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Learn DNA Primer Design for Polymerase Chain Reaction

Primer Blast, Primer Design Tools, Primer3Plus, DNA Primer Design, PCR for Beginner Course


Description

In this Bioinformatics course you will be find out how to DNA Primer Design for polymerase chain reaction. Primer BLAST performs only a specificity check when a target template and both primers are provided. Design primers for single- or multi-insert cloning or for your site-directed mutagenesis experiment (insertion, deletion, replacement) with our primer design tool. Primer3 is a computer program that suggests PCR primers for a variety of applications, for example to create STSs (sequence tagged sites) for radiation hybrid mapping, or to amplify sequences for single nucleotide polymor- phism discovery

Polymerase chain reaction (PCR) steps

Denaturing

Annealing

Extension

Specification of Primer Design

Aim for the GC content to be between 40 and 60% with the 3' of a primer ending in G or C to promote binding

A good length for PCR primers is generally around 18-30 bases.

Try to make the melting temperature (Tm) of the primers between 65°C and 75°C, and within 5°C of each other

A good length for PCR primers is generally around 18-30 bases. Specificity usually is dependent on length and annealing temperature. The shorter the primers are, the more efficiently they will bind or anneal to the target.

However, a primer should not be too long 30-mer primers) or too short. Short primers produce inaccurate, nonspecific DNA amplification product, and long primers result in a slower hybridizing rate. ... One also needs to avoid primer-primer annealing which creates primer dimers and disrupts the amplification process

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