这种方法联合运用了MBD柱层析法及MS-RE法,这就避免了单用MBD引起的非特异性捕获及MS-RE不完全消化引起的假阳性的问题。因此,在快速、简便检测的同时,亦保证了结果的特异性和敏感性。缺点是:需要使用限制性内切酶,因此不同程度地受到内切酶识别位点的限制。
研究甲基化的方法之多,从一个方面说明了甲基化研究难度之大,也从另一个方面说明这些方法都存在着一定的限制。面对具体问题,选择最合适的解决方法就显得尤为重要。首先,根据研究目的选择合适方法:是研究整体水平的甲基化还是特定位点的甲基化,或是要发现全中新的甲基化位点;其次,根据客观条件筛选方法,如:目标的序列是否已知,是定量研究还是定性研究,样本来源及数量如何,是否需要高通量的样本检测方法;最后,全面分析,选取敏感、可靠、经济、简便的方法,以达到理想的效果。随着甲基化研究的不断深入,甲基化分析技术将逐步完善。完善的研究技术将提供强有力的技术支持,从而为表观遗传、胚胎发育、基因印记及肿瘤研究提供一些新的思路。
参考文献 :
[1]Wu C T,Morris J R.Genes,genetics and epigenetics: a correspondence [J].Science,2001,293: 1103-1105.
[2]黄庆,郭颖,府伟灵.人类表观计划[J].生命的化学,2004,24(2): 101-102.
[3]Dahl C,Guldberg P.DNA methylation analysis techniques [J].Biogerontology,2003,4(4): 233-250.
[4]董玉玮,侯进慧,朱必才等.表观遗传学的相关概念和研究进展[J].生命的化学,2005,22(1): 1-3.
[5]武立鹏,朱卫国.DNA 甲基化的生物学应用及检测方法进展[J].中国检验医学杂志,2004,27(7): 468-474.
[6]Riggs A D,Jones P D.5-methylcytosine,gene regulation and cancer [J].Adv Cancer Res,1984,40: 1-30.
[7]Bird A P.CpG-rich islands and the function of DNA methylation [J].Nature,1986,321: 209-213.
[8]Cottrell S E.Molecular diagnostic applications of DNA methylation technology [J].Clin Biochem,2004,Jul,37(7): 595-604.
[9]Jones P A,Baylin S B.The fundamental role of epigenetic events in cancer [J].Nat Rev Genet,2002,Jun.3(6): 415-28.
[10]张永彪,褚嘉�.表观遗传学与人类疾病的研究进展 [J].遗传,2005,27(3): 466-472.
[11]Feinberg A P,Tycko B.The history of cancer epigenetic [J].Nat Rev Cancer,2004,4(2): 143-153.
[12]黄琼晓,金帆,黄荷凤.DNA 甲基化的研究方法学 [J].国外医学遗传学分册,2004,27(6): 354-358.
[13]Nuovo G J,Plaia T W,Belinsky S A,et al.In situ detection of the hypermethylation- induced inactivation of the p16 gene as an early event in oncogenesis [J].Proc Natl Acad Sci USA,1999,96: 12754-12759.
[14]Soares J,Pinto A E,Cunha C V,et al.Global DNA hypomethylation in breast carcinoma: correlation with prognostic factors and tumor ptogression [J].Cancer,1999,85: 112-118.
[15]Shinichi Toyooka,Nobuyoshi Shimizu.Models for studying DNA methylation in human cancer: a review of current status [J].Drug discovery today: Disease Model,2004,1(1): 37-42.
[16]Cui H,Horon I L,Ohlsson R,Hamilton S R,Feinberg A P.Loss of imprinting in normal tissue of colorectal cancer patients with microsatellite instability [J].Nat Med Nov,1998,4(11): 1276-80.
[17]Uhlmann K,Rohde K,Zeller C,et al.Distinct methylation profiles of glioma subtypes.[J] Int Cancer,2003,Aug.10,106(1): 52-59.
[18]Kuo K C,McCune R A,Gehrke C W,et al.Quantitative reversed-phase high performance liquid chromatographic determination of major and modified deoxyribonucleosides in DNA [J].Nucleic Acids Res,1980,8: 4763-4776.
[19]Fraga M F,Uriol E,Borja D L,et al.High-performance capillary electrophoretic method for the quantification of 5-methyl 2-deoxycytidine in genomic DNA : application to plant,animal and human cancer tissues [J].Electrophoresis,2002,23: 1677-1681.
[20]Oefner,P J,Bonn G K,Huber C G,et al.Comparative study of capillary zone electrophoresis and high-performance liquid chromatography in the analysis of oligonucleotides and DNA .[J].Chromatogr,1992,625(2): 331-3401.
[21]邓大君,邓国仁,吕有勇等.变性高效液相色谱 法检测CpG岛胞嘧啶甲基化 [J].中华医学杂志,2001,80(2),158-1611.
[22]Wu J,Issa J,Hermen J,et al.Expression of an exogenous eukaryotic DNA methyl transferase gene induces transformation of NIN3T3 ceils [J].Proc Natl Acad Sci USA,1993,90(19): 8891-8895.
[23]Oakeley E J,Podesta A,Jost J P.Developmental changes in DNA methylation of the two tobacco pollen nuclei during maturation [J].Proc Natl Acad Sci USA,1997,94: 11721-11725.
[24]Oakeley E J,Schmitt F,Jost J P.Quantification of 5-methylcytosine in DNA by the chloroacetaldehyde reaction [J].Biotechniques,1999,27: 744-6,748-50,752.
[25]Frommer M,McDonald L E,Millar D S,et al.A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands [J].Proc Natl Acad Sci USA,1992,89: 1827-1831.
[26]朱燕.DNA 的甲基化的分析与状态检测 [J].现代预防医学,2005,32(9): 1070-1073.
[27]沈佳尧,侯鹏,祭美菊等.DNA 甲基化方法研究现状 [J].生命的化学,2003,23(2): 149-151.
[28]Herman J G,Graff J R,Myohanen S,et al.Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands [J].Proc Natl Acad Sci USA,1996,Sep 3.93(18),9821-9826.
[29]Kuppuswamy M N,Hoffmann J W,Kasper C K,Spitzer S G,Groce S L,Bajaj S P.Single nucleotide primer extension to detect genetic diseases: experimental application to hemophilia B (factor IX)and cystic fibrosis genes [J].Proc Natl Acad Sci USA,1991,88: 1143-1147.
[30]Gonzalgo ml,Jones P A.Rapid quantitation of methylation differences at specific sites using methylation-sensitive single nucleotide primer extension (Ms-SNuPE)[J].Nucleic Acids Res,1997,25: 2529-2531.
[31]Xiong Z,Laird P W COBRA: a sensitive and quantitative DNA methylation assay [J].Nucleic Acids Res,1997,25: 2532-2534.
[32]Maekawa M,Sugano K,Kashiwabara H,et al.DNA