Nucleic Acid SecondarilyStructure

ANDPrimer Selection

Bioinformatics 92-07

http://gcg.nhri.org.tw:8003/gcg-bin/seqweb.cgihttp://gcg.nhri.org.tw:8003/gcg-bin/seqweb.cgi

Primer LengthMinimum - Maximum -----------------------------------------------PCR Product LengthMinimum - 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 PCRSet 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 templateSpecify primer 3' clamp (using IUB ambiguity codes) -----------------------------------------------Primer % G+CMinimum (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+CMinimum (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-combis-96/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 , Israelhttp://bioinformatics.weizmann.ac.il/hydroph/ SAPS - statistical analysis of protein sequenceshttp://www.isrec.isb-sib.ch/software/SAPS_form.html

Exercise 07-1Exercise 07-1Primer Selection

Use the human npm cDNA sequence to design a pair of primers that will copy the

whole coding sequence whentranslated in frame.

THENCheck the specificity of the primers by using BLAST.