引物设计总结word.doc

引物设计总结寡核苷酸的优化设计郑仲承( 中国科学院上海生命科学院生物化学和细胞生物学研究所，上海 200031 )在核酸分子杂交、DNA序列测定和通过PCR放大DNA片段等实验中，都需要使用寡核苷酸作为探针或引物，而对这些反应的质量起最重要影响作用的，就是这些寡核苷酸探针或引物。用优化的寡核苷酸进行实验能够很快得到好的结果，而用不够合适的寡核苷酸时，常常得出似是而非的结果，不仅大大增加了后续实验的工作量，还可能一无所获。怎样优化设计寡核苷酸呢?至少有下列几个方面的问题需要考虑。1. 估测可能形成的DNA或RNA双链的稳定性寡核苷酸，无论是DNA的或者RNA的，都有形成双链结构的潜在可能性，正如下面反复提到的，这种结构对寡核苷酸的作用有很大影响。所以，预测这种结构的稳定性对设计和优化寡核苷酸就很重要。在一个双链结构中，碱基对的相对稳定性是由其邻近碱基决定的。在热动力学中，这样的性质以双链形成时的自由能(G)来表示。现在，大多采用 Breslauer等人提出的，以最接近的相邻核苷酸的动力学数值(自由能)来预测双链稳定性的方法。为简化起见，所有的计算都在25 条件下进行。此时，最接近的相邻核苷酸的自由能是：此主题相关图片如下：G(kcal/mol) 例如，双链d(ACGGCCGT)的G是：G(ACGG)G(AC)G(CG)G(GG)()8.0 kcal/mol此计算方法特别适用于测定其3末端会形成双链的引物的相容性。也可以用来计算发夹环结构的G。不过，这时需要根据环区内核苷酸的数量添加一定的数值。如3个核苷酸时为5.2 kcal/mol；4个时为4.5；5个为4.4；6个是4.3；7和8个为4.1 kcalmo1。2. 选择引物的一般规则设计和选择引物时有5个要素必需注意。2.1 引物的3末端不互补 引物的3末端一定不能有很大的互补性，因为它们的互补会形成引物二聚体，这就会带来很大的问题，例如合成出非专一的产物，极大地减少所期望产物的得量。有实验表明，3末端双链的G是02 kcal/mol时，PCR产量几乎达到百分之百，随着其绝对值的增加产量逐渐下降，在6时只有40%，到8时少于20%，而10时，接近于0。虽然产量还取决于其他参数，如退火温度、引物的专一性等等，但是用Taq聚合酶操作时，由于它的工作能力很强，能够在很短的时间内就识别3末端互补的双链区并发动聚合反应，即使3末端双链的稳定性很差也不能阻碍它的作用，所以这时产量对二聚体的形成就有很大的依赖性。2.2 引物分子内不互补 应当尽量不用会通过释放能量而形成分子内双链结构的寡核苷酸。虽然有些带有发夹环，其G为3 kcal/mol的自身互补引物也可以得到不错的结果，但是如果它的3末端被发夹环占据时就很麻烦，即会引发引物内部的延伸反应，减少了参与正式反应引物的数量。当然，如果发夹环在5末端对反应就没有多大的影响了。2.3 引物的组分、解链温度和长度 普遍认为PCR引物应当有50%的GC/AT比率。其实，这是不对的。以人基因组DNA为模板，用81% AT的引物可以产生单一的，专一的，长250 bp，含有70% AT的产物。完全没有必要复杂地去计算产物和引物的解链温度，PCR引物的GC/AT比率应当等于或高于所要放大的模板的GC/AT比。要知道，更重要的因素是模板与稳定性较小的引物之间解链温度的差异。差异越小，PCR的效率越高。因为DNA的解链温度也取决于它的长度，所以有的研究者喜欢设计很长，而不求它很稳定的引物。可是，引物太长就难以避免形成二聚体和自身互补，因此，一般还是不用为好。如果期待的产物长度等于或小于500 bp，选用短的(1618 mer)的引物：若产物长5 kb，则用24 mer的引物。有人用2023 mer引物得到40 kb的产物。但是，引物较长时，如果不借助引物选择的计算机软件帮助，就很难确定一对引物是否会形成二聚体，是否有自身互补性以及专一性如何。于是，用眼睛选出来的寡核苷酸放大长片段DNA时就会使引物彼此引发而不是延伸模板，得出非专一产物。通过下述的观察内部稳定性原理可以极大地减少这种问题。2.4 引物的内部稳定性 在DNA测序和PCR中最好用5末端稳定(如GC含量较多)，而3末端不太稳定(如AT含量较多)的引物，这种引物的结构可以有效地消除假引发反应。这就是基于引物内部稳定性的经验之谈。其3末端稳定性低的引物在这些反应中能起好作用的原因在于，接近或在3末端上的碱基与非靶位点碱基所形成的配对的稳定程度还不足以引发DNA合成，所以不会产生假产物。因此，为了有效地引发反应，引物的5末端和中央部分必须与靶DNA也形成双链。与此相反，带有稳定的、GC丰富的3末端的寡核苷酸不需要其所有的核苷酸序列都与靶序列配对，只凭借其3末端与靶序列任何位点的牢固配合就可以引发反应，产生非专一产物。如果用3末端低稳定性的引物，反应的最适退火温度范围会不寻常的宽。这就可以不经过事先的最佳化实验就能在最佳条件下进行反应。还要注意的是PCR反应产物的质量还取决于模板(底物的复杂性、Tm、产物长度)和退火的温度与时间。所以，有时3末端稳定的引物也可以满意地进行反应。但是，无论如何，寡核苷酸3末端最后5个核苷酸的稳定性小于9 kcal/mol的，通常就是专一性的探针或引物。寡核苷酸3末端越不稳定，假引发的可能性越低。2.5 引物的唯一性 为了放大单个的、专一性DNA片段，选用的引物序列就应当是唯一的，即在模板中没有重复序列。虽然不会整个引物序列都偏好于和模板中的一个以上位点匹配，但是，通常见到的引物的3末端往往都有67个没有什么个性的核苷酸。如果假引发的位点正好在放大区的内部，那麻烦就大了。由于短的DNA片段有更高的PCR或杂交效率，就容易产生非专一产物。如果用哺乳动物基因组序列作为模板，可以用Alu序列或其他短重复元件来核对想用的引物的互补性。由此也可知，应当避免使用同寡聚物(如AAAAAA)和二核苷酸重复(如ATATAT)。3. 按照氨基酸序列设计寡核苷酸按照多肽的氨基酸序列来设计 PCR引物或杂交探针是最常用的实验手段，尤其是在试图“钓取”一个蛋白质的基因时。此时要注意的问题有：(1)宁可用简并引物，也不用猜测的引物。氨基酸密码子的简并性给予引物设计以可塑性，这比用猜测的密码子要好得多。有人用1 024个简并引物得到很好的结果。但是，应当避免在一个区域内有很高的简并性。但也有简并性低使引物不工作的报道。(2)引物与模板的错配。一般认为，所用引物与模板有15%20%的错配，PCR的效果还能接受。但是，引物3末端的错配比同样错配率的5末端错配会引起更严重的问题。在最后4个碱基中有2个错配的引物，其 PCR产量急剧下降。但是，当核苷酸浓度高时，3末端有错配的引物还能被Tag聚合酶很好地利用。在0.8 mmol/L时，大多数3末端错配引物可以接受，虽然非专一产物比较多，DNA合成的忠实性也下降。即使在低核苷浓度下，还会有少量从错配碱基出发的合成，因此，在开始的PCR循环中把退火时间增加到35分钟，比之于用标准退火时间和高浓度核苷酸能够产生质量更好的所求产物。(3)在用唯一性引物时，建议用0.2 mmol/L或更低的总核苷酸浓度，因为高浓度会增加错误参入的比率。(4)简并寡核苷酸时，PCR应当在比较高的引物浓度下进行，即13 mol/L而不是0.2 mol/L，因为在反应混合物中的大多数寡聚物并不是被用来引发专一的反应，而只是产生高的背景而已。AMV Reverse TranscriptaseTemperature Stability: AMV RT is the preferred reverse transcriptase for templates with high secondary structure due to its stability at higher reaction temperatures (3758C).RT-PCR AnalysisSolutions10X RT Buffer10X PCR Buffer100 mM Tris pH 9.0500 mM KCl1% Triton X-10025 mM MgCl2use at a concentration of 1.5 mMLysis Solution4M GuSCN 250 g guanidine thiocyanate25mM Na citrate 7.0 17.6 ml 0.75M Na citrate pH 7.00.5% Sarkosyl 26.4 ml 10% Sarkosyladd 293 ml Qbefore use, add 72ml bMEWater Saturate Phenolthaw 500 ml phenoladd 0.5 g hydroxyquinolinadd 500 ml Qmix and allow phases to separate at room temperaturerepeat 2 times and store at 4C3M NaOAc 5.224.6 g NaOAc (anhydrous)pH to 5.2 with acetic acidup to 100 ml QProcedurerna isolation Add tissue to 400 microliters lysis solution with bME added just prior to use. If collecting several samples hold tubes on ice. These can be stored for years at -80C. Add 1 microliter glycogen, 30 microliters 3M NaOAc 5.2 and 500 microliters water saturated phenol. Mix by gentle inversion and add 100 microliters chloroform. Mix by inversion and hold on ice for 15 minutes. Spin for 10 minutes at 4C and remove the aqueous phase. Add 500 microliters isopropanol, hold at -20C for 1 hour and spin at 4C for 30 minutes. Wash and dry. Resuspend the pellet in 300 ml Lysis Solution and add 300 ml isopropanol. Hold at -20C for 1 hour and spin at 4C for 30 minutes. Wash and dry. Resuspend the pellet in 10 ml DEPC treated Q and store at -80C.reverse transcription reaction Add 5 microliters RNA to a sterile siliconized eppendorf tube along with 1 microliter of oligo dT (0.25 mg/ml). Heat to 65 for four minutes and immediately transfer to ice. Quickly spin the RNA/oligo dT and add 14 microliters of the RT cocktail:2 microliters 10X RT buffer1 microliter 1 mg/ml BSA1 microliter 20 mM DTT0.5 microliters RNaseIN0.4 microliters 25 mM dNTPs0.2 microliters AMV RT9 microliters Q Incubate at 48 for 30 minutes, dilute 5 fold and store at -20.pcr reaction Dilute cDNA (5 fold) and use between 1-5 microliters of cDNA per PCR reaction. Add the following PCR cocktail per tube:2.5 microliters 10X PCR buffer1.5 microliters 25 mM MgCl21 microliter each primer (10pmol/microliter; 55 Tm)0.2 microliters 25 mM dNTPs0.025 microliters a-32P dATP0.02 microliters Taq polymerase (protocol T.2)14 microliters Q Perform PCR using the following cycle parameters:94 for 4 minutes, (94 for 30 seconds, 55 for 30 seconds, 72 for 30 seconds) x n, 72 for 5 minutes. The number of cycle should be determined empirically to find the linear range of amplification.Ambion Technical Bulletin 176Avoiding DNA Contamination in RT-PCR A frequent cause of concern among investigators performing quantitative RT-PCR is inaccurate data due to DNA contamination in RNA preparations. Although DNA contamination is easily detected by performing a no-RT control, there is no easy remedy. In this technical bulletin, we present data showing levels of DNA contamination in RNA generated by different procedures, and suggest several precautionary measures that can be implemented to reduce the impact of this persistent problem.RT-PCR and Genomic ContaminationRT-PCR is an increasingly popular method for the quantitative analysis of gene expression. With this popularity comes a heightened awareness that most techniques used for total RNA isolation yield RNA with significant amounts of genomic DNA contamination. PCR cannot discriminate between cDNA targets synthesized by reverse transcription and genomic DNA contamination. At Ambion, we can routinely perform PCR from residual genomic DNA present in total RNA samples isolated by most commonly used techniques. To illustrate this problem, we performed RT-PCR on mouse liver RNA isolated by a multi-step guanidinium thiocyanate/acid phenol:chloroform extraction (ToTALLY RNA), a one-step extraction (Tri Reagent), a filter-binding based extraction (RNAqueous), by centrifugation through a CsCl cushion, and by two rounds of oligo d(T) selection using Ambions Poly(A)Pure Kit (see Figure 1a). Regardless of whether reverse transcriptase was added in the reverse transcription step, gene specific product is synthesized in most samples. Among the total RNA samples, the amount of DNA contamination is lowest in the CsCl-pelleted RNA. No signal is apparent in the oligo d(T)-selected sample. The PCR products in the no-RT samples are the result of amplification from trace amounts of genomic contamination. Figure 1. DNA Contamination in RNA Isolated by Five Different Methods. Mouse liver total RNA was isolated according to protocol by five different methods. 0.5 g RNA was used in RT-PCR reactions with Ambions RETROscript Kit. PCR reactions were performed with 5 g RNA. 10 l of each reaction was electrophoresed on a 2% agarose gel and stained with EtBr. Lane RNA Isolation Method 1 One Step RNA Isolation (Tri Reagent) 2 Glass Binding Method (Ambions RNAqueous Kit) 3 Acid Phenol Chloroform Method (Ambions ToTALLY RNA Kit) 4 CsCl cushion 5 Oligo dT Selection (Ambions Poly(A)Pure Kit) 6 H2O Control Differential Enrichment by Oligo d(T) SelectionAlthough two rounds of oligo d(T) selection are sufficient to remove genomic DNA contamination, there are two drawbacks to using this technique to control for DNA contamination. First, oligo d(T) chromatography is expensive and labor intensive for routine analysis. Secondly, a potentially serious problem not usually addressed is that relative amounts of individual transcripts can change with oligo d(T) chromatography, probably as a result of differential polyadenylation between tissues or in response to stimuli. At Ambion, we have found that oligo d(T) selection can even change the apparent abundance of transcripts from genes that are thought to have invariant expression. For example, when we compare the relative enrichment of cyclophilin and GAPDH transcripts by Northern blot analysis of total versus oligo d(T) selected mouse RNA, we see an obvious change in the apparent abundance of these two transcripts. As shown in Figure 2, oligo d(T) selection enriches GAPDH and cyclophilin 17X and 22X, respectively, from kidney RNA, but 21X and 28X from thymus RNA. The source of this variation is unclear, but the implications for quantitation from oligo d(T) selected RNA are impossible to ignore.How can I tell if my RT-PCR product is RNA specific?AnswerInclude a control reaction without reverse transcriptase. Alternatively, a primer set that spans an intron/exon border can be chosen such that the RNA dependent PCR product would be a different size from genomic DNA-dependent PCR productHow much of the first strand reaction should I add to the PCRUse 10% of the first-strand reaction. More than 10% may inhibit the PCR.Figure 2. Differential Enrichment of Specific mRNAs by Oligo dT Chromatography. A Northern blot containing total RNA (1 g) and twice oligo d(T) selected RNA (50 ng) from mouse thymus and kidney was hybridized simultaneously with GAPDH and cyclophilin RNA probes. Hybridization signals were quantitated with a Bio-Rad Molecular Imager. Primer DesignPrimers for quantitative experiments are typically designed to amplify a target between 150 and 600 base pairs. Targets smaller than 200 bp are difficult to resolve on agarose gels, and larger targets place a greater burden on the investigator to optimize PCR conditions. The critical aspect for RT-PCR primer choice with respect to minimizing the problems associated with DNA contamination is to design primers that span at least one intron of the genomic sequence. This will result in a PCR product from genomic contamination that will be larger in size than the product generated from the cDNA. In fact, primers can be designed to span a sufficiently large genomic fragment such that amplification from contaminating DNA may be not be possible. For genes in which the genomic sequence is published, the positions of the splice junctions can be found by retrieving the sequence from the Genbank database at /Genbank/index.html. If the intron - exon structure is unknown, primers can be synthesized in different regions of the cDNA sequence and tried in combinations on both cDNA and genomic DNA. It should be possible to choose a primer combination that yields either no product (additional intron sequence produces too long a target for efficient PCR) or an easily distinguishable product when amplifying from genomic DNA. An additional wrinkle is that pseudogenes exist in the mammalian genome for many genes, including the most commonly used internal controls (-actin, GAPDH, cyclophilin). These sequences, arising from integration of a reverse transcription product into the genome, do not have introns. Thus, the size of a PCR product amplified from a pseudogene may be identical to that produced from a cDNA copy. The only way to identify these products is to perform a no-RT control as shown in Figure 3. The true product from RNA is 361 base pairs. The no-RT control yields both a fragment identical in size to the expected RT-PCR product from the RNA transcript (from a pseudogene), and a 1.2 kb fragment from the legitimate genomic locus. If it is absolutely essential to avoid amplification from these sequences, an amplified fragment from a pseudogene may be sequenced, and primers designed to regions where the sequence varies from the legitimate copy of the gene. As little as a one or two nucleotide difference at the 3 end of a primer binding site can completely inhibit amplification from the pseudogene. Figure 3. DNA Contamination in RNA. Mouse liver total RNA was isolated according to protocol. RT-PCR reactions were performed using Ambions RETROscript Kit and 0.5 g RNA. PCR reactions were performed with 5 g RNA. 10 l of each reaction was electrophoresed on a 2% agarose gel and stained with EtBr. DNase I TreatmentIn a recent informal survey of RT-PCR users, we found that the field is evenly divided by those users who believe that DNase I treatment solves the problem of genomic DNA contamination and those who feel that DNase I treatment is an unacceptable solution. Detractors claim that DNase I treatment and the subsequent inactivation steps compromise the performance of their RT-PCR reactions to an unacceptable degree. Much of the problem these users experience may be traced to the extreme temperatures used to inactivate the DNase I prior to reverse transcription. Huang, et al. (Biotechniques, (1996) 20:(6)1012-1020) report complete inactivation of DNase I by heat denaturation at 75C for 5 minutes. Lower inactivation temperatures do not completely inactivate DNase I, while higher temperatures appear to damage the RNA template. DNase I treatment followed by heat inactivation is a simple enough technique for routine use in systems in which genomic DNA contamination is a problem. The use of high quality, RNase-free DNase is crucial. Two additional conventional methods of reducing contaminating genomic DNA from total RNA preparations are acid phenol extraction, which partitions DNA into the organic phase, and LiCl precipitation, which selectively precipitates RNA from solution (protein and DNA remain in the supernatant). A description of these techniques can be found in Ambions Technical Bulletins #158 and #160. These techniques can be used after DNase I treatment to inactivate the enzyme and precipitate the RNA prior to reverse transcription. Finally, it should be noted that DNase I treatment neither relieves the investigator of the burden of sensible primer design, nor of the necessity to perform the appropriate no-RT controls. In addition to the above techniques, researchers now have a new and convenient option for removal of DNA and DNase I from RNA samples. DNA-free DNase Treatment and Removal Reagents are designed for the removal of contaminating DNA from RNA samples and for the removal of DNase after treatment. As described above, DNase is typically inactivated by heat treatment, and can also be removed from treated preps by phenol extraction. Heat inactivation can present problems, however, as the temperature at which DNase is inactivated also catalyzes RNase-independent RNA strand scission in the presence of divalent cations. Phenol extraction is also avoided by researchers who do not want to work with phenol, or who worry about sample loss. DNA-free avoids both methods of DNase I inactivation by supplying a novel DNase Removal reagent that effectively removes DNase and divalent cations from the reaction mixture. The DNase/cation removal step takes a mere three-minute incubation. No organic extraction, EDTA addition or heat inactivation is required. The DNA-free DNase Treatment and Removal Reagents are provided with RNase-free DNase I, an optimized 10X Reaction Buffer, and the DNase Removal Reagent. The DNA-free Reagents are now also part of the RNAqueous-4PCR Kit, combining the features and benefits of RNAqueous with those of DNA-free. Figure 4. RT-PCR Experiments Using Total RNA DNase-Treated Using DNA-free Reagents. Five l of RNA samples isolated using Ambions RNAqueous Kit were used as templates for reverse transcription reactions; 10% of the resulting cDNA was amplified by PCR using S15 primers. The lanes to the left of the markers are PCR reactions done without reverse transcription, demostrating the lack of genomic DNA contamination in these RNA samples. The lanes to the right of the markers show the S15 RT-PCR product from the indicated samples. In addition to DNA-free, Ambion offers man