SSR技术原理,方法及步骤

.SSR技术1. SSR简介说明：简单重复序列(Simple Sequence Repeat，SSR)，简单重复序(SSR)也称微卫星DNA，其串联重复的核心序列为1-6 bp，其中最常见是双核苷酸重复，即(CA) n和(TG) n每个微卫星DNA的核心序列结构相同，重复单位数目10-60个，其高度多态性主要来源于串联数目的不同。SSR标记的基本原理：根据微卫星序列两端互补序列设计引物，通过PCR反应扩增微卫星片段，由于核心序列串联重复数目不同，因而能够用PCR的方法扩增出不同长度的PCR产物，将扩增产物进行凝胶电泳，根据分离片段的大小决定基因型并计算等位基因频率。在真核生物中，存在许多2-5bp简单重复序列，称为“微卫星DNA”其两端的序列高度保守，可设计双引物进行PCR扩增，揭示其多态性。SSR具有以下一些优点：(l)一般检测到的是一个单一的多等位基因位点；(2)微卫星呈共显性遗传，故可鉴别杂合子和纯合子；(3)所需DNA量少。显然，在采用SSR技术分析微卫星DNA多态性时必须知道重复序列两端的DNA序列的信息。如不能直接从DNA数据库查寻则首先必须对其进行测序。SSR的分类：根据SSR核心序列排列方式的不同，可分为3种类型：1）完全型(perfect)，指核心序列以不间断的重复方式首尾相连构成的DNA。如： ATATATATATATATATATATATATATATATATAT；2）不完全型(imperfect)，指在SSR的核心序列之间有3个以下的非重复碱基,但两端的连续重复核心序列重复数大于3 。如：ATATATATGGATATATATATCGATATATATATATATATGGATATATATAT；3）复合型(compound) ，指2个或2个以上的串联核心序列由3个或3个以上的连续的非重复碱基分隔开,但这种连续性的核心序列重复数不少于5 。如：ATATATATATATATGGGATATATATATATA 。3种类型中完全型是SSR标记中应用较多的一种类型。SSR在植物基因组中的分布：SSR广泛分布于各种真核生物的基因组中，大约每隔1050kb就存在一个SSR。哺乳动物中的SSR的数量大约为植物中的56倍。在植物中，平均23.3kb就有一个SSR；双子叶植物中的SSR数量大于单子叶植物，前者两个SSR之间的平均间距为21.2kb，后者为64.6kb；核DNA中的SSR数量多于细胞质DNA中的SSR，绝大多数单碱基重复型及2碱基重复型SSR存在于非编码区，3碱基重复型多位于编码区。微卫星的利用价值：由于核心序列重复数的不同，等位的SSR位点可呈现出多态性（SSLP，simple sequence length polymorphism）。大多表现为共显性遗传，有的表现为显性遗传。由于SSR DNA两侧序列（离开20bp以上）表现出保守特征，所以可设计出上下游PCR引物，扩增出包含SSR的DNA序列。微卫星分析常用于：遗传图谱构建；种质鉴定；遗传多样性分析；标记辅助选择（MAS，marker- assistant seletion，marker- aided seletion）；基因定位；数量性状基因座（QTL）分析；系谱分析；亲源关系鉴定等。SSR引物的来源：借鉴其他近缘种序列。1）通过筛选文库、测序开发自己的SSR引物。 2）通过核酸数据库查询，从已有序列中搜寻包括SSR的序列并设计引物。SSR分析实验的主要技术环节：提取DNA；PCR扩增；电泳及显色；电泳胶板带型的照相、记录；数据分析处理。其中，PCR产物分离的电泳方法主要有：高浓度琼脂糖电泳（4%胶只能分辨4-6bp差异）；变性聚丙烯酰胺序列胶电泳；非变性聚丙烯酰胺凝胶电泳。 由于扩增的片段短(一般小于300bp)，基因间的差异小(一般为几个bp)，故通常使用分辨率高的聚丙烯酰胺凝胶电泳。在程序上，变性胶虽然比非变性胶麻烦些，但考虑到在非变性胶上会出现人为假象异源双链分子，比如导致SSR杂合子中出现3-4条带，而不是正常的2条带，从而干扰等位位点统计，因此我们建议在SSR分析中均采用变性胶电泳。2. ISSR分子标记的实验原理及操作流程原理：ISSR（inter-simple sequence repeat）标记是一种类似RAPD，但利用包含重复序列并在3或5锚定的单寡聚核酸引物对基因组进行扩增的标记系统，即用SSR引物来扩增重复序列之间的区域。其原理具体是，ISSR标记根据生物广泛存在SSR的特点，利用在生物基因组常出现的SSR本身设计引物，无需预先克隆和测序。用于扩增的引物一般为16-18个碱基序列，由1-4个碱基组成的串联重复和几个非重复的锚定碱基组成，从而保证了引物与基因组DNA中SSR的5或3末端结合，导致位于反向排列、间隔不太大的重复序列间的基因组节段进行PCR扩增。 一、实验材料 不同来源的DNA(30-50ng/ul)。 二、实验设备 PCR仪，PCR管或硅化的0.5ml eppendorf管，电泳装置，凝胶成像仪。 三、试剂 1、ISSR引物：购买成品或根据加拿大British Columbia大学设计的ISSR引物序列（见附录）自己合成2、Taq酶3、10xPCR 缓冲液4、MgCl2：25mmol/L5、dNTP：2.5mmol/L。 四、操作步骤： 1. 在25ul反应体系中，加入 模板DNA 1ul (30-50ng) ISSR引物 1ul (约5pmol) 10xPCR Buffer 2.5ul MgCl2 2ul dNTP 2ul Taq酶 1单位（U） 加ddH2O 至 25ul 混匀稍离心, 加一滴（约20 ul）矿物油。 2. 在PCR仪中预变性94 2分钟, 然后循环: 94 1分钟， 45-68 40秒， 72 1-2分钟，共40轮循环。 3. 循环结束后, 72 10分钟，4保存。 4. 取PCR产物15ul加3ul上样缓冲液(6x)于1.6% 或1.8% 琼脂糖胶上电泳, 稳压50-100（电压低带型整齐， 分辨率高）。 5. 电泳结束，溴化乙锭染色20分钟。 6. 用凝胶成像仪观察、拍照。 操作流程简图：3. SSR GEL and Silver Staining ProtocolPaula Marquardt & Craig EchtPublished in: Echt CS, May-Marquardt P, Hseih M, Zahorchak 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 Research 5985 Highway K Rhinelander, WI 54501 USA Phone: 715-362-1121 Fax: 715-362-1166 e-mail: I. EQUIPMENT:DNA sequencing unit (35 x 45 cm) & 2000V power supplyClampsLg. 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 this 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 electrophoresis / 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 (Stratagene) 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. Change 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% ethanol, 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, dont use too much pressure. This treatment needs to be repeated every 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 disposable 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.)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. 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-packs 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 will 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 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) 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 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 sharktooth 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 stored 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 PREPARATION: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 (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 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 buffer 8.6 ul of 30 ng / ul PGEM 12 ul of SSRP buffer heat 95 for 2 min., ice and 5 ul 1X PE II buffer 2.5 ul of 400 ng / ul of sonicated Poly-dA 7.5 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., 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. 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, 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 insertion. 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 constant temperature 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 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) and silver nitrate (RT), stored in microtubes. -All incubations and washes are performed at room temperature, but the developer solution must be pre-chilled to 4-10 to minimize brown background. -It is important to keep the stop solution at 4-10 as well, for the same reason. -Solutions containing formaldehyde should be handled in a fume 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 formaldehyde, 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 4 mg sodium thiosulfate. Chill developer to 4-10.2. 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. Place 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 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 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 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 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 equal