SSR技術(shù)原理,方法及步驟

1、SSR技術(shù)1. SSR簡(jiǎn)介說(shuō)明：簡(jiǎn)單重復(fù)序列(Simple Sequence Repeat, SSR),簡(jiǎn)單重復(fù)序(SSR)也稱微衛(wèi)星 DNA , 其串聯(lián)重復(fù)的核心序列為 1-6 bp,其中最常見是雙核甘酸重復(fù),即(CA) n和(TG) n每個(gè)微衛(wèi)星DNA的核心序列結(jié)構(gòu)相同,重復(fù)單位數(shù)目 10-60個(gè),其高度多態(tài)性主要來(lái)源于串聯(lián)數(shù)目 的不同.SS刖記的根本原理：根據(jù)微衛(wèi)星序列兩端互補(bǔ)序列設(shè)計(jì)引物,通過(guò) PCR反響擴(kuò)增微 衛(wèi)星片段,由于核心序列串聯(lián)重復(fù)數(shù)目不同,因而能夠用PCR的方法擴(kuò)增出不同長(zhǎng)度的PCR產(chǎn)物,將擴(kuò)增產(chǎn)物進(jìn)行凝膠電泳,根據(jù)別離片段的大小決定基因型并計(jì)算等位基因頻率.在真核生物中,

2、存在許多 2-5bp簡(jiǎn)單重復(fù)序列,稱為 微衛(wèi)星DNA其兩端的序列高度保守, 可設(shè)計(jì)雙引物進(jìn)行 PCR擴(kuò)增,揭示其多態(tài)性.SSR具有以下一些優(yōu)點(diǎn):(l) 一般檢測(cè)到的是一個(gè)單一的多等位基因位點(diǎn)；(2)微衛(wèi)星呈共顯性遺傳,故可鑒別雜合子和純合子；(3)所需DNA量少.顯然,在采用 SSR技術(shù)分析微衛(wèi)星DNA多態(tài)性時(shí)必須知道重復(fù)序列兩端的DNA序列的信息.如不能直接從 DNA數(shù)據(jù)庫(kù)查尋那么首先必須對(duì)其進(jìn)行測(cè)序.SSR的分類:根據(jù)SSR核心序列排列方式的不同,可分為3種類型：1)完全型(perfect),指核心序列以不間斷的重復(fù)方式首尾相連構(gòu)成的DNA.如：ATATATATATATATATATATAT

3、ATATATATATAT ; 2)不完全型(imperfect),指在 SSR 的 核心序列之間有3個(gè)以下的非重復(fù)堿基,但兩端的連續(xù)重復(fù)核心序列重復(fù)數(shù)大于3.如：ATATATATGGATATATATATCGATATATATATATATATGGATATATATAT; 3)復(fù)合型(compound),指2個(gè)或2個(gè)以上的串聯(lián)核心序列由3個(gè)或3個(gè)以上的連續(xù)的非重復(fù)堿基分隔開,但這種連續(xù)性的核心序列重復(fù)數(shù)不少于5.如：ATATATATATATATGGGATATATATATATA .3種類型中完全型是 SSR標(biāo)記中應(yīng)用較多 的一種類型.SSR在植物基因組中的分布 ：SSR廣泛分布于各種真核生物的基因組中,

4、大約每隔1050kb就存在一個(gè)SSR.哺乳動(dòng)物中的 SSR的數(shù)量大約為植物中的 56倍.在植物中,平 均23.3kb就有一個(gè)SSR;雙子葉植物中的 SSR數(shù)量大于單子葉植物, 前者兩個(gè)SSR之間的 平均間距為21.2kb ,后者為64.6kb;核DNA中的SSR數(shù)量多于細(xì)胞質(zhì) DNA中的SSR,絕 大多數(shù)單堿基重復(fù)型及 2堿基重復(fù)型SSR存在于非編碼區(qū),3堿基重復(fù)型多位于編碼區(qū).微衛(wèi)星的利用價(jià)值：由于核心序列重復(fù)數(shù)的不同,等位的SSR位點(diǎn)可呈現(xiàn)出多態(tài)性SSLP, simple sequence length polymorphism .大多表現(xiàn)為共顯性遺傳,有的表現(xiàn)為顯性 遺傳.由于SSR D

5、NA兩側(cè)序列離開 20bp以上表現(xiàn)出保守特征,所以可設(shè)計(jì)出上下游 PCR引物,擴(kuò)增出包含 SSR的DNA序列.微衛(wèi)星分析常用于：遺傳圖譜構(gòu)建；種質(zhì)鑒定； 遺傳多樣性分析；標(biāo)記輔助選擇 MAS, marker- assistant seletion, marker- aided seletion； 基因定位；數(shù)量性狀基因座QTL分析；系譜分析；親源關(guān)系鑒定等.SSR引物的來(lái)源：借鑒其他近緣種序列.1通過(guò)篩選文庫(kù)、測(cè)序開發(fā)自己的SSR引物.2通過(guò)核酸數(shù)據(jù)庫(kù)查詢,從已有序列中搜尋包括SSR的序列并設(shè)計(jì)引物.SSR分析實(shí)驗(yàn)的主要技術(shù)環(huán)節(jié)：提取DNA; PCR擴(kuò)增；電泳及顯色；電泳膠板帶型的照相、記錄；

6、數(shù)據(jù)分析處理.其中,PCR產(chǎn)物別離的電泳方法主要有：高濃度瓊脂糖電泳4%膠只能分辨4-6bp差異；變性聚丙烯酰胺序列膠電泳；非變性聚丙烯酰胺凝膠電泳.由于擴(kuò)增的片段短一般小于300bp,基因間的差異小一般為幾個(gè)bp,故通常使用分辨率 高的聚丙烯酰胺凝膠電泳.在程序上,變性膠雖然比非變性膠麻煩些,但考慮到在非變性膠上會(huì)出現(xiàn)人為假象一異源雙鏈分子,比方導(dǎo)致SSR雜合子中出現(xiàn)3-4條帶,而不是正常的2 條帶,從而干擾等位位點(diǎn)統(tǒng)計(jì),因此我們建議在SSR分析中均采用變性膠電泳.2. ISSR分子標(biāo)記的實(shí)驗(yàn)原理及操作流程原理：ISSR inter-simple sequence repeat標(biāo)記是一種類似

7、 RAPD ,但禾U用包含重復(fù)序 列并在3'或5'錨定的單寡聚核酸引物對(duì)基因組進(jìn)行擴(kuò)增的標(biāo)記系統(tǒng),即用SSR引物來(lái)擴(kuò)增重復(fù)序列之間的區(qū)域.其原理具體是,ISSR標(biāo)記根據(jù)生物廣泛存在 SSR的特點(diǎn),利用在口物基因組常出現(xiàn)的 SSR本身設(shè)計(jì)引物,無(wú)需預(yù)先克隆和測(cè)序.用于擴(kuò)增的引物一般為16-18個(gè)堿基序列,由1-4個(gè)堿基組成的串聯(lián)重復(fù)和幾個(gè)非重復(fù)的錨定堿基組成,從而保證了引物與基因組DNA中SSR的5'或3'末端結(jié)合,導(dǎo)致位于反向排列、間隔不太大的重復(fù)序列間 的基因組節(jié)段進(jìn)行 PCR擴(kuò)增.一、實(shí)驗(yàn)材料不同來(lái)源的 DNA30-50ng/ul.二、實(shí)驗(yàn)設(shè)備PCR儀,PC

8、R管或硅化的0.5ml eppendorf管,電泳裝置,凝膠成像儀.三、試劑1、ISSR引物：購(gòu)置成品或根據(jù)加拿大 British Columbia大學(xué)設(shè)計(jì)的ISSR引物序列見 附錄自己合成2、 Taq 酶3、10xPCR緩沖液4、MgCl 2： 25mmol/L5、dNTP： 2.5mmol/L.四、操作步驟：1 .在25ul反響體系中,參加模板 DNA 1ul (30-50ng)ISSR 弓 I物 1ul (約 5pmol)10xPCR Buffer 2.5ulMgCl 2 2uldNTP 2ulTaq酶1單位U加 ddH2O 至 25ul混勻稍離心,加一滴約20 ul礦物油.45 C-6

9、8 c 40 秒,72 c 1-22 .在PCR儀中預(yù)變性94 C 2分鐘,然后循環(huán)：94 C 1分鐘,分鐘,共40輪循環(huán).3 .循環(huán)結(jié)束后,72C 10分鐘,4c保存.4 .取PCR產(chǎn)物15ul加3ul上樣緩沖液50-100 V 電壓低帶型整潔,分辨率高.5 .電泳結(jié)束,澳化乙錠染色20分鐘.6 .用凝膠成像儀觀察、拍照.操作流程簡(jiǎn)圖：3、 SSR GEL and Silver Staining ProtocolPaula Marquardt & Craig EchtPublished in: Echt CS, May-Marquardt P, Hseih M, Zahorchak

10、R. 1996. Characterization of microsatellite markers in eastern white pine. Genome 39:1102-1108 .Comments can be directed to Paula Marquardt at:USDA Forest Service Research5985 Highway KRhinelander, WI 54501USAPhone: 715-362-1121Fax: 715-362-1166e-mail: pmarquarI. EQUIPMENT:DNA sequencing unit (35 x

11、45 cm) & 2000V power supply ClampsLg. plastic trays (4), about 43 x 50 x 8 cm, and one lidTwo rocking platformsHeat block for microtiter plates. A microplate vortexer is helpful.II. MOLD ASSEMBLY:Notes: Bind silane is toxic and should be used in a chemical fume hood. Wear gloves when handling th

12、is solution. Use a small piece of vinyl tape on a lower outside corner of the acrylease treated glass gel plate to mark the untreated side and also help distinguish the plates. This helps avoid confusion between plates when using offset plates. The tape can remain in place through several electropho

13、resis / washing cycles.1. Wash inner and outer plates well with alconox cleanser. Rinse well with tap water, deionized or distilled water, and ethanol, air dry. Use dedicated sponges for each treatment.2. Using a kimwipe tissue, coat the inner side of notched or offset plate with acrylease (Stratage

14、ne) and allow to dry-I do not treat the top 2 inches of the plate since I feel that the nonstick coating promotes leaking between wells behind the teeth of the comb. Buff well with a kimwipe soaked in ethanol for a clean finish; this takes some elbow grease to get the streaks off of the plate. Chang

15、e gloves before working with bind silane and take care not to cross contaminate the plates with the two treatments. The acrylease treatment only needs to be repeated every four gels or so.3. Prepare fresh 1 ml binding solution by making a solution 0.0005-0.001% bind silane (Sigma #M-6514) in 95% eth

16、anol, 0.5% glacial acetic acid. Apply with a kimwipe and coat the inner side of the larger plate with one ml and allow to dry 4-5 minutes. Wipe the plate with ethanol in one direction and then perpendicular to first direction, don"t use too much pressure. This treatment needs to be repeated eve

17、ry time.III. GEL SOLUTION PREPARATION:Note: Acrylamide is toxic. Wear gloves when handling solution and face mask when weighing out powder. A safer alternative is to buy a premix.1. Rinse all glassware and plastic ware with d.i. water prior to gel solution preparation and pouring, including the disp

18、osable filter unit.2. Gels are 6% acrylamide, 8M urea, 1X TBE. For each gel, mix together 50 g urea, 15 ml 40% 19:1 acrylamide solution, and 31 ml d.i. water. We use a 4.5% gel for fingerprinting reactions.Note: (We store aliquots of premixed 40% Acrylogel solution, Gallard-Schleisinger Ind., at -20

19、 C.)3. Warm and stir the mixture in a beaker of warm water until all the urea is dissolved. Add 1.25 g of amberlite resin and stir 5 min. Filter through a 0.2 uM filter and degas at 25 mg Hg for 5 min. Transfer to graduate cylinder and add 10 ml 10x TBE, bringing volume to 100 ml with d.i. water.4.

20、We have recently started using Burst-Pack from Owl Scientific, which is an acrylamide premix including the buffer and catalysts, for our fluorescent gel work and are very pleased with the quality and reproducibility. This would be an option for the silver staining work as well. The burst-pack"s

21、 eliminate the c hemical weighing and mixing, deionizing, filtering and degassing steps.IV. GEL POURING:1. Immediately prior to pouring, add 500 ul 10% ammonium persulfate (0.1 g + 1 ml d.i. water) to the acrylamide mix in a beaker, gently mixing well. Then add 50 ul TEMED and mix. Polymerization wi

22、ll not start until TEMED has been added. Do not mix the catalysts together before adding to the polyacrylamide solution-this will inhibit polymerization.2. We use the Otter adjustable gel caster (OWL Scientific, Inc.) for pouring gels. In this system the gel is poured horizontally with the top plate

23、 sliding over the bottom plate, without the use of tape, grease or a bottom spacer.a. Place the larger plate onto caster so that it abuts the end wall of the caster. Moisten spacers with water and place them flush to the edge of glass against the caster wall.b. Place the top plate (notched or offset

24、) so that its top edge overlaps the bottom edge of the lower plate by 3-4 cm. Using a 60 ml syringe, slowly dispense the gel solution between the plates, allowing the solution to flow by capillary action. Gently slide the top plate across the bottom plate while dispensing the gel solution along the

25、leading edge of the top plate. If any bubbles form while pouring, try sliding the top plate back to uncover the bubble, then proceed. A more effective method is to drag out the bubbles with a plastic hook (free by request from Promega Corp.)3. Once the gel is poured, insert the flat edge of a sharkt

26、ooth comb (or a casting comb) into the top of the gel to the depth desired for the wells. Place 2-3 clamps along the sides and top to keep plates in tight contact with the spacers and comb while the gel is polymerizing.4. Allow the gel to polymerize at room temperature (RT) for 1 hour. Gel can be st

27、ored at RT over night if steps are taken to prevent it from drying out. To do this, place paper towels dampened with running buffer over the top (remove clamps but leave comb in place) and bottom edges of the gel mold and wrap with plastic wrap. Do not store the gel under buffer.V. SAMPLE PREPARATIO

28、N:Notes: Heat samples immediately prior to loading. Keep the loading dye fresh. Use SSRP loading dye that is less than 2 weeks old. The deionized formamide used in making the loading dye should be less than one month old.1. Denature the sample DNA by adding 1 volume (10 ul) of fresh SSRP loading dye

29、 (10 mM NaOH, 95% formamide, 0.05% bromophenol blue, 0.05% xylene cyanol) to 1 volume of PCR sample in a microtiter plate. Mix well and heat to 95oC for 2 min. Place on ice. 2. Molecular weight standards are PGEM (Promega) and Poly-dA (Pharmacia # 27-7836-01) sonicated to produce a 1 bp ladder. PGEM

30、 is loaded in a well separate from poly A. We do not use poly A for fingerprinting gels. Using a 144-well, microtiter 4X offset comb, load 3 ul of the mix:3.4 ul 1X Perkin Elmer II PCR buffer8.6 ul of 30 ng / ul PGEM12 ul of SSRP bufferheat 95c for 2 min., iceand5 ul 1X PE II buffer 2.5 ul of 400 ng

31、 / ul of sonicated Poly-dA7.5 ul SSRP bufferVI. ELECTROPHORESIS:Note: Adding sodium acetate to the bottom reservoir during electrophoresis (Sheen and Seed, 1988, Biotechniques 6:942-944) produces the same effect as running wedge shaped gels or adding a gradient to the gels themselves (Biggin et. al.

32、, 1983, PNAS 80:3963-3965). In all three methods, the mobility of small DNA fragments is restarded as they approach the bottom of the gel. The sodium acetate method is simpler than the other methods.1. Remove clamps. Clean excess polyacrylamide and urea from the top of plate assembly with d.i. water

33、. Pull the comb out of the mold slowly and evenly, cleaning out the comb area with d.i.water or buffer.2. Add reservoir buffers to the apparatus. The top reservoir buffer is 1X TBE. The bottom buffer reservoir is 2/3X TBE, 1 M sodium acetate. Make 1500 ml bottom buffer for each gel (100 ml 10x TBE,

34、900 ml d.i. water, 500 ml 3 M NaAcetate).3. Pre-electrophorese for 5-10 min to warm the plate so that the comb will easily slide in place. Clean out comb area with buffer. To prevent possible well to well leakage, apply a very light coating of celloseal to the outside surface of the comb, prior to i

35、nsertion. Place plate assembly in gel box and clamp.4. For microtiter format, a hamilton 8 or 12 channel syringe loading device for loading the gels is recommended. Clean out each group of wells immediately prior to loading with the multichannel hamilton syringe. Run the gel at 50 C constant tempera

36、ture and 100 watts limiting power for about 1.5 - 3 hours, depending on size of amplification product. Constant temperature can be maintained with the temperature probe option of the BioRad Power/Pak 3000 power supply.VII. GEL FIXING, STAINING AND COLOR DEVELOPMENT:Notes and tips: -This procedure is

37、 adapted from the Promega Silver Sequence protocol. -Use highest quality reagents. -The fixer, stain and developer are made with 18 mega-ohm water (deionized= d.i.). -The washes are done with distilled water. -We use pre-measured sodium thiosulfate (-20 C ) and silver nitrate (RT), stored in microtu

38、bes. -All incubations and washes areperformed at room temperature, but the developer solution must be pre-chilled to 4-10 C to minimize brown background. -It is important to keep the stop solution at 4-10 C as well, for the same reason. -Solutions containing formaldehyde should be handled in a fume

39、hood. The formaldehyde is aliquoted (RT) -Wear gloves throughout the procedure. - Wearing an apron will prevent silver stains on clothing. -Rocking platforms are better than orbital shakers to achieve even gel staining.1. Freshly prepare staining and developer solutions while gel is running. Add for

40、maldehyde, silver nitrate and sodium thiosulfate to solutions immediately prior to use. To prepare staining solution, combine 2 L d.i. water, 2 g silver nitrate and 3 ml 37% formaldehyde. To prepare developing solution, combine 2 L chilled d.i. water, 60 g sodium carbonate, 3 ml 37% formaldehyde and

41、 4 mg sodium thiosulfate. Chill developer to 4-10 C .2. Remove gel assemble from rig, remove side spacers and carefully separate glass plates with a spatula. The gel will remain attached to the plate coated with bind silane. Cut off the top corner of the high numbered end of gel for orientation.3. P

42、lace gel and plate in a shallow plastic tray containing sufficient fresh fix/stop solution (2 liter 7.5% glacial acetic acid) and gently agitate for 20 minutes or until tracking dye completely disappears. We sometimes use a multiple gel holder from Promega for processing several gels at a time. Two

43、gels require the same volume of solution as one. For three gels, solution volumes are increased by 50%.Note: For overnight storage, the gel can be fixed as above, rinsed with d.i. water and stored in fresh fixer without agitation.4. Rinse the gel 3 times for 2 minutes each with distilled water with

44、gentle agitation.5. Add staining solution to gel and gently agitate for 30 minutes, decant and precipitate silver. The silver in the used staining solution is precipitated with NaCl (10 g /2 L) for recycling.6. Rinse gel with distilled water for 5 seconds (from time gel is placed in distilled water

45、to time into developer, i.e. a quick dip). Longer rinses result in weaker signal. If the rinse goes too long, repeat step 5 with the staining solution.7. Transfer the gel to 800 mls of chilled developing solution and gently agitate with a rocking motion until the bands first become visible, usually

46、within in a few minutes. Decant the used developer, add the remaining chilled developer and continue to gently agitate until the bands have reached desired intensity. Over-development will result in a brown background with low contrast bands rather than a pencil gray background with sharp bands.8. To terminate the developing reaction, add an