Primer Pair Matching
Primers work in pairs – forward primer and reverse primer. Since they are used in the same PCR reaction, it shall be ensured that the PCR condition is suitable for both of them.
One critical feature is their annealing temperatures, which shall be compatible with each other. The maximum difference allowed is 3℃. The closer their Tanneal are, the better.
Summary ~ when is a “primer” a primer?
Summary ~ Primer Design Criteria
1.Uniqueness: ensure correct priming site;
2.Length: 17-28 bases.This range varies;
3.Base composition: average (G+C) content around 50-60%; avoid long (A+T) and (G+C) rich region if possible;
4.Optimize base pairing: it’s critical that the stability at 5’ end be high and the stability at 3’ end be relatively low to minimize false priming.
5.Melting Tm between 55-80 ℃ are preferred;
6.Assure that primers at a set have annealing Tm within 2 – 3 ℃ of each other.
7.Minimize internal secondary structure: hairpins and dimmers shall be avoided.
Computer-Aided Primer Design
Primer design is an art when done by human beings, and a far better done by machines.
Some primer design programs we use:
- Oligo: Life Science Software, standalone application
- GCG: Accelrys, ICBR maintains the server.
- Primer5: MIT, standalone / web application
http://www.bbioo.com/Soft/2005/114.htm
- BioTools: BioTools, Inc. ICBR distributes the license.
- Others: GeneFisher, Primer!, Web Primer, NBI oligo program, etc.
Melting temperature calculation software:
- BioMath: http://www.promega.com/biomath/calc11.htm
Task
Design a pair of primers for sequence “NM_203378” in NCBI GenBank, so that the coding sequence of human myoglobin will be amplified using PCR reaction.
Between 156..620
Primer3
Exercise
Repeat the above task.
Pick out a pair of primers you think best.
Assume you are only given myoglobin instead of the accession number. What do you do?
