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Websites useful when designing primers or oligos
These tools make the process of primer design (or oligo design) much easier and more reliable.

The combination of Primer3Plus, SNP Check and Reverse e-PCR is particularly helpful. Genomic Primers is especially useful if, for example, primer pairs for multiple exons in a gene sequence are required.


Primer3Plus

Performs automated primer design around a specifiied sequence, using default or customised parameters. Square brackets can be placed around the sequence (e.g. exon) to mark it as the target amplified region. The user should indicate to the algorithm the wider region to be flanked by primers by enclosing it within "<" and ">" symbols.

Genomic Primers

Can be used to obtain primer sequences for multiple exons (in a genomic DNA sequence) at once. It uses the Primer3 program for the design of each primer pair.

DNA Oligo Calculator

Can be used to calculate annealing temperatures of specific oligonucleotide primers such as those designed manually. Note that Primer3Plus provides annealing temperature information within its output data and provides automatic forward and reverse primer pairing based on calculated melting temperatures.

SNP Check

Provided by the National Genetics Reference Laboratory at Manchester, UK. It provides a useful check that the primer sequences chosen do not contain any SNPs that could potentially interfere with primer annealing and thus cause allele drop-out during PCR.

Reverse e-PCR

Based at NCBI. It carries out a virtual PCR using the user's chosen primer sequences and shows whether the primers are likely to generate non-specific products. It is easiest to use the "Table Input" method at this website. It is a good test of primer specifity to change the default settings to permit 2 mismatches and 2 gaps during virtual primer annealling.

 
A repository of user-submitted pairs of primer sequences for quantitative real-time PCR.
 
 
 
 
Molbiol-tools

A page of links to many specialised additional online PCR design tools, provided by a university in Ontario, Canada.

 

For more details and for a user's guide to online primer design tools, see Part 3 of Essential Medical Genetics 6th Edition