特殊醫(yī)學(xué)檢驗(yàn)學(xué)實(shí)驗(yàn)操作手冊(cè)

1、特殊醫(yī)學(xué)檢驗(yàn)學(xué)實(shí)驗(yàn)操作手冊(cè)班級(jí)：姓名：學(xué)號(hào)：班別：課程總覽周別日期進(jìn)度備注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 (cel

3、l 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. 課程介紹：主要讓學(xué)生能操作新的分子檢驗(yàn)方法2. 課程

4、目標(biāo)：讓學(xué)生能了解生物技術(shù)學(xué)與操作過程，更進(jìn)一步使其了解實(shí)驗(yàn)上每一項(xiàng)操作過程的意義。3. 學(xué)業(yè)成績(jī)之計(jì)算方式： 1. 平常成績(jī)占70%（含實(shí)驗(yàn)報(bào)告）。2.考試成績(jī)占 30%。實(shí)驗(yàn)、genomic DNA extraction4. 實(shí)驗(yàn)報(bào)告于實(shí)驗(yàn)完隔周上課時(shí)繳交，每組一份，內(nèi)容包括: 封面 (含課程名稱、組別、組員學(xué)號(hào)姓名、授課老師、日期)、標(biāo)題 (5%) 實(shí)驗(yàn)?zāi)康?(10%) 材料 (10%) 儀器 (10%) 操作流程與注意事項(xiàng) (20%) 實(shí)驗(yàn)結(jié)果 (20%) 實(shí)驗(yàn)討論 (15%) 補(bǔ)充數(shù)據(jù) (10%)，以計(jì)算機(jī)打字，格式: 字型以中文標(biāo)楷體、英文Times new roman、大標(biāo)題以1

5、6號(hào)字粗體、內(nèi)容以14號(hào)字標(biāo)準(zhǔn)體，行距27pt、段落間隔0.5行，邊緣留2cm。不需膠裝。實(shí)驗(yàn)二、A Rapid and Simple Procedure of Sex Identification Using the Polymerase Chain ReactionSex identification is a frequently encountered problem in various fields of medicine, including forensic medicine and sports medicine. However, by conventional metho

6、ds, 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 ide

7、ntification 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 identificati

8、on.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,0.5ml primerpair (X),0.5ml primer pair (Y),0.1 ml Ampil-Taq Gold II (DNA polymerase),2 ml M

9、g2+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 for 1min.,and

10、 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 fragment size of

11、 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 use not only two s

12、eparate 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 identification. First, it

13、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 special skill. There

14、fore, 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 application of a rapid and

15、 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, Takenaka O, Nakagome

16、 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, Metzger DA, Higuchi

17、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 Genet 82, 271-2748. R

18、eceived on March 1, 1997 and accepted on April 10, 1997Locus PrimersProduct SizeCommentsAmelogenin(96)5-CCCTGGGCTCTGTAAAGAATAGTG-35-ATCAGAGCTTAAACTGGGAAGCTG-3X = 106 bpY = 112 bpmay be multiplexed with STRsAmelogenin(96)5-ACCTCATCCTGGGCACCCTGG-35-AGGCTTGAGGCCAACCATCAG-3X = 212 bpY = 218 bpmay be mul

19、tiplexed with STRs or D1S80Amelogenin (99)5-CTGATGGTTGGCCTCAAGCCTGTG-35-TAAAGAGATTCATTAACTTGACTG-3X = 977 bpY = 788 bpmay be analyzed with agarose gelsCentromeric alphoid repeat (98)5-TATTTGGACTCTCTCTGAGGA-3 (X3)5-TTCTACTACAAGGGTGTTGCA-3 (X4)5-GTGTATTCACCTCCGGGAG-3 (Y3)5-ACAAAAGGTTCAATTCTGTGAG-3 (Y4

20、)X = 157 bpY = 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 bpY = 130 bpseparate PCR reactions were performedZFX/ZFY zinc finger gene (103,104

21、)5-CTGGAGAGCCACAAGCTGAC-35-TTGCTGTGGACTGCCAAGAG-3X/Y = 209bpafter HaeIII cutsX = 172 +37Y = 88 +84 +37reverse dot blot typing assay; may be used with AmpliType PM and HLA-DQA1SRY 93 (800, 1185)5-ATAAGTATCGACCTCGTCGGAAG-35-GCACTTCGCTGCAGAGTACCGAAG-3 Y = 93 bpcan be multiplexed with amelogenin實(shí)驗(yàn)三、利用dH

22、PLC檢測(cè)ACE多型性在心血管疾病的關(guān)系一、 前言：dHPLC是最近發(fā)展出來用以檢驗(yàn)核酸突變之方法，其自動(dòng)化及靈敏度超出過去的突變分析方法如SSCP、DGGE等，未來之臨床應(yīng)用性極高。而心臟血管疾病在現(xiàn)代社會(huì)已經(jīng)成為極其普遍的慢性疾病，ACE多型性在此類疾病占有非常重要的角色，其涉及了預(yù)防、診斷、治療及預(yù)后的多種功能，藉由這個(gè)基因的檢測(cè)讓學(xué)生學(xué)習(xí)以新一代的dHPLC分析基因的多型性，可以充分提升學(xué)生在分子檢驗(yàn)方面的競(jìng)爭(zhēng)力。二、 原理：dHPLC的基礎(chǔ)是離子配對(duì)逆相層析法(Ion-Pair Reverse Phase chromatography)。其固定相是帶有18個(gè)碳烷基的非極性親合性管柱，

23、流動(dòng)相為親水性的Acetonitrile。分析的DNA先與離子配對(duì)試劑TEAA( triethyl-ammonium acetate)混合，TEAA會(huì)與DNA的磷酸基形成離子配對(duì)，在流動(dòng)相中將帶帶負(fù)電的DNA包覆，露出非極性的烷基端，形成以非極性表面為主的混合分子，因而吸附在非極性的固定相上，這個(gè)混合分子在分離管柱的滯留主要決定于TEAA中帶正電離子和DNA中帶負(fù)電離子的結(jié)合力。若DNA 鏈越長(zhǎng)，就帶有越多磷酸基，因此有較多的TEAA結(jié)合使其在管柱滯留的時(shí)間越長(zhǎng)，藉由增加流動(dòng)相中Acetonitrile 濃度，使具有親水性的Acetonitrile將DNA 置換，造成DNA 和TEAA的脫離，

24、進(jìn)而使DNA 分子隨流動(dòng)相析出而達(dá)到分離效果。ACE基因的intron 16具有插入與刪除多型性的區(qū)域，利用PCR擴(kuò)增其片斷之后，藉由dHPLC分析出其長(zhǎng)短片段，得知其多型性的變化。三、 方法與步驟A. 樣本收集：血液樣本采用人類靜脈血液，收集在EDTA真空采血管，在一天之內(nèi)進(jìn)行分析。B. DNA抽取：將利用小量基因體DNA萃取套組進(jìn)行萃取，DNA純度將以分光光度計(jì)在260nm/280nm高于1.8才可以使用。C. PCR放大ACE intron16：primer序列，順向 5-CTGGAGACCACTCCCATCCTTT CT-3與反向5 -GATGTGGCCATCA CATTCGTCAGA

25、T-3，每一擴(kuò)增的樣本共20ml包含:5 ml Genomic DNA,2 ml 10X Taq buffer,0.4 ml 10mM dNTP,0.4ml ACE primer pair,0.1 ml Ampil-Taq Gold II (DNA polymerase), 2 ml Mg2+10.1 ml ddH2O.PCR條件是先以9410分鐘進(jìn)行熱解離，再進(jìn)入放大循環(huán)，循環(huán)條件為94 1分鐘、58 1分鐘及72 2分鐘，進(jìn)行30個(gè)循環(huán)。產(chǎn)物以2% agarose膠體電泳確認(rèn)，再進(jìn)行PCR產(chǎn)物的純化，準(zhǔn)備上機(jī)。D. dHPLC分析DNA多型性：dHPLC將與行政院衛(wèi)生署桃園醫(yī)院商借，上機(jī)前

26、先以分子量標(biāo)準(zhǔn)品進(jìn)行校正，確定毎一分子量的流出時(shí)間，再將樣本一一注入進(jìn)行分析。四、 預(yù)期結(jié)果我們將可以得到每一個(gè)樣本的ACE intron 16的多型性，這個(gè)多型性將有兩種結(jié)果來說明，一是agarose膠體電泳，二是dHPLC的圖形，并且由dHPLC的結(jié)果可以較精確定出各片段之分子量，有可能找到新的多型性類別或是突變。五、 討論ACE多型性已被證明與心血管疾病有密切關(guān)系，傳統(tǒng)作法是以agarose電泳分析其片斷長(zhǎng)短，是一簡(jiǎn)單也容易操作的方法，然而其分辨率受到相當(dāng)多的限制，同時(shí)操作的時(shí)間長(zhǎng)，對(duì)臨床檢驗(yàn)來說不是一可以經(jīng)常使用之方法。dHPLC改善了電泳需要一一以手動(dòng)方式注入樣本，并且耗費(fèi)時(shí)間進(jìn)行電

27、泳的方式，改以自動(dòng)針注樣本、管柱層析與自動(dòng)偵測(cè)方式獲得結(jié)果，因此這檢驗(yàn)機(jī)器將是未來分子檢驗(yàn)的重要工具，對(duì)學(xué)生將有很大的幫助。實(shí)驗(yàn)四、ABO基因型檢驗(yàn)ABO血型系統(tǒng)是第一個(gè)被發(fā)現(xiàn)的人類遺傳標(biāo)記系統(tǒng)，在輸血、親子鑒定、人類學(xué)研究、法醫(yī)物證檢驗(yàn)等領(lǐng)域中有著非常重要的應(yīng)用價(jià)值。對(duì)于ABO血型，傳統(tǒng)的檢驗(yàn)方法是對(duì)抗原或抗體進(jìn)行檢驗(yàn)，在法醫(yī)物證中最為常用的方法是檢驗(yàn)抗原物質(zhì)。由于ABO血型抗原是糖脂或糖蛋白，因此檢驗(yàn)時(shí)易受抗原活性的強(qiáng)弱、抗體的特異性以及微生物的污染等因素的影響。對(duì)于性犯罪中的混合斑檢驗(yàn)，傳統(tǒng)血清學(xué)方法有很大的局限性，使這一難題長(zhǎng)期得不到很好的解決。19901993年，Yamamoto16

28、報(bào)導(dǎo)了ABO基因的苷酸序列，查清了各等位基因的差異，能夠測(cè)定ABO基因。從目前所報(bào)導(dǎo)的文獻(xiàn)來看，測(cè)定ABO基因型的方法主要有3類：(1)PCR產(chǎn)物限制性片段長(zhǎng)度多態(tài)性(AmpRFLP)7，8；(2)用等位基因特異寡核苷酸引物(ASOP)擴(kuò)增9；(3)單鏈構(gòu)象多態(tài)性(SSCP)10。本文選擇PCR增殖條件進(jìn)行四primer放大，再用限制性內(nèi)切酶KpnI和AluI分別酶解擴(kuò)增產(chǎn)物，電泳分離、檢驗(yàn)ABO基因型，簡(jiǎn)便可靠。材料和方法 (一)主要試劑(1) Primers:ABO-1: 5-CACCGTGGAAGGATGTCCTC-3ABO-2：5-AATGTCCACAGTCACTCGCC-3ABO-3

29、：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的反應(yīng)體系中進(jìn)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中進(jìn)行擴(kuò)增。*擴(kuò)增程序?yàn)椋?5變性30s，60結(jié)合30s，72延伸30s，30個(gè)cycles結(jié)束后再72外延伸3min。(三)擴(kuò)增產(chǎn)物的酶解擴(kuò)增產(chǎn)物的酶

30、解在PCR反應(yīng)體系中進(jìn)行，直接加入2.5U KpnI和2.5U AluI，37酶解1h。(四)擴(kuò)增產(chǎn)物的檢測(cè)用2% Agarose電泳，EtBr染色，檢測(cè)擴(kuò)增產(chǎn)物。結(jié)果ABO基因位點(diǎn)定位于人類染色體9q34上，該基因有A，B，O 3個(gè)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位核苷酸有替換。由于其在cDNA序列上的差異，能夠用PCR方法分析ABO血型系統(tǒng)的基因型。根據(jù)第258位核苷酸(G)缺失和第703位核苷酸替換(GA)產(chǎn)生出兩個(gè)酶切位點(diǎn)，設(shè)計(jì)出4個(gè)引物：ABO-1位于cDNA第232位，ABO-2位于基因內(nèi)非編碼插入序列，ABO-3位于cDNA第661位，ABO-

31、4位于cDNA第789位。用primer ABO-1，ABO-2進(jìn)行擴(kuò)增，產(chǎn)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，從而產(chǎn)生KpnI的限制酶切位點(diǎn)(GGTACC)，O基因擴(kuò)增產(chǎn)物經(jīng)酶切后產(chǎn)生171和28bp的片段，而A或B基因產(chǎn)物不能被KpnI酶切。用primer ABO-3，ABO-4進(jìn)行擴(kuò)增，產(chǎn)生128bp的片段。由于A，B基因的cDNA第700位核苷酸的替換(GA)，從而產(chǎn)生出AluI的限制酶切位點(diǎn)(AGCT)，B基因擴(kuò)增產(chǎn)物經(jīng)酶切后產(chǎn)生88和40bp的片段。A基因擴(kuò)增產(chǎn)物128bp不能被AluI酶切。本文經(jīng)過反復(fù)摸索

32、，選擇最佳擴(kuò)增條件進(jìn)行4引物復(fù)合擴(kuò)增，產(chǎn)物在PCR反應(yīng)體系中進(jìn)行酶解，操作方便，結(jié)果穩(wěn)定。如圖1所示ABO基因的6種基因型。由于本方法是直接測(cè)定基因型，雜合子AO，BO很容易與純合子AA，BB區(qū)分開來，增加了分型，提高了個(gè)人識(shí)別能力，增強(qiáng)了ABO血型在辦案中的應(yīng)用價(jià)值。參考文獻(xiàn)1. Yamamoto F, Clausen H, White T, et al. Molecular genetic basis of the histo-blood ABO system. Nature, 1990,345:2292332. Yamamoto F, Hakomori S. Sugar-nucleoti

33、de 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: Fucal 2Gala 3Gal-Nac transferase (histo-blood group A t

34、ransferase) 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, Yamamoto M, et al. Molecular genetic analysis of the

35、 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 JCI, Chang JG, et al.ABO genotyping by poly-merase cha

36、in 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 alleles on forensic-type specimens using rapid ABO genoty

37、ping. 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實(shí)驗(yàn)五、利用流式細(xì)胞儀分析外圍血液中的CD3+CD4+淋巴球?qū)嶒?yàn)?zāi)康? 學(xué)習(xí)如何操作細(xì)胞免疫抗體熒光染色，并且以流式細(xì)胞儀分析外圍血液白血球中的特定表面抗原細(xì)胞。本次分析為其中之T淋巴球中的CD3+CD4+的細(xì)胞。材料:3ml syringeEDTA tube

38、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處理步驟1. 針筒先抽出少許的肝素在自自愿者靜脈取出2ml全血充分混合。2. 檢體與抗體配制PBS as c

39、ontrolCD3 onlyCD4 onlyCD3+CD4Blood100ul100ul100ul100ulPBS20ul10ul10ul-CD3+-FITC-10ul-10ulCD4+-PE-10ul10ul3. 充分混合后避光下室溫放置15分鐘。4. 加入2ml 1X lyse buffer。5. 充分混合后避光下室溫放置10分鐘。6. 離心 (2800rpm*5min, at 4)。7. 丟棄上清液加入2ml的PBS充分混合后重復(fù)步驟6。8. 再一次PBS清洗。9. 加入1ml PBS-FCS (PBS含2% fetal calf serum)充分混合后準(zhǔn)備上機(jī)。實(shí)驗(yàn)六、以流式細(xì)胞儀偵測(cè)

40、細(xì)胞周期原理：細(xì)胞含有成套的染色體，當(dāng)細(xì)胞分裂時(shí)，細(xì)胞的染色體會(huì)經(jīng)由DNA復(fù)制的過程從原先的兩套變成四套，經(jīng)由有絲分裂的流程(mitosis)將細(xì)胞一分為二。這種周期性的分裂流程可以利用流式細(xì)胞儀分析，原理上就是利用一些可以嵌入DNA的熒光染劑進(jìn)行染色，DNA含量越多的細(xì)胞所嵌入之染劑就越多，個(gè)別細(xì)胞的DNA含量即可藉由流式細(xì)胞儀偵測(cè)其嵌入之染劑多少得到定量。實(shí)驗(yàn)操作流程1 材料與溶液：Colorectal carcinoma cell line SW480或HT-29 (1*106 cells)(含四種處理: 正常、去血清、20nM Vincristine, 1mM hydroxyurea)ice-cold PBS (phosphate buffer saline)15 ml 離心管70% 酒精 (-20)p200 tip, p1000 tippropidium iodide/RNase A solution (50ug/ml:10ug/ml)2 儀器：flowcytometry,