1、Non-Radioactive NucleicAcid Labeling and Detection 非放射性的核酸標(biāo)記及檢測(cè)技術(shù),羅氏診斷產(chǎn)品（上海）有限公司 應(yīng)用科學(xué)部,主要內(nèi)容,背景知識(shí) 非放射性核酸標(biāo)記方法 非放射性標(biāo)記的核酸雜交 非放射性核酸檢測(cè)方法,Isotope Lab,Benefits,對(duì)使用者健康無影響 更高的靈敏度 compared to radioactivity 更快獲得結(jié)果 compared to isotopic procedures 探針可以至少保存一年 多種用途 by wide product range 方便操作 of handling,Advantages

2、of Non-radioactive Labeling and detection:,Detection of Fragile X Hybridization with 32P-labeled probe,Target: 10g human genomic DNA Probe: RPL-labeled Hybr.: Home Brew Exp.: o.n.,1 2 3 4 5 6 7 8 9 10 11 12 13,Detection of Fragile X Hybridization with DIG-labeled probe,Target: 10g human genomic DNA

3、Probe: PCR labeled Hybr.: Roche protocol Exp.: 20 min,1 2 3 4 5 6 7 8,Non-Radioactive vs. Radioactive (放射性與非放射性比較),地高辛 Digoxigenin,Digitalis purpurea,Digoxigenin occurs exclusively in D. purpurea and D. lanata 地高辛唯一來源于毛地黃和毛花毛地黃中,What is DIG ?,類固醇半抗原物質(zhì) 可標(biāo)記核酸或者蛋白 可選擇多種檢測(cè)方法: 顯色，熒光或者化學(xué)發(fā)光 快速安全靈敏的同位素代替品,F

4、eatures of DiGoxigenin,Structure of DIG-Modified Nucleotides Coupled via 堿穩(wěn)定 Ether Bond,Available: DIG-UTP DIG-dUTP DIG-ddUTP,UTP,Spacer,O,DIG,Ether bond,Structure of DIG-dUTP Coupled via 堿不穩(wěn)定,O,UTP,Spacer,DIG,Ester bond,標(biāo)記技術(shù),非放射法標(biāo)記核酸,酶法標(biāo)記: Klenow進(jìn)行隨機(jī)引物標(biāo)記 DNA Pol I / DNase I進(jìn)行缺口平移標(biāo)記 PCR 方法進(jìn)行標(biāo)記（Taq D

5、NA Polymerase,/Pwo DNA Polymerase or Expand)* 寡核苷酸 3-標(biāo)記或用末端轉(zhuǎn)移酶加尾標(biāo)記DNA* 用SP6-, T3- or T7 RNAPolymerases*進(jìn)行體外轉(zhuǎn)錄標(biāo)記RNA 用AMV/M-MuLV合成cDNA C. therm/Taq, C. therm/Pwo or C. therm/Expand進(jìn)行RT-PCR,* Labeling mixes or kits available from Roche Applied Science,非放射性隨機(jī)引物標(biāo)記法,dNTPs,DIG-dUTP,Single-Stranded Template

6、 +,+,Klenow-Enzyme,Labeled Probe,DIG High Prime和傳統(tǒng)隨機(jī)引物標(biāo)記法的對(duì)比,DIG High Prime DIG Standard R.P.,Labeled DNA (ng),2500,2000,1500,1000,500,0,0,200,400,600,800,1000,min,PCR法標(biāo)記探針,隨機(jī)引物法標(biāo)記探針,模板：完整質(zhì)粒DNA,Taq Polymerase Specific Primers, (e.g. for multiple cloning site),Standard Direct Detection Assay,Labeling

7、 of two different probes by RPL Spotting of dilution series and direct detection with chemiluminescence Calculation of dilution series according to 1g of template DNA Result: For use in hybridization it is important, that the 0.3 pg spot (Probe 1) ideally the 0.1 pg spot (Probe 2) is visible,Amount

8、of DIG-labeled DNA per spot,10,3,1,0.3,0.1,0 pg,Control Probe 1 Probe 2,探針純化,Purification of labeled probe prior to hybridization It is not necessary to purify labeled probes from unincorporated DIG-dUTP If a probe causes background problems, e.g. if the template DNA was isolated with a home brew me

9、thod apply the High Pure PCR Product Purification Kit,寡聚核苷酸標(biāo)記法3端標(biāo)記和加尾,+ DIG-ddUTP + Terminal transferase,+ dATP + DIG-dUTP + Terminal transferase,template independent synthesis of DNA-tail of 40-50 nt length with several DIG-molecules incorporated,3- OH,5,Oligonucleotide with specific sequence,addit

10、ion of a single DIG-ddUTP nucleotide,非放射性RNA標(biāo)記,RE,pSPT 18/19,insert,promoter,轉(zhuǎn)錄載體，含有噬菌體啟動(dòng)子 和插入DNA片斷,用某一限制性內(nèi)切酶將載體線性化,+ATP,CTP,GTP,UTP,+DIG-UTP +SP6, T3 or T 7 RNA polymerase,進(jìn)行體外轉(zhuǎn)錄 defined length in a 2 h incubation at 37C,RNA synthesis by in vitro transcription from phage promoter,每ug的DNA模板最多能生成20ug

11、RNA,地高辛(DIG)標(biāo)記試劑盒,DIG DNA Labeling Kit DIG-High Prime DIG Oligonucleotide 3-End Labeling Kit. (2nd Gen) DIG Oligonucleotide 5-End Labeling Set DIG Oligonucleotide Tailing Kit. (2nd Gen) DIG RNA Labeling Kit (SP6/T7) PCR DIG Probe Synthesis Kit,Hybridization 雜交,標(biāo)記效率檢測(cè) 雜交原理 有關(guān)雜交的產(chǎn)品,DIG-Quantification

12、Teststrip(定量測(cè)試條),應(yīng)用DIG-Quantification Teststrips檢測(cè) DNA and RNA 探針的標(biāo)記效率,DIG RNA 標(biāo)記 1h,DIG DNA Control-Strip,DIG DNA 標(biāo)記 1h,備注: 次方法不能對(duì)DIG標(biāo)記的寡核苷酸或PCR 探針定量,3,10,30,100,300,pg,不同的方法標(biāo)記探針用于雜交,Positively Charged Nylon Membrances (正電荷尼龍膜),DIG Easy Hyb Buffer,優(yōu)點(diǎn): 無毒性 (尿素取代甲酰胺) 無DNase 和RNase , 無菌 Southern和North

13、ern-雜交 作為質(zhì)控檢測(cè) 在室溫下穩(wěn)定 即用試劑 可用于所有的雜交反應(yīng),Hybridization Bags with Spout 帶管口的雜交袋,去除氣泡的步驟： 盡可能去袋中的除氣泡 擠壓小部分的液體到管口內(nèi) 蓋上蓋子,檢測(cè)方法,Detection 地高辛(DIG)檢測(cè)方法,NBT/BCIP顯色檢測(cè) 化學(xué)發(fā)光檢測(cè) 熒光檢測(cè),檢測(cè)DIG標(biāo)記的核酸,You need: Anti-DIG-Antibody coupled to Alkaline Phosphatase A substrate for Alkaline Phosphatase, Color substrate: NBT/BCIP

14、 (membranes and ISH) Chemiluminescent substrates: CSPD or CDP-Star (nylon membranes only) 原位雜交: Anti-DIG-Fluorescein Anti-DIG-Rhodamine,檢測(cè)DIG標(biāo)記的核酸 化學(xué)發(fā)光底物,X-Ray Film,-,C,-,G,-,T,-,G,-,A,-,T,-,A,-,G,-,C,-,A,-,C,-,U,-,A,-,T,P,P,Chemiluminescent Detection,CSPD / CDP-Star,Antibody-Conjugate,Labeled-Probe

15、,Target DNA,Membrane,Digoxigenin,Alkaline Phosphatase,Substrate,Substrate,標(biāo)記和檢測(cè)試劑盒,DIG-High Prime DNA Labeling and Detection Starter Kit I (顯色法) DIG-High Prime DNA Labeling and Detection Starter Kit II (化學(xué)發(fā)光法) DIG Northern Starter Kit DIG DNA Labeling and Detection Kit DIG Nucleic Acid Detection Kit

16、,NBT/BCIP Color Substrates and Reaction Products(顯色檢測(cè)),fragment size (bp),4907 2176 1766 1230 1033 653 517 453 394 298, 298 234, 234 220 194, 194,同源Southern雜交中顯色法檢測(cè)DNA,醋酸纖維膜,尼龍膜 (uncharged membrane),fragment size (bp),4907 2176 1766 1230 1033 653 517 453 394 298, 298 234, 234 220 194, 194,300,100,30

17、,10,3,1,300,100,30,10,3,1,DNA loaded per lane (pg),DNA loaded per lane (pg),Target DNA: Dilution buffer: Probe: Detection:,pBR 328 digested separately with EcoRl, Bg/l and Hinfl; 50 g/ml Herring sperm DNA 10 mM Tris-HCL, 1 mM EDTA, pH 8 Digoxigenin-labeled pBR328 DNA NBT/BCIP 16 hours,Chemiluminesce

18、nce Substrates and Reaction Products(化學(xué)發(fā)光檢測(cè)),同源Southern雜交 化學(xué)發(fā)光檢測(cè)的靈敏度,pBR328-fragments,100,Target: pBR328 digested with BamHl, Bgl l and Hinfl, 100; 10; 1 pg loaded per lane Probe: 20 ng/ml DIG-labeled pBR328 DNA (random priming),Polaroid-film, 5 min. exp.,X-ray-film, 3 min. exp.,fragment size (bp),4

19、907 2176 1766 1230 1033 653 517 453 394 298 (d) 234(d),10,1,100,10,1, 70 fg,不同發(fā)光 底物CSPD/ CDP-STAR 比較,典型的Southern雜交,用Southern-Blot 雜交法檢測(cè) 同源單拷貝基因,(Courtesy of Thomas Lahaye, Univ. Aachen, Germany),Target: Probe: Hybridization: Washes: Detection: Exposure.,10 mg DNA of 6 different barley cultivars, cut

20、 with EcoRI, separated on 0.8% agarose gel, followed by capillary transfer Single-copy DNA sequence of barley labeled by PCR DIG EASY HYB, 38C o.n. 2 l PCR product/ml 2 x 5 min. room temp. 2 x SSC, 0.1% SDS 2 x 15 min. 68C; 0.5 x SSC, 0.1% SDS CSPD 8 min.,r,典型的Northern雜交,(Courtesy of C. Kraemer, Ins

21、titute for Molecular Genetics, University of Mainz, Germany),Detection of the DmX Gene from Drosophila Melanogaster on a Northern Blot,Target: Probe: Exposure:,1 g poly (A)+ RNA extracted from D. melanogaster adult (lane 1) and 500 ng poly (A)+ RNA from larvae (lane 2), separated on a MOPS - formaldehyde gel followed by capillary transfer Antisense RNA 5 min. ( 32P-labeled probe 14 days !),11.5 kb,1 2,典型原位雜交,原位雜交,(Courtesy of N. Batel, Baltimore, USA),Detection of Developmentally Regulated Gene Expression in Wh