[分享]冷泉港实验室PCR Primer上关于real-time的一节
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关于REAL-TIME引物设计和探针选择， 附有三种常用探针的介绍网址，希望对大家有用
1.The design step: the selection of PCR primers and probes
2.Choice of detection method (SYBR Green or a fluoresent nucleic acid probe) for the real time PCR.
Generally, real time PCR with SYBR green is used to optimize primers and is principally used to detect PCR products in a research setting. TaqMan probes and molecular beacons provide further specificity, necessary for diagnostic PCR and also to enable multiplexing and reaction.
The design of a real-time PCR assay has more parameters than conventional PCR. Computer software is available to aid the design of real time PCR. e.g. Primer Express, and Beacon Designer. These programs include all of the design parameters required.
1.The first step in the design of a PCR is to choose a target region of the gene. If the target sequence contains regions of variability, then a alignment of sequences is necessary to find conserved areas, such as 5'NCR of enteroviruse.
2.When selecting Primers, they should define a 100- to 200-bp amplicon. The melting temperature(Tm) of primers should be 7-10 less than that of the probe in order to achieve probe binding prior to primer binding, which is crucial for the produciton of fluorescence. The best Tm for the primers is 55-60. There are many criteria in the computer programs for optimal primer and probe design. In general ,the primers should be 20-25 bp long and fulfill general PCR requirements of having minimal anti-complementarity sequeces and a maximum of two GCs at the 3' end.
3. Once the primers have been selected, the target sequeces need to be checked for secondary structures at the annealing temperature. This is best performed by putting the whole target sequece into the Mfold/Zuker program(http://www.bioinfo.rpi.edu/~zukerm/rna/) and entering the proposed annealing temperature and reaction conditions. The resulting structure should have little secondary structure and more particularly, no folding where either primers or probe will bind.
4. The probe is designed in a similar way as the primers to fulfill general PCR requirements and to have a Tm 7-10 higher than that of the primer. Molecular beacons have 5- to 7-bp stem sequences attached to the probe. These are GC-rich and, again, should form a stable hairpin structure in the Mfold program using the same parameters for the taarget as described above(www.molecular-beacons.org/). In some cases, a quenching effect of a 5' G in a fluorescent probe has been described. However, initially a 5'G was in the standard stem of molecular beacons, as deduced from one of the first reports of application of molecular beacons, and no problems were encountered.
5.Finally, the primers and probe should have their specificity checked for homology with other sequences using the BLAST program.If significant homology is detected, it may be necessary to redesign the primer pairs.
Taqman probes: http://www.premierbiosoft.com/tech_notes/TaqMan.html
Scorpion primers: http://www.premierbiosoft.com/tech_notes/Scorpion.html
Molecular Beacons: http://www.molecular-beacons.org/Introduction.html
1.The design step: the selection of PCR primers and probes
2.Choice of detection method (SYBR Green or a fluoresent nucleic acid probe) for the real time PCR.
Generally, real time PCR with SYBR green is used to optimize primers and is principally used to detect PCR products in a research setting. TaqMan probes and molecular beacons provide further specificity, necessary for diagnostic PCR and also to enable multiplexing and reaction.
The design of a real-time PCR assay has more parameters than conventional PCR. Computer software is available to aid the design of real time PCR. e.g. Primer Express, and Beacon Designer. These programs include all of the design parameters required.
1.The first step in the design of a PCR is to choose a target region of the gene. If the target sequence contains regions of variability, then a alignment of sequences is necessary to find conserved areas, such as 5'NCR of enteroviruse.
2.When selecting Primers, they should define a 100- to 200-bp amplicon. The melting temperature(Tm) of primers should be 7-10 less than that of the probe in order to achieve probe binding prior to primer binding, which is crucial for the produciton of fluorescence. The best Tm for the primers is 55-60. There are many criteria in the computer programs for optimal primer and probe design. In general ,the primers should be 20-25 bp long and fulfill general PCR requirements of having minimal anti-complementarity sequeces and a maximum of two GCs at the 3' end.
3. Once the primers have been selected, the target sequeces need to be checked for secondary structures at the annealing temperature. This is best performed by putting the whole target sequece into the Mfold/Zuker program(http://www.bioinfo.rpi.edu/~zukerm/rna/) and entering the proposed annealing temperature and reaction conditions. The resulting structure should have little secondary structure and more particularly, no folding where either primers or probe will bind.
4. The probe is designed in a similar way as the primers to fulfill general PCR requirements and to have a Tm 7-10 higher than that of the primer. Molecular beacons have 5- to 7-bp stem sequences attached to the probe. These are GC-rich and, again, should form a stable hairpin structure in the Mfold program using the same parameters for the taarget as described above(www.molecular-beacons.org/). In some cases, a quenching effect of a 5' G in a fluorescent probe has been described. However, initially a 5'G was in the standard stem of molecular beacons, as deduced from one of the first reports of application of molecular beacons, and no problems were encountered.
5.Finally, the primers and probe should have their specificity checked for homology with other sequences using the BLAST program.If significant homology is detected, it may be necessary to redesign the primer pairs.
Taqman probes: http://www.premierbiosoft.com/tech_notes/TaqMan.html
Scorpion primers: http://www.premierbiosoft.com/tech_notes/Scorpion.html
Molecular Beacons: http://www.molecular-beacons.org/Introduction.html
