PCR & Sequencing Primer Design
Primer Design Programs
There are a number of different software packages that can be used to create PCR (and sequencing) primers. They all provide essentially the same set of
features and any of them can be used to design primers that will work.
Different research applications may benefit from particular algorithms or special features in one particular program.
GCG has a well respected tool known simply as
PRIME .
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A similar program know as PRIMER3 [Steve Rozen, Helen J. Skaletsky
(1996)] is available on a WWW server at the Whitehead Institute, MIT:
Primer3
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There are a whole host of other primer design applications for every conceivable type of computer. Since many of these utilize some proprietary (i.e.. secret) algorithms to test the suitability of primers for PCR, I will
not attempt to evaluate them here.
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The Macintosh computer applications MacVector and GeneWorks , provided by the RCR, both include simple and elegant PCR primer design functions. Many people find these programs to be much easier to use than GCG for primer design.
Some other PCR primer design programs on the Web:
PCR & Sequencing Primer Design
The design of PCR (and sequencing) primers is relatively simple from a
computational point of view: just search along a sequence and find short
sub-sequences that fit certain criteria. However, since the
molecular biology of PCR is very complex, the nature of these criteria is not at all obvious.
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Rules for choosing PCR primers are a rough combination of educated guesses
and old fashioned trial-and-error.
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Some general guidelines are:
- primers should be at least 15 base pairs long,
- have at least 50% G/C content
- anneal at a temperature in the range of 50 to 65 degrees C.
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Usually higher annealing temperatures (Tm) are better (i.e. more specific for your desired target).
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In addition, the forward and reverse primer should anneal at approximately the same temperature (allowing perhaps 3 or 4 degrees C of difference between them).
However, none of the published formulas for calculating annealing temperatures has been proven to give better than a rough "ballpark" estimate i.e. plus or minus about 5 or even 10 degrees C, so how well can you trust an estimate of the difference between the Tms of two primers?
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Specificity is of course related to the uniqueness of the region that you are
attempting to amplify.
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Repeated sequences can be amplified - but only if unique
flanking regions can be found where primers can bind
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Primers that match multiple sequences will give multiple products.
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Next, you have to consider the formation of self-annealing regions within each
primer (i.e. hairpin and foldback loops) as well as direct annealing between two primers to form the dreaded "primer dimers".
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It is also possible that primers will bind to regions within the sequence fragment that is being amplified.
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Once you get into multiplex PCR reactions with several sets of different primers working simultaneously in a single amplification tube making different products, forget it; no software can realistically predict what is going to happen.
PRIME
The GCG program PRIME
is a good tool for the design of primers for PCR and sequencing.
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Like all GCG programs, it takes some effort to understand PRIME
well enough to use it effectively.
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Selection of the basic parameters is quite simple and accomplished through a series of interactive questions (or parameters can be set in the command line).
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For PCR primer pair selection, you can choose a target range of the template
sequence to be amplified.
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For DNA sequencing primers, you can specify positions
on the template that must be included in the sequencing.
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In selecting appropriate primers, PRIME allows you to specify a variety of constraints on the primer and amplified product sequences.
- upper and lower limits for primer and product melting temperatures
- primer and product GC contents.
- a range of acceptable primer sizes
- a range of acceptable product sizes.
- required bases at the 3' end of the primer (3' clamp)
- maximum difference in melting temperatures between a pair of PCR primers
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PRIME uses a simulated annealing test to check individual primers for
self-complementarity and to check the two primers in a PCR primer pair for
complementarity to each other.
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Using this same annealing test, PRIME optionally can screen against non-specific primer binding on the template sequence and on
any repeated sequences you specify.
