实时定量PCR引物和探针设计操作步骤Primer Express软件

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 of the parameters.

引物和探针的排序及选择

Primer Express is now ready to find Primers and Probes. Click the "Primers" tab, select "Options" and "Find Primers/Probes Now". The software will display the progress in the small window below the sequence.

** Please disregard the "Optimal Primer Pairs Only" checkbox and the "Penalty" heading. By checking the Optimal Primer Pairs Only box, you will be severely limiting the range of your search, since the parameters it employs are not based on TaqMan? design guidelines. The Penalty score assigned to your Primer & Probe set is based on factors such as amplicon length. Since the default TaqMan? design parameters keep amplicons under 150 bp, this can be disregarded as well.

Primer/probe sets will be listed when the search is complete. Scroll to the right to view the Probes. Click on the "Start" heading under probes to sort probes by sequence. This will group similar probes, simplifying the search.

探针的选择

Select a probe that is less than 30 bp in length and contains more C's than G's. The probes displayed are on the sense strand only. If the probes displayed do not have more C's than G's, then you will need to use the complement probe (as illustrated in this example). If you need to use the complement, make sure that the probe selected here does not have a C at the 3' end of the probe (otherwise, the complement will have a G at the 5' end ? which