Primer Selection and Matching for Amplification of Viral Genomes

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  *This tool will find valid primer candidates from input genomes and pair the primers to create amplicon candidates.Do not refresh screen after you click 'submit' button.

Primer & Amplicon Selection Conditions:

Primer size (bp) Maximum number of nucleotides in primer  Default: 20
Primer overlap length (bp) Number of bases primers can overlap by in design phase  Default: 19
Minimum GC percentage Minimum GC content of each primer  Default: 45
Maximum GC percentage Maximum GC content of each primer  Default: 55
Minimum Tm (C) Lower limit of primer melting temperatures  Default: 56
Maximum Tm (C) Upper limit of primer melting temperatures  Default: 65
Maximum number of degenerate bases allowed (bp) Maximum number of IUPAC bases allowed in final primer  Default: 3
Maximum number of monomer bases allowed (bp) Number of bases in a mononucleotide run within a primer  Default: 4
Primer concentration (nM) Concentration at which primer will be synthesized (used for Tm calculation)  Default: 50
Salt concentration (mM) Salt concentration in primer stock (used for Tm calculation)  Default: 50
Valid base minimum frequency The minimum frequency of a given base at a given position in the alignment at which the base is considered valid  Default: 0.1
Valid degenerate maximum frequency The upper limit of conservation in an alignment at a given position, above which the base is called for the majority at that position. Below this, a dengenerate base is called.  Default: 0.85
Maximum self-complement bases allowed within a single primer (bp) Number of bases within a primer that are self-complimentary  Default: 10
Valid gap minimum frequency The frequency of genomes within an alignment that must contain a gap at a given position for that gap to be represented in the consensus  Default: 0.8
Primer blast minimum hit frequency Minimum frequency of hits for a given primer back to the genomes in the alignment  Default: 0.8
Maximum amplicon length (bp) Upper limit for amplified product length  Default: 900
Minimum amplicon length (bp) Lower limit for amplified product length  Default: 500
Amplicon overlap length (bp) Desired overlap between adjacent amplicons in the panel  Default: 100
Maximum degenerate primer combination Maximum number of unique primer combinations as determined by the permutations of degenerate bases within a primer   Default: 8
Maximum complement bases in primer pair Number of bases that can be complimentary between a forward and reverse primer in a pair  Default: 15
Genome name (Single word without space or symbol[-, _, #, ...], e.g. HIV) Single word (no spaces or symbols) to indicate template. This title will appear in primer name.  

Upload genome multi-fasta file (Required)
(With extension .fasta only. No spaces No symbols in filename):
File used to make alignment, with extension .fasta.  

After you click "Submit", the results will be sent to you via email
Your Email Address

  **Please Note: We have no resource to support PriSM. If you cannot get results via email two days after submitting fasta file, that means PriSM cannot find valid primer sets for your fasta or your fasta's format has something wrong. Please double check.

Copyright © Special Project Group, Broad Institute of MIT and Harvard     Last Modified 2009-06-23