Improved hybridization conditions for DNA 'fingerprints' probed with M13.
Improved hybridization conditions for DNA 'fingerprints' probed with M13.
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改进了用 M13 探测的 DNA“指纹”的杂交条件。
DOI：
10.1093/nar/16.9.4161
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发表时间：
1988
影响因子：
14.9
通讯作者：
Aquadro,CF
中科院分区：
文献类型：
--
作者：
Westneat,DF;Noon,WA;Reeve,HK;Aquadro,CF
野生型M13噬菌体DNA已被证明可以检测人类和其他几种哺乳动物中高度可变的小卫星序列（DNA指纹）（1）。我们在这里报告新的杂交条件，显着提高分辨率和一致性的DNA指纹与M13探针获得。Vassart et al. (1)使用基于脱脂乳的”印迹”杂交混合物，发现与膜（硝酸纤维素或尼龙）产生不一致的杂交和通常高水平的背景杂交。这可能反映了不同批次和/或品牌的脱脂奶粉成分的差异。这些问题通过使用基于SDS、BSA和磷酸钠的预杂交和杂交条件来消除（2）。这些条件以类似于”印迹”的方式起作用，因为在杂交期间不存在脊椎动物载体DNA（例如来自鲑鱼精子的DNA）。这种新的协议同样适用于尼龙或硝酸纤维素膜，并大大提高了用M13 DNA作为小卫星探针获得的DNA指纹。在我们的实验室中，使用Jeffreys的33.15小卫星探针的这种方案也产生了改进的DNA指纹。我们推荐以下Southern印迹方案：Southern转移：电泳后，将凝胶浸泡在1.5 M NaCl、1.5 M NaOH中两次，每次15分钟，然后在1 M乙酸铵、0.04 M NaOH中洗涤两次，每次15分钟。在放置膜之前（硝化纤维素（Schleicher和Schuell）或尼龙（Zetabind，AMF Cuno）），将其在水中润湿5-10分钟，然后用1 M乙酸铵润湿5分钟。在1 M乙酸铵/0.04M NaOH中进行转移过夜，干燥过滤器并在80 ℃下烘烤2小时。将过滤器在5xSSC中短暂润湿，然后置于含有7%SDS、ImM EDTA的热封袋中（pH 8.0），0.263 M Na 2 HPO 4和1%牛血清白蛋白（级分V）[0.5 M Na 2 HPO 4（pH 7.2）储备液由134 g Na 2 HPO 4·7 H2O和约4 ml 85%H3PO4/L组成]。我们使用10 ml用于400 cm 2过滤器。预杂交在60 ℃下进行过夜.杂交在相同的溶液中于60 ℃下进行24-48小时，加入32 μ l。标记探针（我们使用随机引物探针）。洗涤：对于M13探针：1）在2xSSC、0.1%SDS中在室温下洗涤两次，每次15分钟，然后2）在相同溶液中在60 ℃下洗涤一次，每次15分钟. 3)在包裹以暴露于胶片之前，在室温下在lxSSC中短暂冲洗过滤器。对于Jeffreys的33.15探针：与上述洗涤1相同，但洗涤2和3的温度都升高到65 ℃，洗涤3持续30分钟。暴露时间通常为1天，使用2个增感屏，没有增感屏为6-7天。在后一种洗涤条件下，我们检测到靛蓝色白颊雀（Passerina cyaneal，Jeffreys的33.15探针（在M13中克隆的重复核心序列）和野生型M13）中的小卫星的非常少的交叉杂交。使用对33.15探针的核心序列具有特异性的探针应允许检测与随机引物M13 DNA检测到的小卫星序列不同的小卫星序列。
Wildtype M13 phage DNA has been shown to detect highly variable minisatellite sequences (DNA fingerprints) in humans and several other mammals (1). We report here new hybridization conditions that significantly improve the resolution and consistency of DNA fingerprints obtained with the M13 probe. The conditions suggested by Vassart et al.(1), using the skim milk based" blotto" hybidization cocktail, were found to give inconsistant hybridization and often high levels of background hybridization to the membrane (nitrocellulose or nylon). This may reflect variation in the components of different lots and/or brands of dried skim milk used. These problems are eliminated by the use of prehybridization and hybridization conditions based on SDS, BS A and sodium phosphate (2). These conditions function in a similar manner to" blotto" in that no vertebrate carrier DNA (such as that from salmon sperm) is present during hybridization. This new protocol works equally well with nylon or nitrocellulose membranes and has substantially improved the DNA fingerprints obtained with M13 DNA as a minisatellite probe. The use of this protocol with Jeffreys' 33.15 minisatellite probe has also yielded improved DNA fingerprints in our laboratory. We recommend the following Southern blot protocol: Southern transfer: After electrophoresis, soak gel in 1.5 M NaCl, 1.5 M NaOH twice for 15 min. each, followed by two 15 min. washes in 1 M ammonium acetate, 0.04 M NaOH. Before placing the membrane (nitrocellulose (Schleicher and Schuell) or nylon (Zetabind, AMF Cuno)) on the gel, it is wetted in water for 5-10 min. followed by 1 M ammonium acetate for 5 min. Tranfer is carried out overnight in 1 M ammonium acetate/0.04 M NaOH and the filter is dried and baked at 80 C for 2 hr. Prehybridization: Wet the filter in 5xSSC briefly, then place in a heatseal bag containing 7% SDS, lmM EDTA (pH 8.0), 0.263 M Na2HPO4 and 1% bovine serum albumin (fraction V)[0.5 M Na2HPO4 (ph 7.2) stock is composed of 134 g of Na2HPO4-7H2O and approximately 4 ml of 85% H3PO4 per liter]. We use 10 ml for a 400 cm^ filter. Prehybridization is carried out at 60 C overnight. Hybridization is carried out in the same solution at 60 C for 24-48 hr with the addition of 32p. labeled probe (we used random-primed probes). Washes: For M13 probe: 1) Twice each for 15 min. in 2xSSC, 0.1% SDS at room temperature, followed by 2) one 15 min. wash in the same solution at 60 C. 3) The filter is rinsed briefly at room temperature in lxSSC before being wrapped for exposure to film. For Jeffreys' 33.15 probe: As above for wash 1, but the temperature of washes 2 and 3 are both raised to 65 C and wash 3 lasts for 30 min. Exposure times are typically 1 day with 2 intensifying screens and 6-7 days with no screens. Under the latter wash conditions, we detect very litde cross-hybridization of minisatellites in indigo buntings (Passerina cyaneal by Jeffreys' 33.15 probe (a repeated core sequence cloned in Ml3) and wildtype M13. Use of probes specific to the core sequence of the 33.15 probe should allow detection of minisatellite sequences distinct from those detected by random-primed M13 DNA.