NGS 是一種識別和確認未知致病菌的前景廣闊的技術，然而其在生物防御和公共健康應用等方面的時效性，卻往往因為缺乏快速、有效、可靠的自動DNA樣品制備方法而受到限制。為了突破這種限制，Kim 等設計了一種基于流體分布元件的數字微流體(DMF) 平臺，使得多子系統模塊能夠進入自動NGS庫樣品制備系統。通過一種新型毛細管接口，可以實現液體在DMF設備與外部流體模塊間的高重復性轉移，使得流路連續，且小滴樣品能夠在一個完整的系統內得到處理。這里，該技術強調了DMF元件平臺和NGS 樣品制備流程中自動運行毛細管接口的效用。將毛細管接口與一種嵌入式非接觸電導檢測器連接，DMF設備實現了目標分析物從樣品流到小液滴的閉環自動片段采集。NGS 中重復次數最多的緩沖液交換與樣品清洗通過使用一種磁珠解決，實現了DMF平臺上平均DNA回收率為(80±4.8)%。
先天性糖基化缺陷是由于機體缺少糖基化作用，主要影響到N 相關途徑造成的。至少40% 的先天性糖基化缺陷病人在診斷過程中沒有得到分子水平的確認。Jones 等通過已經導致先天性糖基化缺陷基因的下一代測序驗證的研究，從分子水平提高了診斷先天性糖基化缺陷的水平。12例未知樣本致病突變的先天性糖基化缺陷病人作為陽性對照進行NGS驗證，分別采用RainDance 與Fluidigm 平臺( dPCR) 進行序列富集和目標擴增，SOLiD平臺用于測序和擴增產物。進而通過NextGENe 進行生物信息學分析。結果表明，12個陽性對照的致病突變通過NGS都得以確認。在病人診斷過程中，NGS的發展使實驗室診斷多基因與分析逐個基因相比成本消耗更低、速度更快、效率更高。臨床實驗室的下一代測序數據分析結果同樣支持這項技術，推廣使用這項技術至關重要。
4 總結與展望
生物學的基礎研究和分子技術的前進伴隨著更精確和更靈敏的測量技術發展。值得一提的是，dPCR具有測量獨立性與無需任何校準物的特點。因此，該技術是潛在的核酸測量基準方法，并從原理上為核酸計量提供了保證。相比其他方法，dPCR作為絕對定量方法能準確定量目標DNA和提供可靠的定量數據。商業化dPCR 儀器( 如Fluidigm 公司的BioMark System)的大量出現進一步推動和擴大了該技術的發展和應用范圍。
dPCR技術及其應用凸顯了單分子定量技術的潛力。不久，微流體dPCR就會突破反應速度和體積的限制，實現自動化和高通量的應用。核酸測序將是基于dPCR的單分子擴增技術最重要的應用領域。dPCR的克隆擴增可以減少下一代測序的時間和成本，并使個人基因組測序得以實現。我們期望在不遠的將來，這項技術的發展將對單分子核酸擴增領域產生深遠影響，在分子生物學和醫學等基礎研究和應用方面發揮更大的作用。
參考文獻
[1] Keer J T , Birch L. Essentials of Nucleic Acid Analysis: A Robust Approach. London: Royal Society of Chemistry, 2008
[2] Zhang C, Xing D. Single-molecule DNA amplification and analysis using microfluidics.Chem Rev, 2010, 110 (8): 4910-4947
[3] Vogelstein B, Kinzler K W. Digital PCR. Proc Natl Acad Sci USA,1999,96(16): 9236-9241
[4] Bhat S, Herrmann J, Armishaw P, et al. Single molecule detection in nanofluidic digital array enables accurate measurement of DNA copy number. Anal Bioanal Chem, 2009, 394(2): 457-467
[5] Bustin S A, Benes V, Garson J A, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem , 2009, 55 (4 ): 611-622
[6] Burns M, Burrell A, Foy C. The applicability of digital PCR for the assessment of detection limits in GMO analysis. European Food Research and Technology, 2010, 231 (3 ): 353-362
[7] Sykes P J , Neoh S H , Brisco M J, et al. Quantitation of targets for PCR by use of limiting dilution. Biotechniques, 1992,13(3): 444
[8] Olga K, Irina L, James B, et al. Nanoliter scale PCR with TaqMan detection. Nucleic Acids Res, 1997,25 (10 ): 1999-2004
[9] Devin D, Hai Y, Giovanni T, et al. Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations . Proc Natl Acad Sci USA, 2003,100 (15): 8817-8822
[10] Diehl F, Li M, Dressman D, et al. Detection and quantification of mutations in the plasma of patients with colorectal tumors. Proc Natl Acad Sci USA, 2005, 102 (45): 16368-16373
[11] Frank D, Meng L, Yiping H, et al. BEA Ming: single-molecule PCR on micro particles in water-in-oil emulsions. Nat Methods, 2006, 3 (7): 551-559
[12] Liu J , Hansen C, Quake S R. Solving the " world-to-chip" in terface problem with a microfluidic matrix. Anal Chem,2003, 75(18): 4718-4723
[13] Dube S, Qin J , Ramakrishnan R. Mathematic alanalysis of copy number variation in a DNA sample using digital PCR on a nanofluidic device. PLoS One , 2008, 3 (8): e2876
[14] Heyries K A, Carolina T, Michael C, et al. Megapixel digital PCR. Nat Methods, 2011,8 (8): 649-651
[15] Sanders R, Jim F, Huggett, et al. Evaluation of digital PCR for absolute DNA quantification. Anal Chem, 2011, 83(17): 6474-6484
[16] Heid C A, Stevens J, Livak K L, e t al. Real time quantitative PCR. Genome Res, 1996, 6 (10): 986-994
[17] Morrison T B, Weis J J, Wittwer C T. Quantification of low-copy transcripts by continuous SYBR Green I monitoring during amplification. Biotechniques, 1998, 24(6): 954-958 , 960 , 962
[18] 趙錦榮, 白玉杰, 王勝春, 等.新型MGB探針在沙眼衣原體實時PCR檢測中的應用. 生物化學與生物物理進展, 2003, 30(3): 466– 470 Zhao J R, Bai Y J , Wang S C, et al. Prog Biochem Biophys, 2003,30(3): 466-470
[19] Corbisier P, Somanath B, Lina P, et al. Absolute quantification of genetically modified MO N810 maize(Zea mays L.) by digital polymerase chain reaction.Anal Bioanal Chem, 2009, 396 (6):2143-2150
[20] Terry C F, Shanahan D J , Ballam L D, et al. Real-time detection of genetically modified soya using Lightcycler and ABI 7700 platforms with TaqMan , Scorpion, and SYBR GreenⅠ chemistries. J AOAC international, 2002, 85 (4): 938- 944
[21] Pohl G, Shih I M. Principle and applications of digital PCR. Expert Re v Mo l Di agn , 2 00 4, 4(1 ): 41- 47
[22] Oehler V G, Qin J, Ramakrishnan R, et al. Absolute quantitative detection of ABL tyrosine kinase domain point mutations in chronic myeloid leukemia using a novel nanofluidic platform and mutation-specific PCR. Leukemia, 2008, 23(2): 396-399
[23] Chan M, Mei W C, Ting W L, et al . Evaluation of nanofluidics technology for high-through put SNP genotyping in a clinical setting. J Mol Diagn, 2011, 13 (3): 305-312
[24] Ropers H H. New perspectives for the e lucidation of genetic disorders . Am J Hum Genet,2007, 81(2 ): 199-207
[25] Lupski J R. Genomic rearrangements and sporadic disease. Nat
Genet, 2007, 39: S43-S47
[26] Slamon D J, Clark G M , Wong S G, et al . Human breast cancer:correlation of relapse and survival with amplification of the HER-2 /neu oncogene .Science, 1987, 235 (4785): 177
[27] Qin J, Jones R C, Ramakrishnan R. Studying copy number variations using a nanofluidic platform. Nucleic Acids Res, 2008,36 (18): e116
[28] Lo Y M D. Noninvasive prenatal detection of fetal chromosomal aneuploidies by maternal plasman ucleic acid analysis: a review of the current state of the art. BJOG: An International J Obstetrics&Gynaecology, 2009, 116 (2): 152-157
[29] Lo Y M D, Fiona M F, Chan K C A, et al. Digital PCR for the molecular detection of fetal chromosomal aneuploidy. Proc Natl Acad Sci USA, 2007, 104(32): 13116 -13121
[30] Fan H C, Quake S R. Detection of aneuploidy with digital polymerase chain reaction. Anal Chem, 2007, 79 (19): 7576-7579
[31] Lo Y M, Nancy B Y T , Rossa W K C, et al. Plasma placental RNA allelic ratio permits noninvasive prenatal chromosomal aneuploidy detection. Nat Med , 2007, 13 (2): 218-223
[32] Lun F M, Allen C K, Yeung L T, et al. Microfluidics digital PCR reveals a higher than expected fraction of fetal DNA in maternal plasma. Clin Chem, 2008, 54(10): 1664-1672
[33] 郭奇偉,周裕林.唐氏綜合征的無創產前診斷研究進展. 中國優生與遺傳雜志, 2010,18(4): 137-139
Guo Q W, Zhou Y L. Chin J Birth Health & Hered, 2010, 18(4):137-139
[34] BioMark. BioMark Advanced Development Protocol 10. Absolute quantitation using the digital array; Fluidigm (Fluidigm Corporation, S.F)
[35] White A K, Michael V,Oleh I P, et al. High-through put micro fluidic single-cell RT-qPCR. Proc Natl Acad Sci USA, 2011,108(34): 13999-14004
[36] Spurgeon S L, Jones R C, Ramakrishnan R. High throughput gene expression measurement with real time PCR in a microfluidic dynamic array. PLoS One , 2008, 3(2): e1662
[37]Warren L,David B, Irving L W ,et al.Transcription factor profiling in individual hematopoietic progenitors by digital RT-PCR. Proc Natl Acad Sci USA , 2006,103(47):17807-1 7812
[38] Guo G , Huss M, Tong G Q, et al. Resolution of cell fated ecisions revealed by single-cell gene expression an alysis from zygote to blastocyst. Dev Cell , 2011,18 (4): 675-685
[39] Ottesen E A , Jong W H, Stephen R Q, et al. Microfluidic digital PCR enables multigene analysis of individual environmental bacteria.Science ,2006,314 (5804): 1464-1467
[40] Tadmor A D, Elizabeth A O , Jared R L, et al. Probing in dividual environmental bacteria for viruses by using microfluidic digital PCR. Science, 2011,333(6038): 58-62
[41] Braslavsky I, Hebert B, Kartalov E, et al. Sequence information can be obtained from single DNA molecules. Proc Natl Acad Sci USA,2003, 100 (7): 3960-39 64
[42] Eid J, Fehr A, Gray J, et al. Real-time DN A sequencing from single polymerase molecules . Science, 2009, 323 (5910): 133-138
[43] Harris T D, Buzby P R, Babcock H, et al. Single-molecule DNA sequencing of a viral genome. Science, 2008, 320(5872 ): 106 -109
[44] White R A , Blainey P C, Fan H C, et al . Digital PCR provides sensitive and absolute calibration for high throughput sequencing.BMC Genomics, 2009, 10:116
[45] Zernant J , Schubert C, Im K, et al. Analysis of the ABCA4 gene by next-generation sequencing . Invest Ophthalmol Vis Sci, 2011,52(11): 8479-8487
[46] Kim H , Bartsch M S, Renzi R F, et al. Automated digital microfluidic sample preparation for next-generation DNA sequencing. J Lab Autom , 2011, 16(6): 405-414
[47] Jones M A, Bhide S, Chin E, et al. Targeted polymerase chain reaction-based enrichment and next generation sequencing for diagnostic testing of congenital disorders of glycosylation. Genet Med, 2011,13 (11): 921-932