CN102016069A - 母体血浆中无细胞胎儿dna的两步富集 - Google Patents
母体血浆中无细胞胎儿dna的两步富集 Download PDFInfo
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Abstract
本发明提供了在来自母体的生物试样中富集胎儿核酸的方法。还提供了检测胎儿、肿瘤或赘生物核酸中标志物存在或不存在的方法。所述方法可包括用DNA酶处理所述生物试样,并任选地,对经处理的试样进行全基因组扩增。所述生物试样可为,例如,血样。
Description
尽管胎儿DNA存在于母体血浆中,但其水平相对较低,不能进行常规产前遗传分析。不愿拘于任何理论,我们相信部分胎儿DNA被包裹,并因此受保护而免于血浆内切核酸酶的威胁。我们提出一种结合了两种不同方法的新型富集技术。首先用DNA酶处理分离的血浆以有效减少污染性无细胞母体DNA片段的百分比。随后胎儿序列的增加使用修改的全基因组扩增(WGA)技术实现。
方法:将血浆从母体全血(n=24)中分离,然后用浓度逐渐增加的DNA酶处理。在进行修改的WGA实验方法之前使用Qiagen DNA提取小型试剂盒(Qiagen DNA extraction mini kit)对经DNA酶处理的DNA进行进一步加工以富集较小的DNA片段。胎儿序列的存在使用实时Taqman PCR(RT-PCR)测定β-珠蛋白和DYS1序列水平来加以确证。
结果:在所有DNA酶处理后检测出胎儿DNA序列。在WGA之前和之后均确证具有男性胎儿的全部试样(n=10)中达成了对男性胎儿的正确检测。除了正确的性别确定外,进行了最高量的DNA酶处理以及修改的WGA实验方法的试样(n=7)显示了胎儿序列的显著富集,达到了50%的胎儿DNA。
结论:结果确认了血浆中的胎儿DNA受到保护,并对DNA酶处理导致的降解具有抗性。这些初步数据还提示可达成最理想水平的DNA酶处理,其允许使用WGA进行进一步富集。观察到DNA酶主要消除母体序列,这提示无细胞胎儿DNA与其母体对应物的包裹方式不同,从而允许对胎儿序列的优先富集。
背景介绍
产前遗传诊断一直依赖侵入性方法,如羊膜腔穿刺术或绒毛膜取样(chorionic villous sampling,CVS)。尽管这些方法多年来提供了可靠的结果,其仍旧对胎儿具有些微的风险(1)。自从在母体血浆中发现了可扩增的胎儿无细胞核酸(2),就有许多研究以确定临床非侵入性产前遗传测试的潜力为目标。尽管这些研究中的许多都显示出很大的希望,但是胎儿DNA的小存在量使其难以在临床上实施。因此,我们着眼于改善胎儿DNA的分离和富集方法。不愿拘于任何理论,相信这些循环的核苷酸是胎儿细胞经历凋亡的结果(3)。在血浆中(已知含有核酸酶)的无细胞DNA和RNA的相对稳定性表明这些核酸在具膜小泡(membrane bound vesicle)中进行循环,所述小泡的形成是程序性细胞死亡的结果(4)。这些胎儿DNA片段亦可在大小上区别于母体片段,胎儿片段(<300bp)通常小于母体片段(>500bp)(5;JorgezC等,2007)。
我们描述了一种新型两步富集胎儿片段的方法。第一阶段涉及用DNA酶处理总母体血浆(母体和胎儿DNA片段均包含在内)。鉴于胎儿片段更加稳定,并可能由具膜的凋亡小体(apoptotic body)包裹,我们假设DNA酶处理会耗尽全部(未包裹的)母源序列。第二阶段涉及修改的全基因组扩增(WGA)实验方法,其设计为扩增较小的片段,推定为胎儿片段。
方法
经过IRB的批准和书面同意,收集了总共24个全血试样(10个确证为怀有男性,平均胎龄是18 1/7周,范围为11 4/7到25 2/7周;12个确证为怀有女性,平均胎龄是20 1/14周,范围为9 6/7到37 4/7周;以及2个未怀孕的对照,一个男性和一个女性)。对于每一个,对ACD真空采集管(vecutainer)中抽取的大约30ml的血液进行处理,通过在800g下10min的初始离心将血浆与细胞级分分离。去除血浆级分,并再一次在16000g下离心10min以进一步去除任何污染的细胞颗粒。随即将该级分以800μl的等分试样冷冻,之后解冻以供同时批量处理(simultaneous batch process)。将每份800μl的血浆试样在室温下解冻,随后对其进行不同浓度的(未处理、1μl、5μl、10μl、30μl的1单位/μl)DNA酶(Promega,Cat#M6101)处理。将试样在37℃下温育1小时,然后添加终止溶液。随后使用Qiagen QiAamp Blood Mini Kit(Cat#51106)对试样进行DNA提取,并洗脱得到终体积100μl。随后使用稍作修改的GenomePlexComplete Whole Genome Amplification(WGA)Kit(Sigma,Cat #WGA2-50rxn)的实验方法施用于七个母体血样(四个确证为男性胎儿,而三个确证为女性胎儿(4 confirmed males,and 3 confirmed females))。我们对该方法进行了修改,首先由于靶序列的预期大小省略了生产商建议的断裂温育。其次将扩增步骤的循环数增加到20,而不是建议的14。扩增的试样储存于4℃,直至进行RT-PCR分析以供检测并量化β-珠蛋白(BGLO354F:GTG CAC CTG ACT CCT GAG GAG A,BGLO455R:CCT TGA TAC CAA CCT GCC CAG)(6)和DYS1(DYS1F:TCC TGC TTA TCC AAA TTC ACC AT,DYS1R:ACT TCC CTC TGA CAT TAC CTG ATA ATT G)(7)(Applied Biosystems 7700,Foster City,CA)。富集水平基于按照DYS1对β-珠蛋白的比例计算出的%胎儿DNA来确定。β-珠蛋白代表分离DNA的总量(母体的和胎儿的),而在确证为怀有男性胎儿时,DYS1代表试样中存在的胎儿DNA的量。
结果
在对未怀孕对照试样进行初始DNA酶处理之后,β-珠蛋白和DYS1的水平作为添加酶的量的函数均有所减少。两者均被DNA酶处理有效的消除(图1)。在母体试样中,尽管检测出的β-珠蛋白水平下降,但这些水平即使经更加严苛的DNA酶处理仍保持不变(对于男性胎儿母体的情况为916.5±91.2Geq/ml,而对于女性胎儿母体的情况为610.5±389.62Geq/ml)。未在任何已知怀有女性胎儿的情况下检测到假阳性的DYS1水平。然而,在已知怀有男性胎儿的情况中(n=10),在所有的处理增量下均100%的检测出了DYS1序列(0μl:127.4±72.3Geq/ml,1μl:71.4±57.3Geq/ml,5μl:71±58.6Geq/ml,10μl:57.1±46.6Geq/ml,30μl:154±179.6Geq/ml)。
对七个经DNA酶处理的母体试样(三个女性胎儿和四个男性胎儿)进行了WGA。尽管在所有试样中检测出了胎儿序列,但是仅在接受30μl DNA酶处理的试样中观察到了胎儿DYS1序列水平的增加(0μl:1979±2083.9Geq/ml,1μl:1.62±1.6Geq/ml,5μl:723.5±875.7Geq/ml,10μl:0.83±1.22Geq/ml,30μl:7025±4381Geq/ml)。如此表明更加严格的DNA酶处理对将母体序列(基于β-珠蛋白)消减到不以能够在与DYS1序列扩增的竞争中胜出的水平存在的程度而言是必要的。在接受30μl DNA酶继以修改的WGA实验方法的试样中,我们能够达成均值为49.96%的胎儿DNA。在未经DNA酶处理而仅进行了修改的WGA的试样中,试样间的平均胎儿DNA百分比为11.16%。经1、5、10μl的1单位/μl DNA酶处理的试样在WGA后分别具有平均值为0.01%、3.5%和0.01%的胎儿DNA。在WGA前,所述试样具有从0.05%到0.17%范围的百分比。
讨论
我们的结果说明无细胞胎儿DNA对由DNA酶造成的降解具有抗性,支持了无细胞胎儿DNA包裹于具膜小泡中的假说。我们有效地去除了母体序列,以及任何可能导致假阳性结果的污染序列。无细胞胎儿的DNA水平在患者试样中相对对照试样持续不变。这一发现确证胎儿序列对降解具有抗性,且与母体序列相比以不同的方式受到保护或包裹。β-珠蛋白水平代表总DNA(母体的和胎儿的),因此无法检测出β-珠蛋白的完全消化并不令人惊讶,因为其一部分很可能是胎儿的。胎儿序列似乎具有独特的分子特征,将其与母体序列区分开来,并使富集成为可能。检测出所有的男性案例且无假阳性表明DNA酶处理在消除不需要的母体序列中具有新用途,所述母体序列在循环中进一步发生了降解。
GenomePlexComplete Whole Genome Amplification Kit(Sigma-Aldrich)通过首先随机断裂(fragment)DNA,并随即在其末端附加共有序列来扩增基因组DNA,所述共有序列是用于通过聚合酶链式反应(PCR)扩增所有片段。我们消除了所述方法中的断裂步骤以阻止较大的母体序列被断裂从而随后被扩增。这潜在地为小的事先存在(pre-existing)的断裂胎儿序列在扩增过程中提供了优势。WGA增加了经受最严格DNA酶处理的试样中胎儿对母体的比例。在WGA前,胎儿DNA的平均%在所有处理水平下均小于1%。然而,在经受30μl DNA酶以及修改的WGA实验方法的试样中,得到了50%的胎儿DNA,表明其为在母体血浆中富集无细胞胎儿DNA的可行方法。基于β-珠蛋白水平,DNA酶似乎减少了试样中存在的母体序列的量,使胎儿序列得以扩增。母体核酸的降解阻止了其在PCR反应中与胎儿序列的扩增竞争。经受较温和(1、5、10μl)DNA酶处理的试样在WGA后与未经DNA酶处理的试样相比较显示出较低的胎儿对母体的比例。一个可能的解释是,在这些试样中,较大的母体序列被降解,但未完全被消除,产生替代原WGA实验方法中的断裂步骤的效果,从而允许对所述母体序列更有效的扩增。
我们描述的组合方法允许我们克服两个限制非侵入性产前DNA遗传测试的主要难题:胎儿对母体比例低,以及胎儿DNA的量小。总体而言,该新型两步富集方法在扩增后的选择性富集方面显示了很大的潜力。
鸣谢
NIH研究基金
参考文献
1.Tabor A,Philip J,Madsen M,Banq J,Obel EB,Pedersen B.Randomised controlled trial of genetic amniocentesis in 4606 low-risk women.Lancet 1986;8493:1287-93.
2.Lo YM,Corbetta N,Chamberlain PF,Rai V,Sargent IL,Redman CW and Wainscoat JS.Presence of fetal DNA in matemal plasma and serum.Lancet 1997;350:485-7.
3.Bischoff FZ,Lewis DE,Simpson JL.Cell-free fetal DNA in maternal blood:kinetics,source and structure.Human Reproductive Update 2004;11:59-67.
4.Orozco AF,Bischoff FZ,Horne C,Popek E,Simpson JL,Lewis DE.Hypoxia-induced membrane-bound apoptotic DNA particles:potential mechanismof fetal DNA in maternal plasma.Ann N YAcad Sci.2006;1075:57-62.
5.K.C.Allen Chan,Jun Zhang,Angela B.Y.Hui,Nathalie Wong,Tze K.Lau,Tse N.Leung,Kwok-Wai Lo,Dolly W.S.Huang,and Y.M.Dennis Lo.Size Distributions of Maternal and Fetal DNA in Maternal Plasma.Clinical Chemistry 2004;500:88-92.
Carolina J.Jorgez,Farideh Z.Bischoff.Improving Utility of Circulating DNA for Prenatal Genetic Testing:Fragment Size and Purity.2007
6.Y M Lo,M S Tein,T K Lau,C J Haines,T N Leung,P M Poon,J S Wainscoat,P J Johnson,A M Chang,and N M Hjelm.Quantitative analysis of fetal DNA in maternal plasma and serum:implications for noninvasive prenatal diagnosis.Am J Hum Genet.1998;62:768-75.
7.Tuangsit Wataganara,Erik LeShane,Antonio Farina,Geralyn M.Messerlian,Thomas Lee,Jacob A.Canick,Diana W.Bianchi.Maternal Serum Cell-Free DNA Levels are Increased in Cases of Trisomy 13 but not 18.Hum Genet 2003;112:204-8
Claims (30)
1.一种富集胎儿核酸的方法,包括
用包含具有DNA酶活性的试剂的组合物处理含有无细胞胎儿核酸的母体生物试样,
其中在处理前所述生物试样中胎儿核酸的第一百分比低于处理后所述生物试样中胎儿核酸的第二百分比。
2.权利要求1的方法,其中所述生物试样是母体的血样。
3.权利要求1的方法,其中所述生物试样是母体的血浆或血清试样。
4.权利要求1的方法,其中所述具有DNA酶活性的试剂是DNA酶。
5.权利要求1的方法,其中所述具有DNA酶活性的试剂是DNA酶且所述DNA酶的量为约10到200单位/μl。
6.权利要求1的方法,其中所述第二百分比为约10%到50%。
7.权利要求1的方法,其中所述第二百分比为至少10%、20%、30%、40%或50%。
8.权利要求1的方法,其中在处理前所述生物试样中母体核酸的第一百分比高于处理后所述生物试样中母体核酸的第二百分比。
9.权利要求1的方法,进一步包括在处理后扩增所述生物试样中的胎儿核酸。
10.权利要求1的方法,进一步包括通过全基因组扩增(Whole Genome Amplification,WGA)方法在处理后扩增所述生物试样中的胎儿核酸。
11.权利要求10的方法,其中进行所述WGA方法不用断裂温育。
12.一种由权利要求1的方法获得的混合物。
13.一种由权利要求1的方法获得的混合物,其中所述混合物含有至少10%、20%、30%、40%或50%的胎儿核酸。
14.一种在胎儿核酸中检测标志物存在或不存在的方法,包括
用包含具有DNA酶活性的试剂的组合物处理含有无细胞胎儿核酸的母体生物试样,
在处理后扩增所述生物试样中的胎儿核酸,和
在扩增过程中或之后在胎儿核酸中检测标志物的存在或不存在。
15.权利要求14的方法,其中所述生物试样是血浆或血清试样。
16.权利要求14的方法,其中所述试剂是DNA酶。
17.权利要求14的方法,其中所述扩增通过全基因组扩增(WGA)方法进行。
18.权利要求17的方法,其中进行所述WGA方法不用断裂温育。
19.权利要求14的方法,其中扩增后胎儿核酸的百分比为至少10%、20%、30%、40%或50%。
20.一种自凋亡或坏死细胞富集无细胞核酸的方法,包括
用包含具有DNA酶活性的试剂的组合物处理含有来自凋亡或坏死细胞的无细胞核酸的生物试样,
其中在处理前来自凋亡或坏死细胞的无细胞核酸的第一百分比低于处理后所述生物试样中来自凋亡或坏死细胞的无细胞核酸的第二百分比。
21.权利要求20的方法,其中所述生物试样是血样、血浆试样或血清试样。
22.权利要求20的方法,其中所述具有DNA酶活性的试剂是DNA酶。
23.权利要求20的方法,其中所述具有DNA酶活性的试剂是DNA酶且所述DNA酶的量为约10到200单位/μl。
24.权利要求20的方法,其中所述无细胞核酸来自肿瘤细胞或赘生性细胞(neoplastic cell)。
25.权利要求20的方法,进一步包括通过全基因组扩增(WGA)方法在处理后扩增所述生物试样中的无细胞核酸。
26.一种在肿瘤或赘生物核酸(neoplastic nucleic acid)中检测标志物的存在或不存在的方法,包括
用包含具有DNA酶活性的试剂的组合物处理含有来自肿瘤或赘生性细胞的无细胞核酸的生物试样,
在处理后扩增所述生物试样中的无细胞核酸,和
在扩增过程中或之后检测肿瘤或赘生物核酸中标志物的存在或不存在。
27.权利要求26的方法,其中所述生物试样是血样、血浆试样或血清试样。
28.权利要求26的方法,其中所述试剂是DNA酶。
29.权利要求26的方法,其中所述扩增通过全基因组扩增(WGA)方法进行。
30.权利要求26的方法,其中进行所述WGA方法不用断裂温育。
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PCT/US2009/032614 WO2009097511A2 (en) | 2008-01-30 | 2009-01-30 | Two stage enrichment of cell-free fetal dna in maternal plasma |
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CN109312332A (zh) * | 2016-04-06 | 2019-02-05 | 韦恩州立大学 | 自子宫颈内管获取的绒毛外滋养层细胞的胎儿dna的分离与分析 |
Families Citing this family (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ES2564656T3 (es) * | 2009-10-26 | 2016-03-28 | Lifecodexx Ag | Medios y métodos para el diagnóstico no invasivo de la aneuploidía cromosómica |
DK2516680T3 (en) * | 2009-12-22 | 2016-05-02 | Sequenom Inc | Method and kits to identify aneuploidy |
US10131947B2 (en) * | 2011-01-25 | 2018-11-20 | Ariosa Diagnostics, Inc. | Noninvasive detection of fetal aneuploidy in egg donor pregnancies |
GB2488358A (en) * | 2011-02-25 | 2012-08-29 | Univ Plymouth | Enrichment of foetal DNA in maternal plasma |
IN2015DN03186A (zh) | 2012-10-19 | 2015-10-02 | Univ Wayne State | |
US9644232B2 (en) | 2013-07-26 | 2017-05-09 | General Electric Company | Method and device for collection and amplification of circulating nucleic acids |
GB2524948A (en) * | 2014-03-07 | 2015-10-14 | Oxford Gene Technology Operations Ltd | Detecting Increase or Decrease in the Amount of a Nucleic Acid having a Sequence of Interest |
AU2015329709B2 (en) | 2014-10-10 | 2020-09-10 | Wayne State University | Methods and compositions relating to assays of fetal extravillous trophoblast cells |
ES2963004T3 (es) | 2015-09-09 | 2024-03-22 | Drawbridge Health Inc | Dispositivos para la recopilación, estabilización y conservación de muestras |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1997003405A2 (en) * | 1995-07-13 | 1997-01-30 | Philips Electronics N.V. | Method and system for data repetition between logically successive clusters |
CN1452665A (zh) * | 2000-07-10 | 2003-10-29 | 赛姆格有限公司 | 在母体样品中鉴定胎儿dna的诊断方法 |
CN1930303A (zh) * | 2003-10-08 | 2007-03-14 | 波士顿大学信托人 | 染色体异常的产前诊断方法 |
CN1978668A (zh) * | 2006-12-18 | 2007-06-13 | 中国人民解放军第三军医大学第三附属医院 | 从孕妇血浆中检测胎儿父系dna单核苷酸差异的方法 |
CN101016562A (zh) * | 2006-02-08 | 2007-08-15 | 陈熹 | 一种富集孕妇血浆中胎儿游离dna的方法 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7718367B2 (en) * | 2005-03-18 | 2010-05-18 | The Chinese University Of Hong Kong | Markers for prenatal diagnosis and monitoring |
-
2009
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- 2009-01-30 RU RU2010135816/10A patent/RU2010135816A/ru not_active Application Discontinuation
- 2009-01-30 US US12/865,381 patent/US20110183338A1/en not_active Abandoned
- 2009-01-30 BR BRPI0906641A patent/BRPI0906641A2/pt not_active IP Right Cessation
- 2009-01-30 MX MX2010008374A patent/MX2010008374A/es active IP Right Grant
- 2009-01-30 ES ES09705752T patent/ES2389038T3/es active Active
- 2009-01-30 CN CN2009801116591A patent/CN102016069A/zh active Pending
- 2009-01-30 WO PCT/US2009/032614 patent/WO2009097511A2/en active Application Filing
- 2009-01-30 CA CA2713545A patent/CA2713545A1/en not_active Abandoned
- 2009-01-30 JP JP2010545199A patent/JP2011510660A/ja not_active Withdrawn
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1997003405A2 (en) * | 1995-07-13 | 1997-01-30 | Philips Electronics N.V. | Method and system for data repetition between logically successive clusters |
CN1452665A (zh) * | 2000-07-10 | 2003-10-29 | 赛姆格有限公司 | 在母体样品中鉴定胎儿dna的诊断方法 |
CN1930303A (zh) * | 2003-10-08 | 2007-03-14 | 波士顿大学信托人 | 染色体异常的产前诊断方法 |
CN101016562A (zh) * | 2006-02-08 | 2007-08-15 | 陈熹 | 一种富集孕妇血浆中胎儿游离dna的方法 |
CN1978668A (zh) * | 2006-12-18 | 2007-06-13 | 中国人民解放军第三军医大学第三附属医院 | 从孕妇血浆中检测胎儿父系dna单核苷酸差异的方法 |
Non-Patent Citations (5)
Title |
---|
AIHUA YIN ET AL: "correlation of maternal plasma total cell-free DNA and fetal DNA levels with short term outcome of first-trimester vaginal bleeding", 《HUMAN REPRODUCTION》 * |
DOROTHY J. HUANG ET AL: "Improvement of methods for the isolation of cell-free fetal DNA from maternal plasma comparison of a manual and an automated method", 《ANN. N.Y. ACAD. SCI.》 * |
ERIN K. HANSON ET AL: "Whole genome amplification strategy for forensic genetic analysis using single or few cell equivalents of genomic DNA", 《ANALYTICAL BIOCHEMISTRY》 * |
FARIDEH Z.BISCHOFF ET AL: "Cell-free fetal DNA in maternal blood kinetics, source and structure", 《HUMAN REPRODUCTION UPDATE》 * |
RAVINDER DHALLAN ET AL: "Methods to increase the percentage of free fetal DNA recovered from the maternal circulation", 《JAMA》 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109312332A (zh) * | 2016-04-06 | 2019-02-05 | 韦恩州立大学 | 自子宫颈内管获取的绒毛外滋养层细胞的胎儿dna的分离与分析 |
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ES2389038T3 (es) | 2012-10-22 |
CA2713545A1 (en) | 2009-08-06 |
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