PowerPoint 簡報

Bioinformatics 92-
ucleic Acid Secondari
Structure
AND
Primer Selection
http://gcg.nhri.org.tw:8003/gcg-bin/seqweb.cgi
Primer Length
Minimum Maximum -
---------------------------------------------PCR Product Length
Minimum Maximum -
---------------------------------------------Maximum number of primers or PCR
products in output (range 1 thru 2500)
Primer DNA concentration (nM)
(range .1 thru 500.0) Salt concentration (mM) (range .1 thru
500.0) -
---------------------------------------------Select:
forward primers, only
reverse primers, only
primers on both strands for PCR
Set maximum overlap (in base pairs)
between predicted PCR products
Forward strand primer extension must
include position
Reverse strand primer extension must
include position
----------------------------------------------
Reject duplicate primer binding sites on
template
Specify primer 3' clamp (using IUB
ambiguity codes)
----------------------------------------------Primer % G+C
Minimum (range 0.0 thru 100.0)
Maximum
----------------------------------------------Primer Melting Temperature
(degrees Celsius)
Minimum (range 0.0 thru 200.0)
Maximum
----------------------------------------------Maximum difference between
melting temperatures of two
primers in PCR (degrees Celsius)
(range 0.0 thru 25.0)
----------------------------------------------Product % G+C
Minimum (range 0.0 thru 100.0)
Maximum
----------------------------------------------Product Melting Temperature
(degrees Celsius)
Minimum (range 0.0 thru 200.0)
Maximum
Useful bookmarks for probe and primer design:
http://www.operon.com/oligos/toolkit.php
Use free online Tm calculators to see what the Tm for primer and probe sequences are. We
use the Operon calculator, as it also has a good tool to check possible primer-dimerization
sequences. Reach it from www.operon.com then select DNA Synthesis, then select Oligo
Toolkit.
http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi
This is a link to Primer3 software. It is software that allows for primer design and also helps
picking an internal oligo sequence to these primers (a probe sequence). Like most primer
design algorithms, it has the disadvantage of not taking into account secondary structure
issues that are paramount in primer/probe design for real-time PCR. With that caveat in mind,
it is a good place to start the design process, if you are not inclined to do it by eye (i.e.
scanning the sequence yourself). Then you can check your sequences at the folding site
(described below).
http://www.ncbi.nlm.nih.gov/BLAST/
This is the site for the Basic Local Alignment Search Tool from the National Center for
Biotechnology Information. Use this site for checking specificity of probe and primer
sequences.
RNA folding programs on the Web
mfold version 3.0 by Zuker and Turner at Washington Univ. of St.
Louis
http://mfold2.wustl.edu/~mfold/rna/form1.cgi
SStructView: RNA Secondary Structure Java Applet that
visualizes RNA structures as calculated by mfold
http://smi-web.stanford.edu/projects/helix/pubs/gene-combis96/eg-rnafold.html
RNA secondary structure prediction with GenBee at the
Belozersky Institute, Moscow State University, Russia
http://www.genebee.msu.su/services/rna2_reduced.html
Protein Hydrophobicity Server: Bioinformatics Unit, Weizmann
Institute of Science , Israel
http://bioinformatics.weizmann.ac.il/hydroph/
SAPS - statistical analysis of protein sequences
http://www.isrec.isb-sib.ch/software/SAPS_form.html
Exercise 07-1
Primer Selection
Use the human npm cDNA sequence to design
a pair of primers that will copy the
whole coding sequence when
translated in frame.
THEN
Check the specificity of the primers by using BLAST.