Documentation for pcr_overlap
Last updated:  21 Oct 1999

Program Description:
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This program will take large tracks of sequence data in FASTA file format, and produce PCR products in 
overlapping seqments to span the entire region.  This program is written to divide up the given 
sequence, based on the users criteria for PCR product size and overlap between adjacent segments, and 
pass this data to the PCR primer selecting program 'Primer3', written at the Whitehead Institute.  
Primer3 then chooses primer sets based on specific selection criteria.   The output file is a list of 
primer sets consisting of the primer sequence, melting temperature (Primer3 calculated), primer set 
"quality" (lower is better;Primer3 calculated), primer positions, primer lengths, PCR product length, 
and amount of overlap between adjacent fragments.  

Usage:  pcr_overlap

**Note:  You need to install the program Primer3 on your system before running pcr_overlap.pl.  
Primer3 can be found at the website:  http://www-genome.wi.mit.edu/


Program Input:
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pcr_overlap has been made completely command-line input format (no more interactive input). 

pcr_overlap     [-file] [-gene] 
                <-start> 
                <-size> 
                <-overlap> 
                <-nouniv> 


[-file] required fasta input file where you want to select your primers. 
[-gene] "basename" which will be added to all of your primer names. 

optional input: 
<-start>  = start position for picking primers in your sequence 
<-size>  =  average size of pcr product (default is 1000+-15%).  If you give it a size it will always have a range of +- 15% 
<-overlap> = average overlap size between fragments (default is 180+-30%)  again if you give a value range will be +-30% 
<-nouniv> = this is  a switch.  If you DO NOT want universal tails on your sequences, use it.  (default is with universal tails) 

Output: 
"basename".primers.fasta = fasta file output of primers numbered using nonmenclature (0 = for, 1 = rev) 
"basename".primers.txt = text output of primer pairs with additional information

#  The amount of overlap is calculated from the 5' end of a fragment to the 3' end of the previous
(adjacent) fragment and takes into account the primer lengths.  Generally, we have found that when
sequencing off of these PCR products with dye-primer sequencing an overlap 150-200 bp is best.  #
 

#  The range between the maximum overlap and minimum overlap defines the search region when looking
for a primer on the 5' end of each product.

#  If the program fails to pick primers across your entire region you can change the input parameters
    Generally increasing the -overlap helps the most.  You can also restart the program from the failure point
    using the -start parameters.

#  The universal 5' tails are used for DNA sequencing of the PCR product.  These tails

are compatible with the Perkin-Elmer Dye Primer sequencing kits.