特殊医学检验学实验操作手册

1、 特殊医学检验学实验操作手册班级：姓名：学号：班别：课程总览周别日期进度备注12008/2/25Introduction22008/3/3Genomic DNA extraction from peripheral blood32008/3/10Sex identification (I) (PCR)42008/3/17Sex identification (II) (Gel electrophoresis)report 152008/3/24DNA polymorphisms (ACE PCR)62008/3/31DNA polymorphisms (Gel electrophoresis)

2、; ABO blood typing (PCR)report 272008/4/7ABO blood typing (PCR gel electrophoresis); RFLP82008/4/14ABO blood typing (RFLP gel electrophoresis)report 392008/4/21Middle exam. 102008/4/28Flowcytometry for blood cell type112008/5/5Data analysisreport 4122008/5/12Cell cycle analysis of cultured cells (ce

3、ll fixation)132008/5/19Cell cycle analysis of cultured cells (flowcytometry analysis)142008/5/26Data analysisreport 5152008/6/2Apoptosis (Cell lysis and DNA fragment extraction)162008/6/9Apoptosis (Gel electrophoresis)report 6172008/6/16final reports182008/6/23final exam.1. 课程介绍：主要让学生能操作新的分子检验方法2. 课

4、程目标：让学生能了解生物技术学与操作过程，更进一步使其了解实验上每一项操作过程的意义。3. 学业成绩之计算方式： 1. 平常成绩占70%（含实验报告）。2.考试成绩占 30%。实验、genomic DNA extraction4. 实验报告于实验完隔周上课时缴交，每组一份，内容包括: 封面 (含课程名称、组别、组员学号姓名、授课老师、日期)、标题 (5%) 实验目的 (10%) 材料 (10%) 仪器 (10%) 操作流程与注意事项 (20%) 实验结果 (20%) 实验讨论 (15%) 补充数据 (10%)，以计算机打字，格式: 字型以中文标楷体、英文Times new roman、大标题以

5、16号字粗体、内容以14号字标准体，行距27pt、段落间隔0.5行，边缘留2cm。不需胶装。实验二、A Rapid and Simple Procedure of Sex Identification Using the Polymerase Chain Reaction Sex identification is a frequently encountered problem in various fields of medicine, including forensic medicine and sports medicine. However, by conventional met

6、hods, the accurate judgment of sex is sometimes troublesome or requires special skill for sample preparation and identification. Recent advances of DNA analyzing techniques could shorten the procedure and presenting reliable results without exquisite skill (Witt & Erickson 1988). We can perform sex

7、identification easily and accurately using the polymerase chain reaction (PCR) of the sex chromosome specific locus (Pascal et al. 1991). Therefore, a simple protocol DNA extraction (Walsh et al.1991) and optimized polymerase chain reaction within 6 hours from the sample collection for sex identific

8、ation. MATERIALS AND METHODSDNA extraction : Viogene Blood & Tissue Genomic DNA Extraction Miniprep System.Each amplified sample was performed in 20 l mixture:1 ml Genomic DNA, 2 ml 10X Taq buffer, 0.8 ml 10mM dNTP,0.5 ml primer pair (X),0.5 ml primer pair (Y),0.1 ml Ampil-Taq Gold II (DNA polymeras

9、e), 2 ml Mg2+13.1 ml ddH2O.The primers：5-AATCATCAAATGGAGATTTG-3 (X1)、5-GTTCAGCTCTGTGAGTGAAA-3 (X2)、5-ATGATAGAAACGGAAATATG-3 (Y1) 、5-AGTAGAATGCAAAGGGCTC-3 (Y2) for X-Y Centromeric alphoid repeat were synthesized to amplify the target DNA. PCR conditions: denaturation at 94 for 1 min., annealing at 56

10、 for 1min., and extension at 72 for 1min. for 35 cycles. Electrophoresis: 10-15 l of amplified products was electrophoresed in 2% of agarose gels at 100V for 1 to 1.5 hours, and the amplified fragments were directly visualized with ethidium bromide under UV light. RESULTS AND DISCUSSIONThe amplified

11、 fragment size of the X chromosome was 130bp and that in the Y chromosome was 170bp. For sex identification, amplification of the X-and Y-specific sequences is inevitable, and PCR targeting alpha satellite sequences were first introduced by Wit and Erick-son (1988). However, in that method, we must

12、use not only two separate PCRs for identification, but sometimes obtain false results because the PCR for the Y chromosome is less sensitive than that for the X chromosome. Therefore, misjudgment of male as female sometimes occurs. There are several advantages of using this procedure for sex identif

13、ication. First, it is rapid requiring only 40 minutes for sample preparation, 2.5 hours for PCR, and 2.5 hours for electrophoresis. Secondly, sample collection is easy to perform because only one drop of saliva is needed. In addition, this test is accurate, the result easy to read, and requires no s

14、pecial skill. Therefore, it is suitable for screening of sex identification in many fields of medicine. REFERENCES1. Pascal O, Aubert D, Gilbert E, Moisan J P (1991) Sexing of forensic samples using PCR. Int J Leg Med 104: 205-2072. Mannucci A, Sullivan K M, Ivanov P L, Gill P (1994) Forensic applic

15、ation of a rapid and quantitative DNA sex test by amplification of the X-Y homologous gene amelogenin. Int J Leg Med 106: 190-1933. Nakahori Y, Hamano K, Iwaya M, Nakagome Y (1991a) Sex identification by polymerase chain reaction using X-Y homologous primer. Am J Med Genet 39: 472-4734. Nakahori Y,

16、Takenaka O, Nakagome Y (1991b) A human X-Y homologous region encodes Amelogenin. Genomics 9: 264-2695. Salido EC, Yen P H, Koprivnikar K, Yu L-H, Shapiro L J (1992) The human enamel protein gene amelogenin is expressed from both the X and the Y chromosomes. Am J Human Genet. 50: 303-3166. Walsh PS,

17、Metzger DA, Higuchi R (1991) Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. Biotechnique 10, 506-5137. Witt M. and Erickson RP (1988) A rapid method for detection of Y-chromosomal DNA from dried blood specimens by the polymerase chain reaction. Hum G

18、enet 82, 271-2748. Received on March 1, 1997 and accepted on April 10, 1997 Locus PrimersProduct SizeCommentsAmelogenin (96)5-CCCTGGGCTCTGTAAAGAATAGTG-3 5-ATCAGAGCTTAAACTGGGAAGCTG-3X = 106 bp Y = 112 bpmay be multiplexed with STRsAmelogenin (96)5-ACCTCATCCTGGGCACCCTGG-3 5-AGGCTTGAGGCCAACCATCAG-3X =

19、212 bp Y = 218 bpmay be multiplexed with STRs or D1S80Amelogenin (99)5-CTGATGGTTGGCCTCAAGCCTGTG-3 5-TAAAGAGATTCATTAACTTGACTG-3X = 977 bp Y = 788 bpmay be analyzed with agarose gelsCentromeric alphoid repeat (98)5-TATTTGGACTCTCTCTGAGGA-3 (X3) 5-TTCTACTACAAGGGTGTTGCA-3 (X4) 5-GTGTATTCACCTCCGGGAG-3 (Y3

20、) 5-ACAAAAGGTTCAATTCTGTGAG-3 (Y4)X = 157 bp Y = 200 bpboth X- and Y-sequences were coamplifiedCentromeric alphoid repeat (100)5-AATCATCAAATGGAGATTTG-3 (X1) 5-GTTCAGCTCTGTGAGTGAAA-3 (X2) 5-ATGATAGAAACGGAAATATG-3 (Y11) 5-AGTAGAATGCAAAGGGCTC-3 (Y22)X = 170 bp Y = 130 bpseparate PCR reactions were perfo

21、rmedZFX/ZFY zinc finger gene (103,104)5-CTGGAGAGCCACAAGCTGAC-3 5-TTGCTGTGGACTGCCAAGAG-3X/Y = 209bp after HaeIII cuts X = 172 +37 Y = 88 +84 +37reverse dot blot typing assay; may be used with AmpliType PM and HLA-DQA1SRY 93 (800, 1185)5-ATAAGTATCGACCTCGTCGGAAG-3 5-GCACTTCGCTGCAGAGTACCGAAG-3 Y = 93 bp

22、can be multiplexed with amelogenin实验三、利用dHPLC检测ACE多型性在心血管疾病的关系一、 前言：dHPLC是最近发展出来用以检验核酸突变之方法，其自动化及灵敏度超出过去的突变分析方法如SSCP、DGGE等，未来之临床应用性极高。而心脏血管疾病在现代社会已经成为极其普遍的慢性疾病，ACE多型性在此类疾病占有非常重要的角色，其涉及了预防、诊断、治疗及预后的多种功能，藉由这个基因的检测让学生学习以新一代的dHPLC分析基因的多型性，可以充分提升学生在分子检验方面的竞争力。二、 原理：dHPLC的基础是离子配对逆相层析法(Ion-Pair Reverse Pha

23、se chromatography)。其固定相是带有18个碳烷基的非极性亲合性管柱，流动相为亲水性的Acetonitrile。分析的DNA先与离子配对试剂TEAA( triethyl-ammonium acetate)混合，TEAA会与DNA的磷酸基形成离子配对，在流动相中将带带负电的DNA包覆，露出非极性的烷基端，形成以非极性表面为主的混合分子，因而吸附在非极性的固定相上，这个混合分子在分离管柱的滞留主要决定于TEAA中带正电离子和DNA中带负电离子的结合力。若DNA 链越长，就带有越多磷酸基，因此有较多的TEAA结合使其在管柱滞留的时间越长，藉由增加流动相中Acetonitrile 浓度，

24、使具有亲水性的Acetonitrile将DNA 置换，造成DNA 和TEAA的脱离，进而使DNA 分子随流动相析出而达到分离效果。ACE基因的intron 16具有插入与删除多型性的区域，利用PCR扩增其片断之后，藉由dHPLC分析出其长短片段，得知其多型性的变化。三、 方法与步骤A. 样本收集：血液样本采用人类静脉血液，收集在EDTA真空采血管，在一天之内进行分析。B. DNA抽取：将利用小量基因体DNA萃取套组进行萃取，DNA纯度将以分光光度计在260nm/280nm高于1.8才可以使用。C. PCR放大ACE intron16：primer序列，顺向 5-CTGGAGACCACTCCCA

25、TCCTTT CT-3与反向5 -GATGTGGCCATCA CATTCGTCAGAT-3，每一扩增的样本共20ml包含:5 ml Genomic DNA, 2 ml 10X Taq buffer, 0.4 ml 10mM dNTP,0.4 ml ACE primer pair,0.1 ml Ampil-Taq Gold II (DNA polymerase), 2 ml Mg2+10.1 ml ddH2O.PCR条件是先以9410分钟进行热解离，再进入放大循环，循环条件为94 1分钟、58 1分钟及72 2分钟，进行30个循环。产物以2% agarose胶体电泳确认，再进行PCR产物的纯化，

26、准备上机。D. dHPLC分析DNA多型性：dHPLC将与行政院卫生署桃园医院商借，上机前先以分子量标准品进行校正，确定毎一分子量的流出时间，再将样本一一注入进行分析。四、 预期结果我们将可以得到每一个样本的ACE intron 16的多型性，这个多型性将有两种结果来说明，一是agarose胶体电泳，二是dHPLC的图形，并且由dHPLC的结果可以较精确定出各片段之分子量，有可能找到新的多型性类别或是突变。 五、 讨论ACE多型性已被证明与心血管疾病有密切关系，传统作法是以agarose电泳分析其片断长短，是一简单也容易操作的方法，然而其分辨率受到相当多的限制，同时操作的时间长，对临床检验来说

27、不是一可以经常使用之方法。dHPLC改善了电泳需要一一以手动方式注入样本，并且耗费时间进行电泳的方式，改以自动针注样本、管柱层析与自动侦测方式获得结果，因此这检验机器将是未来分子检验的重要工具，对学生将有很大的帮助。实验四、ABO基因型检验ABO血型系统是第一个被发现的人类遗传标记系统，在输血、亲子鉴定、人类学研究、法医物证检验等领域中有着非常重要的应用价值。对于ABO血型，传统的检验方法是对抗原或抗体进行检验，在法医物证中最为常用的方法是检验抗原物质。由于ABO血型抗原是糖脂或糖蛋白，因此检验时易受抗原活性的强弱、抗体的特异性以及微生物的污染等因素的影响。对于性犯罪中的混合斑检验，传统血清学

28、方法有很大的局限性，使这一难题长期得不到很好的解决。19901993年，Yamamoto16报导了ABO基因的苷酸序列，查清了各等位基因的差异，能够测定ABO基因。从目前所报导的文献来看，测定ABO基因型的方法主要有3类：(1)PCR产物限制性片段长度多态性(AmpRFLP)7，8；(2)用等位基因特异寡核苷酸引物(ASOP)扩增9；(3)单链构象多态性(SSCP)10。本文选择PCR增殖条件进行四primer放大，再用限制性内切酶KpnI和AluI分别酶解扩增产物，电泳分离、检验ABO基因型，简便可靠。材料和方法 (一)主要试剂(1) Primers:ABO-1: 5-CACCGTGGAAG

29、GATGTCCTC-3ABO-2：5-AATGTCCACAGTCACTCGCC-3ABO-3：5-GTGGAGATCCTGACTCCGCTG-3ABO-4：5-GTAGAAATCGCCCTCGTCC-3(2) Taq DNA聚合酶(3) dNTP(4) 限制酶KpnI(5) 限制酶AluI (二) DNA的增殖：PCR在50l的反应体系中进行：2 ml DNA, 5 ml buffer, 5 ml 4 mM dNTP, 1 ml primer, 1 ml Taq DNApolymerase, 最后加入35 ml H2O，置于PCR machine中进行扩增。*扩增程序为：95变性30s，60结

30、合30s，72延伸30s，30个cycles结束后再72外延伸3min。(三)扩增产物的酶解扩增产物的酶解在PCR反应体系中进行，直接加入2.5U KpnI和2.5U AluI，37酶解1h。(四)扩增产物的检测用2% Agarose电泳，EtBr染色，检测扩增产物。结果ABO基因位点定位于人类染色体9q34上，该基因有A，B，O 3个等位基因，O基因缺失第258位核苷酸(G)，A基因和B基因在第700位核苷酸有替换。由于其在cDNA序列上的差异，能够用PCR方法分析ABO血型系统的基因型。根据第258位核苷酸(G)缺失和第703位核苷酸替换(GA)产生出两个酶切位点，设计出4个引物：ABO-

31、1位于cDNA第232位，ABO-2位于基因内非编码插入序列，ABO-3位于cDNA第661位，ABO-4位于cDNA第789位。用primer ABO-1，ABO-2进行扩增，产生200bp(A或B)或199bp(O)的片段。199bp片段是由于cDNA第258位核苷酸(G)缺失造成的，从而产生KpnI的限制酶切位点(GGTACC)，O基因扩增产物经酶切后产生171和28bp的片段，而A或B基因产物不能被KpnI酶切。用primer ABO-3，ABO-4进行扩增，产生128bp的片段。由于A，B基因的cDNA第700位核苷酸的替换(GA)，从而产生出AluI的限制酶切位点(AGCT)，B基

32、因扩增产物经酶切后产生88和40bp的片段。A基因扩增产物128bp不能被AluI酶切。本文经过反复摸索，选择最佳扩增条件进行4引物复合扩增，产物在PCR反应体系中进行酶解，操作方便，结果稳定。如图1所示ABO基因的6种基因型。由于本方法是直接测定基因型，杂合子AO，BO很容易与纯合子AA，BB区分开来，增加了分型，提高了个人识别能力，增强了ABO血型在办案中的应用价值。参考文献1. Yamamoto F, Clausen H, White T, et al. Molecular genetic basis of the histo-blood ABO system. Nature, 1990

33、,345:2292332. Yamamoto F, Hakomori S. Sugar-nucleotide donor specificity of histo-blood group A and B transferases is based on amino acid substitution. J Bio Chem, 1990,265:19257192623. Yamamoto F, Marken J, Tsuji T, et al. Cloning and characterization of DNA complementarity to human DUP-GaINAc: Fuc

34、al 2Gala 3Gal-Nac transferase (histo-blood group A transferase) mRNA. J Bio Chem, 1990,265(2):114611514. Yamamoto F, McNeill PD, Yamamoto M, et al. Molecular genetic analysis of the ABO blood group system: 1. Weak subgroups: A3 and B3 alleles. Vox Sanguinis, 1993,64:1161195. Yamamoto F, McNeill PD,

35、Yamamoto M, et al. Molecular genetic analysis of the ABO blood group system: 3.AX and B(A) alleles. Vox Sanguinis, 1993,64:1711746. Yamamoto F, McNeill PD, Yamamoto M, et al. Molecular genetic analysis of the ABO blood group system: 4. Another type of O allele. Vox Sanguinis, 1993,64:1751787. Lee JC

36、I, Chang JG, et al.ABO genotyping by poly-merase chain reaction. J Forensic Sci,1992,37(5):126912758. Herrin G. A simplified amplification procedure for two regions of the glycosyl transferase (ABO blood group) gene. J Forensic Sci, 1996,41(1):1381419. Crouse C, Vincek V. Identification of ABO allel

37、es on forensic-type specimens using rapid ABO genotyping. Bio Techniques, 1995,18(3):47848310. Akane A, Yoshimura S, Yoshida M, et al. ABO genotyping following a single PCR amplification. J Forensic Sci, 1996,41(2):272274实验五、利用流式细胞仪分析外围血液中的CD3+CD4+淋巴球实验目的: 学习如何操作细胞免疫抗体荧光染色，并且以流式细胞仪分析外围血液白血球中的特定表面抗原细

38、胞。本次分析为其中之T淋巴球中的CD3+CD4+的细胞。材料:3ml syringeEDTA tube or heparin tube75% EtOHTourniquetFlowcytometry tube1X FASC lyse bufferRefrigerated centrifugePhosphate buffer saline (PBS)Fetal calf serumAnti-CD3-FITC conjugated antibodyAnti-CD4-PE conjugated antibodyRacksPipettman and pipette tipsFlowcytometry处理

39、步骤1. 针筒先抽出少许的肝素在自自愿者静脉取出2ml全血充分混合。2. 检体与抗体配制PBS as controlCD3 onlyCD4 onlyCD3+CD4Blood100ul100ul100ul100ulPBS20ul10ul10ul-CD3+-FITC-10ul-10ulCD4+-PE-10ul10ul3. 充分混合后避光下室温放置15分钟。4. 加入2ml 1X lyse buffer。5. 充分混合后避光下室温放置10分钟。6. 离心 (2800rpm*5min, at 4)。7. 丢弃上清液加入2ml的PBS充分混合后重复步骤6。8. 再一次PBS清洗。9. 加入1ml PBS

40、-FCS (PBS含2% fetal calf serum)充分混合后准备上机。实验六、以流式细胞仪侦测细胞周期原理：细胞含有成套的染色体，当细胞分裂时，细胞的染色体会经由DNA复制的过程从原先的两套变成四套，经由有丝分裂的流程(mitosis)将细胞一分为二。这种周期性的分裂流程可以利用流式细胞仪分析，原理上就是利用一些可以嵌入DNA的荧光染剂进行染色，DNA含量越多的细胞所嵌入之染剂就越多，个别细胞的DNA含量即可藉由流式细胞仪侦测其嵌入之染剂多少得到定量。实验操作流程1 材料与溶液：Colorectal carcinoma cell line SW480或HT-29 (1*106 cells)(含四种处理: 正常、去血清、20nM Vincristine, 1mM hydroxyurea)ice-cold PBS (phosphate buffer saline)15 ml 离心管70% 酒精 (-20)p200 tip, p1000 tippropidium iodide/RNase A solution (50ug/ml:10ug/ml)2 仪器：flowcytom