繼續增加長度。最終的結果是獲得所有可能獲得的、不同長度的DNA片段。目前最普遍最先進的方法,是將雙脫氧核糖核苷酸進行不同熒游標記。將PCR反應獲得的總DNA通過毛細管電泳分離,跑到最末端的DNA就可以在雷射的作用下發出熒光。由於ddATP, ddGTP, ddCTP, ddTTP(4種雙脫氧核糖核苷酸)熒游標記不同,計算機可以自動根據顏色判斷該位置上鹼基究竟是A,T,G,C中的哪一個[2]

高級方法和de novo測序法[編輯]

霰彈槍定序法[編輯]

霰彈槍定序法Shotgun sequencing,又稱鳥槍法)是一種廣泛使用的為長DNA測序的方法,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[編輯]

新一代測序[編輯]

隨著人們對低成本測序的需求與日俱增,推動了高通量測序(或稱為新一代測序)的發展,這些技術對測序過程多路復用,同時產生上千或上百萬條序列[3][4]。高通量測序技術的目的是降低DNA測序的成本,這個成本比同樣可實現測序的染料終止法來得低得多[5]。超高通量測序過程中可同時運行高達500,000次的邊合成邊測序[6][7][8]

需要根據多個片段序列所重疊的區域將它們全部組裝起來。
新一代測序方法的比較 [9][10]
方法 單分子實時測序(Pacific Bio) 離子半導體(Ion Torrent sequencing) 焦磷酸測序(454) 邊合成邊測序(Illumina) 邊連接邊測序(SOLiD sequencing) 鏈終止法(Sanger sequencing)
讀長 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
精確度 99.999% consensus accuracy; 87% single-read accuracy[14] 98% 99.9% 98% 99.9% 99.9%
每次運行可獲取讀段數 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
每次運行耗時 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
每百萬鹼基所耗成本(美元) $0.33-$1.00 $1 $10 $0.05 to $0.15 $0.13 $2400
優勢 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.
劣勢 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生物科學和焦磷酸測序法[編輯]

454測序法由454生物科學發明,是一個類似焦磷酸測序法的新方法。2003年向GenBank提交了一個腺病毒全序列[21],使得他們的技術成為Sanger測序法後第一個被用來測生物基因組全序列的新方法。454使用類似於焦磷酸測序的方法,有著相當高的讀取速度,大約為5小時可以測兩千萬鹼基對[22]

正在開發的測序法[編輯]

Nanopore DNA sequencing[編輯]

參見[編輯]

注釋[編輯]

  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. ^ 引用錯誤:無效<ref>標籤;未為name屬性為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. [頁碼請求]
  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.  編輯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.  編輯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