实时定量PCR引物和探针设计操作步骤PrimerExpress软件.doc

word文档整理分享 参考资料 实时定量PCR引物和探针设计操作步骤Primer Express软件Primer Express 是实时定量PCR引物和探针设计的专用软件。遵守以下三个原则有助于快速建立定量PCR反应体系：1.所有扩增按照同样的原则设计 (Primer Express)；2.所有PCR反应在ABI PRISM ?7000/7900上使用同样的热循环条件；3.所有反应使用相同的PCR试剂。引物和探针的设计原则下述原则的重要程度由上往下越来越低，请尽量满足编号靠前的条件。它们中有的已经在Primer Expre软件中设置成缺省值，有的则需要在选择引物和探针时由设计者加以运用。如果是设计SYBRGreen 引物，也要选择TaqMan Primer and Probe design并遵守这些规则，但是只需要合成引物就可以了。TaqMan 探针：1. 保持G-C含量在30-80%之间。2. 避免同一碱基重复过多。特别是G，不可超过4个及以上。3. 5 end不能是G。4. 尽量使探针中的Cs多于Gs。如果不能满足，则使用互补链上的探针。5. 对于单探针反应，用Primer Express?软件计算出来的Tm值应当在68-70 °C 之间。引物：1. 在探针确定以后再选择引物。2. 引物要尽可能地接近探针，但是不要重叠。3. 保持G-C含量在30-80%之间。4. 避免同一碱基重复过多。特别是G，不可超过4个及以上。5. 用Primer Express?软件计算出来的Tm值应当在58-60 °C之间。6. 3 end 的5个碱基中G and/or C碱基的总数不能超过2个。实时TaqMan 引物和探针设计Begin by opening Primer Express and selecting File, New, and TaqMan? Primer Probe Design. The following screen will appear. You can close the TaqMan? Primer Probe Data box as shown. 输入或插入序列 Import or paste a sequence into the window (Import shown). To paste a sequence from a Word or text file, first copy it to the clipboard. Be sure to only select the sequence (including numbers or annotations is OK); do not include extraneous information such as accession numbers etc. Next, select Edit and Paste. The sequence will appear in the Sequence screen of Primer Express. Or, to Import a Sequence, click the Import DNA File button as shown. The software will then ask you to locate the sequence file. Select it from a folder, hard drive, disk, or desktop. Again, no annotations should be present in this sequence. A file is then imported after selecting the file location. 保存输入的序列 Select File and Save to give the sequence a name. This will be displayed in the File Name Box and will save the sequence in the Archive Folder. 引物和探针设计参数Click the Parameters tab. This displays the Universal default parameters used to search for suitable TaqMan? primer probe sets for real-time assays. It is strongly recommended that you do not adjust any