DNA Strider (very old, Mac only)(download from: http://endeavor.med.nyu.edu)
MacVector (Mac only, RCR has a site license)
OMIGA (Windows only,RCR has a site license)
Sequencher (Mac and Windows,RCR has a site license)
Gene Construction Kit (Mac only)
Vector NTI (Mac and Windows)
Plasmid Premier (Windows only)
DNA Strider is simple, but still elegant
Vector NTI
Vector NTI puts the plasmid map at the center of all program functions
PCR Primer DesignThe 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.
All primers design software uses approx-imately the same criteria and computing algorithms. Graphical output is not necessary.
Molecular Biology of PCRThe fundamental Molecular Biology of PCR is not well understood.
We know what happens in a descriptive sense, but not the physical chemistry/thermodynamics
The rules for choosing PCR primers are a rough combination of educated guesses and old fashioned trial-and-error.
None of the published formulas for calculating annealing temperatures has been proven to give better than a rough estimate
The PCR ProcessIn a nutshell, PCR works like this:
DNA and two primers are combined in a salt solution with dNTPs and a heat stable DNA polymerase enzyme
The primers match some sequence in the target DNA
The solution is rapidly heated to DNA denaturing temperatures (~95°C) and cooled to a temperature where the polymerase can function
Each thermal cycle generates copies of the sequence between the primers, so the total number of fragments amplifies in an exponential fashion: 2, 4, 8,16, 32, 64, etc.
Primer Design Rulesprimers should be at least 15 base pairs long
have at least 50% G/C content
anneal at a temperature in the range of 50-65 degrees C
Usually higher annealing temperatures (Tm) are better (i.e. more specific for your desired target)
forward and reverse primer should anneal at approximately the same temperature
Primer Problems
primers should flank the sequence of interest
primer sequences should be unique
primers that match multiple sequences will give multiple products
repeated sequences can be amplified - but only if unique flanking regions can be found where primers can bind
primers can have self-annealing regions within each primer (i.e. hairpin and foldback loops)
pairs of primers can anneal to each other to form the dreaded "primer dimers"
Differential Primers
-New challenges for PCR primer design
.gene-specific primers (for multi-gene families)
.identify specific species or strains of organisms
molecular diagnostics/detectors
.Consensus primers
amplify a gene from all of a diverse group of organsims (eg. bacterial 16-S rDNA)
-Need to work with multiple alignments and find differential or conserved regions
Other Technologies
-Multiplex PCR
GeneScan (ABI)
-PCR related technologies
Primer extension
Taqman (ABI)
Orchid
Pyrosequencing
Ligase chain reaction
Oligos for microarrays
GCG PRIME
The GCG program PRIME is a good tool for the design of primers for PCR and sequencing
For PCR primer pair selection, you can choose a target range of the template sequence to be amplified
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
Other Features of PRIME
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.
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.
Primer Design on the Web
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