分子遺傳學(xué)理論與技術(shù)基礎(chǔ)-chapter-iii-technology-basis第二部分

1、Chapter Ⅲ technology basis of molecular genetics,Summary about the technology for gene research,Ⅰ. isolation and purify of nuceic acid,1.Types of nucleic acid in biont: karyota’s: chromosome DNA

2、 mitochondria DNA RNA monera’s: chromosome DNA plasmid DNA RNA virus: DNA or RNA (that is enveloped by protein s

3、hell ),2.Principle of isolation and purify ①to keep nucleic acid molecular structure complete②to avoid nucleic acid sample polluted by protein or other nucleic acid ③to decrease the capacity of enzyme inhibitor in sa

4、mple,3.To isolate and purify animal chromosome DNA (鄂p271)⑴ tissue sources of genome DNA: a: fresh tissue or operating sample p274 b: blood sample P274 c: cultured cells P271,⑵ basic r

5、eagents and their function: 鄂p271-273 Antithrombotics (抗凝劑)① 2%EDTA EDTA 2g(乙二胺四乙酸) p274 (鄂p274) NaCl 0.85g add H2O to 100 ml *②ACD solution citric acid 0.48g

6、 (檸檬酸) sodium citrate 1.32g(檸檬酸鈉) glucose 1.47g add H2O to 100 ml Volume ration: blood:ACD=6:1,,,Buffer PBS （磷酸鹽緩沖液） 鄂P48

7、8 TBS （Tris-硼酸緩沖液） 鄂P487Cytolysis solution (細(xì)胞裂解液) 鄂P27210mM Tris-Cl (PH7.4):【this solution is regulated to PH7.4 by HCL solution (鄂486表)】10mM EDTA：【that is th

8、e inhibitor for DNase】150mM NaCl0.4%SDS(十二烷基硫酸鈉)【to destroy biomembrane and be added finally】Quantitative volume to 1000ml by 10mM Tris-Cl finally,,,Protase K : to degrade protein into oligo

9、peptids. the work solution is 20mg/ml,storage at -20℃.Phenol : protein denaturant of biochemical pure can be used directly. （鄂P485 盧P51） chemical pure have to be redistilled.Chlor

10、oform(氯仿)：it cooperate with phenol to speed separate the water phase from organic phase.,NH4Ac: DNA sedimentation reagent.Ethanol: to make DNA molecular dehydrate and separate out .Ether(乙醚)：to remo

11、ve phenol and chloroform from DNA sample .TE solution: it is DNA storage solution 鄂P486 10mM Tris-Cl 1mM EDTA,,⑶technology steps for isolat

12、ing genome DNA from human blood sample: (need large volume DNA ) 鄂P274 ①to get leucocyte cells from blood sample: fresh blood sample: ACD solution=6:1,mixed, 1300g centrifuge for 15min ,draw away the up laye

13、r of serum, then take up the yellowness cell layer of sediment into a new tube, avoid the red cell mixed (may repeat again)②to destroy cell membrane, releasing genome DNA:--suspend leucocyte cells in cytolysis solution

14、15ml for 1h,and waterbathing at 37 ℃,③to degrade protein of chromatin and liberating DNA molecules :--add 20mg/ml of protase K to 100ug/ml terminal concentration ,mixed to homogenous ,keep in vibrator(振蕩儀) at 50 ℃ for 3

15、h④to remove protein and extract DNA into water phase:--cold to RT ,add equal volume of solution (phenol / chloroform =1:1),vibrate softly to make water phase and organic phase mixed, centrifuge 5000g for 15min, then ex

16、tract solution is separated into 3 phases* in tube, to draw out the water phase carefully into a new tube with big bore pipette tube(0.3cm).repeat again to purify DNA.,⑤DNA sedimentation --add 1/5 volume of 10M NH4Ac,

17、mixed, then add 2 times of Ethanol(無水乙醇)，mixed, then some DNA fibers appeared in solution.⑥wash and store DNA sample --pick DNA fibers with a specific glass bar, transfer into a 70% Ethanol in a enppendorf tube for wa

18、shing, centrifuge 5000g for 5 min by table centrifuge, remove the supernatant, then drying DNA sediment at RT, then add TE solution to lyses DNA sample, storage at 4 ℃ in refrigerator,⑷to isolate minim DNA from fresh blo

19、od (少量DNA樣品制備) chelex-100 to extract the DNA (the example for blood )①add 200ul blood into 1.5ml tube, then plus sterilized water (or blood buffer ) 600 μl into tube, vortex.②1000r/min centrifuge for five min, throw

20、 away supernatant, repeat steps of 1 and 2 for 2-3 times .③add 200 μl chelex-100 solution of percent five into precipitate, 56℃ water bath for thirty min.④vortex 5-10 seconds, 100 ℃ water bath about eight min, take out

21、 of , vortex 5-10 seconds. ⑤10000r/min centrifuge for five min, supernatant contained DNA sample for PCR reaction.,⑸to isolate total RNA from karyota’s cells: 鄂P274①total RNA : mRNA tRNA rRNA

22、 hnRNA every tool small RNA unstable and degrade easily ,so it is a difficult work to extract RNA②RNase: it can catalysis every RNA degradation; the activity of RNase is very strong an

23、d stable; RNA sample can be polluted by RNase in many way, that is the main reason for isolating RNA failure .,,③attention: when do the work to isolate RNA Environment must be cleared and work system must be asepti

24、c.The aseptic appliance must be handled by DEPC(焦磷酸二乙酯)，that is the inhibitor for RNase.The work man have to work in flu mask and gloves (口罩和手套)④technology steps: 見鄂P275,⑹quantitative determination for nucleic acid

25、①ultraviolet absorptivity characteristic :For nucleic acid largest absorb wavelength 260nm least absorb wavelength 230nm Absorb wavelength: DNA ＞RNA Largest absorb : themine (T)—264.5

26、nm (DNA) urocil (U) — 259nm (RNA),,,②to examine OD value at 260nm for counting the density of nucleic acid ：the larger of OD value, the more density for the sample。 When the OD260 value=1

27、(or A260=1), the density of sample is : double strand DNA (dsDNA)=50ug/ml single strand DNA (ssDNA) or RNA=40ug/ml single strand oligonucleotide=20ug/ml③to estimate the pure degree of sample by rati

28、o of A260/A280: pure DNA =1.8 pure RNA=2.0 when sample containing Pr impurity＜1.8,,,④to examine the density of DNA or RNA sample by ultraviolet photometer :Take 5ul

29、DNA sample solution, add ddH2O to 1ml volume (diluting for 200 times) ,mixed homogeneous , then transfer into the colorimetric cup(比色杯) of photometer .To preheated photometer, then to correct the zero point with a cup o

30、f pure ddH2O.Regulate to 260 nm and 280nm wavelength position separately.,Density of DNA sample=OD260 x Diluted times x 50ug/ml for example: OD260=1.51 OD280=0.95 Density=1.51 × 200 × 50ug/1000ul=1

31、5.1ug/ul OD260/OD280=1.51/0.95=1.59To analysis the pure degree of sample : OD260/OD280＞1.8 sample contain more RNA impurity . OD260/OD280＜1.8 sample conta

32、in more Pr impurity.,Ⅱ electrophoresis technology,1.Concept the technology is to push some electric material move directionally in electric field medium with a apparatus ,the

33、n to qualitative or quantitative analysis target molecules.,2.Principle Electrophoresis sample molecular is the amphiline electrolyte (兩性電解質(zhì)),Under acidic: basic ionization, with positive electron, move to negativ

34、e polar (black line).Under basic: acidic ionization, with negative electron, move to positive polar (red line).Mobility : that is the mobile distance of charge particle in unit time ,under some electric field strength.

35、,Influence factors :①self-property of electrophoresis sample molecular (公式略).*②electric field medium: medium’s property and density, the medium work as a molecular sieve(篩).③electric field strength :In some vol

36、tage limits, the mobility is direct ratio with voltage .Under surperstrong voltage, the electrophoresis resolution (分辨率) decrease suddenly .,④ionic strength of electrophoresis buffer .To keep stable PH value limit, ot

37、herwise no good electrophoresis result . TAE (tris-醋酸-buffer) general buffer TBE (tris-硼酸-buffer) TPE (tris-硫酸-buffer),,3.Multiforms of electrophoresis te

38、chnology Electrophoresis apparatus: high voltage form (3000v) middle voltage form (300v)Electrophoresis tank: plate form 平臺式 vertical slab form 垂直板式 disc form 圓盤柱式 membrane b

39、ridge form 薄膜橋式,,,Electrophoretic medium agarose Gel polyacrylamide Gel acetic acid fibmembrane filter paper urea polyacrylamide Gel Technology types : gradient gel

40、electrophoresis denaturing gel electrophoresis SDS electrophoresis double direction electrophoresis isoelectric focusing electrophoresis,,,4.Indicator reagents: Some indicator with color

41、 for indicating sample mobile process. 鄂P489 Bromophenol blue ,Bb(溴酚藍(lán)) xylol-celason, Xc (二甲苯青)5.Stain reagents :It can stain target sample for electrophoretic band pattern analysis. Pr stain rea

42、gent: extensive stain reagent: to stain all of Pr . specific stain reagent: to stain a specific Pr.,,,Nucleic acid stain reagent : ethidium bromide, EB(溴乙錠) —stain DNA or RNA

43、 stain principle: 鄂 P485Storage solution EB 10mg at RT ddH2O 1ml *be careful to toxicant.working solution 100ml H2O *mixed to use onetime only. 10ul EB st

44、orage solution,,,6.Polyacrylamide gel electrophoresis (PAGE)①fit field: vertical slab gel disc gel to isolate Pr or nucleic acid ②main reagents:Acrylamide (Acr): 丙烯酰胺Bisa

45、crylamide(Bis): N N’-亞基雙丙烯酰胺AP: 過硫酸銨TEMED：N N N’ N’-四甲基乙二胺,,③Gel density and DNA resolution range PAG density (%w/v) DNA resolution (bp) Bb indicator relative bp 3.5(Acr+Bis) 100—2000

46、 1005.0 80-500 658.0 60-400 4512.0 40-200

47、3015.0 25-150 1520.0 6-100 12,,,,④preparation of PAGa: to compound solutions 30%PAG storage solution : Acr

48、29g, Bis 1g, dH2O add to 100ml ,mixed and lyses keep in brown bottle, at 4℃.TBE buffer(PH8.0)(5x): Tris 54g,Boric acid 27.5g, 0.5M EDTA 20ml, dH2O add to 1000ml ,

49、 lyses keep at 4℃.10%AP solution :AP 1g, dH2O add to 10ml , deliquescence(潮解) easily, closed stock, stock at 4℃ only one month.,TEMED primary solution: closed stock at 4℃. Gel loading buf

50、fer:(鄂P489) formamid 9.5ml, EB 2mg, 0.5M EDTA 520ul,mixed stock at 4℃ .Marker DNA: loading buffer 60ul, TBE(1x) 60ul, phix 174/hacIII,RF DNA 2ul ,mixed in enppondolf tube , stock at

51、 4℃ .b: to wash the appliance of electrophoresis, then drying . (electrophoresis tank gel plate, glass plate etc)C: to do gel casting(凝膠灌制)：鄂P285表14-2,,First to compute total volume of PAG solutio

52、n that is needed in this experiment. preparation of PAG work solution (100ml total volum)PAG work solution(%) 3.5 5.0 8.0 12.0 20.030%PAG stock solution 11.6 16.6 26.6

53、 40.0 66.6ddH2O 67.7 62.7 52.7 39.3 12.7TBE(5x) 20.0 20.0 20.0 20.0 20.010% AP solution

54、 0.7 0.7 0.7 0.7 0.7To add TEMED 0.005 ml finally ,mixed.Then casting gel solution rapidly, continuously, stay to polymer is formed ready.,,,,⑤electrophoresis process (run gel ) Atte

55、ntion: initiation: to make electrophoresis apparatus, pour electrophoresis buffer ready. first, connect with power, then, turn on electric curr

56、ent . termination: first turn off electric current ,then, break power.,,,⑥r(nóng)esult analysis : ?peel off gel plate . ? stain . ?observation in dark room. ?take picture.

57、7.Agarose gel electrophoresis :①fit field : plate form .isolate sample: Pr ; nucleic acid (DNA RNA).,②Gel density and DNA resolution range(鄂P282)Agarose density (% w/v) DNA resolution (kb) 0.3

58、 5-60 0.6 1-20 0.7 0.8-10 0.9 0.5-7 1.2