oligo 7 使用介绍.ppt

Oligo 7Primer Analysis Software
Rules for PCR Primer Selection
and Presentation of the Software
Ⓒ 2010 Molecular Biology Insights, Inc.
Contents
1. PCR Primer Selection Criteria - explanation of primer stability calculations - what makes a good PCR primer - other factors besides individual primer characteristics influencing PCR reaction
2. Oligo 7 Analyze Features - the Sequence window - general information (the Key Info) - duplex and hairpin formation - data about primers (Composition and Melting Temperatures) - false priming sites and homology analysis - oligonucleotide stability graph (Tm, DG) - internal stability and its importance - sequence frequency - other DNA analysis options: ORF, restriction sites, DNA calculator
3. Search Options - search for primers and probes - other searches - batch processing
4. Oligonucleotide Database - multiplexing
5. Concluding Remarks
Primer Stability CalculationsAn introduction to the nearest neighbor method
One of the most important characteristics of a primer is its the melting temperature and stability of various its regions. The most accurate Tm calculation provides the nearest neighbor method. The primer Tm formula is plex:
Tm (°C) = DH/(DS + R * ln[C]) + log [K+]/(1+*[K+]) –
where DH and DS are the enthalpy and entropy for helix formation, respectively, R is the molar gas constant ( cal/°C * mol), and C is concentration of the primer/probe (that is always used in several-fold excess than the target DNA), but puter does all the work. To plexity of duplex stability calculations it is easier to explain it by showing how free energy (DG, another measure of DNA helix stability), as the formula is simpler:
DG = DH – TDS
(T is the temperature in °K). Although is looks simple enough, it is not so simple if you actually do it. Here’s the example of DG calculation of a 4-mer TCGA, hybridizing to a longer strand like this:
Now, the DG can be calculated like this surprisingly long formul