Brief Overview of Primer Design

Selection of oligonucleotide primers is useful for polymerase chain reaction (PCR), oligo hybridization and DNA sequencing. Proper primer design is actually one of the most important factors/steps in successful DNA sequencing.

Various bioinformatics programs could aid your selection of primer pairs from a template sequence.

It is also often useful to search the nucleic acid sequence databases to see if your chosen oligonucleotide occurs in known sequences. (The blast and fasta programs are unsuitable for searching databases with oligos less than about 30 bp.)

Finally there may well already be suitable characterised primers, so check the relevant databases.

What primer design criteria are there?

The default values of a program's parameters can be altered by you, the user. If a program fails to select any appropriate primers review the program summary output and consider altering criteria.

Although the criteria for selecting appropriate primers varies between programs, some primer characteristics are rather crucial in designing proper primers, such as:

Size	ideal size: 20-25 nucleotides long; generally: 18-30 nucleotides long
3' end base	should be a G or a C
Melting temperatures (Tm)	50-65 degrees Celcius
GC content	40-60%
self-complementarity	should be avoided to minimize primer secondary structure and primer dimer formation
Similarity	should have 100% match with the template

A longer list of criteria can be found at following sites:

• Alkami Biosystems
• Choosing Primers for sequencing
• Design of Primers for Automated Sequencing

What Primer Prediction & Analysis Programs are availabe at CBI?

• eprimer3 - Picks PCR primers and hybridization oligos
• primersearch - Searches DNA sequences for matches with primer pairs
• stssearch - Search a DNA database for matches with a set of STS primers

Can I do Electronic PCR at CBI?

Yes, of course. There are

• forward_ePCR - Forward Electronic PCR
• reverse_ePCR - Reverse Electronic PCR
• isPCR - In-Silico PCR