EMBOSS: prima


Program prima

Function

Selects primers for PCR and DNA amplification

Description

prima analyzes a template DNA sequence and chooses primer pairs for the polymerase chain reaction (PCR) and primers for DNA sequencing. For PCR primer pair selection, you can choose a target range of the template sequence to be amplified. For DNA sequencing primers, you can specify positions on the template that must be included in the sequencing. You can allow prima to choose primers from the whole template or limit the choices to a particular set of primers listed in a file.

In selecting appropriate primers, prima considers a variety of constraints on the primer and amplified product sequences. You either can use the program's default constraint values or modify those values to customize the analysis. You can specify upper and lower limits for primer and product melting temperatures and for primer and product GC contents. For primers, you can specify a range of acceptable primer sizes, any required bases at the 3' end of the primer (3' clamp), and a maximum difference in primer melting temperatures for PCR primer pairs. For PCR products, you can specify a range of acceptable product sizes.

The terms forward primer and reverse primer are used in the remainder of this document and in the program output. Forward primers are complementary to sequences on the reverse template strand and create copies of the forward strand by primer extension. Conversely, reverse primers are complementary to sequences on the forward template strand and create copies of the reverse strand by primer extension.

The name prima is a truly European one. In searching for yet another primer program name, we discovered that not only can "prima" be pronounced in English as "Primer", but it has additional meanings in other languages. In Italian it means "first", in German it means "super", and in Spanish it means "cousin".

Usage

Here is a sample session with prima.

% prima
Input sequence: embl:eclaci
Specify a Target Range? [N]: 
Minimum Primer Tm (deg Celsius) [53]: 
Maximum Primer Tm (deg Celsius) [58]: 
Minimum product length [100]: 
Maximum product length [300]: 
Output file [eclaci.prima]: 

Command line arguments

   Mandatory qualifiers (* if not always prompted):
  [-sequence]          sequence   Sequence USA
  [-targetrange]       bool       Specify a Target Range?
*  -targetstart        integer    Target start position.
*  -targetend          integer    Target end position.
   -minprimertm        float      Minimum Primer Tm (deg Celsius)
   -maxprimertm        float      Maximum Primer Tm (deg Celsius)
*  -minprodlen         integer    Minimum product length
*  -maxprodlen         integer    Maximum product length
  [-outf]              outfile    Output file name

   Optional qualifiers: (none)
   Advanced qualifiers:
   -overlap            integer    Minimum overlap of sequences
   -minprimerlen       integer    Minimum primer length
   -maxprimerlen       integer    Maximum primer length
   -minpmgccont        float      Minimum primer GC fraction
   -maxpmgccont        float      Maximum primer GC fraction
   -minprodgccont      float      Minimum product GC fraction
   -maxprodgccont      float      Maximum product GC fraction
   -saltconc           float      Salt concentration (mM)
   -dnaconc            float      DNA concentration (mM)
   -list               bool       Force list-style output

   General qualifiers:
  -help                bool       report command line options. More
                                  information on associated and general
                                  qualifiers can be found with -help -verbose


Mandatory qualifiers Allowed values Default
[-sequence]
(Parameter 1)
Sequence USA Readable sequence Required
[-targetrange]
(Parameter 2)
Specify a Target Range? Yes/No No
-targetstart Target start position. Any integer value Start of sequence
-targetend Target end position. Any integer value End of sequence
-minprimertm Minimum Primer Tm (deg Celsius) Any integer value 53
-maxprimertm Maximum Primer Tm (deg Celsius) Any integer value 58
-minprodlen Minimum product length Any integer value 100
-maxprodlen Maximum product length Any integer value 300
[-outf]
(Parameter 3)
Output file name Output file <sequence>.prima
Optional qualifiers Allowed values Default
(none)
Advanced qualifiers Allowed values Default
-overlap Minimum overlap of sequences Any integer value 50
-minprimerlen Minimum primer length Any integer value 18
-maxprimerlen Maximum primer length Any integer value 22
-minpmgccont Minimum primer GC fraction Number from 0.300 to 0.700 .40
-maxpmgccont Maximum primer GC fraction Number from 0.300 to 0.700 .55
-minprodgccont Minimum product GC fraction Number from 0.300 to 0.700 .40
-maxprodgccont Maximum product GC fraction Number from 0.300 to 0.700 .55
-saltconc Salt concentration (mM) Number from 1.000 to 100.000 50
-dnaconc DNA concentration (mM) Number from 1.000 to 100.000 50
-list Force list-style output Yes/No No

Input file format

Nucleic acid sequence USA.

Output file format

Here is the output from the example run:




INPUT SUMMARY
*************

Prima of ECLACI
PRIMER CONSTRAINTS:
PRIMA DOES NOT ALLOW PRIMER SEQUENCE AMBIGUITY OR DUPLICATE PRIMER ENDPOINTS
Primer size range is 18-22
Primer GC content range is 0.40-0.55
Primer melting Temp range is 53.00 - 58.00 C
PRODUCT CONSTRAINTS:
Product GC content range is 0.40-0.55
Salt concentration is 50.00 (mM)
DNA concentration is 50.00 (nM)
Considering all suitable Primer pairs with Product length ranges 100 to 300




PRIMER/PRODUCT PAIR CALCULATIONS & OUTPUT
*****************************************

3 pairs found


                Forward                                 Reverse

[1]
    10 AGTCAATTCAGGGTGGTGAA      29        154 ATGTAATTCAGCTCCGCCAT      173
       Tm  56.24 C  (GC 50.00%)                Tm  57.14 C  (GC 50.00%)
             Length: 20                              Length: 20
             Tma:    40.56 C                         Tma:    40.83 C


       Product GC: 53.23%
       Product Tm: 55.12 C
       Length:     124


[2]
   266 TTGTCGCGGCGATTAAATCTC     286       510 GTACCGTCTTCATGGGAGAAA     530
       Tm  57.88 C  (GC 42.86%)                Tm  55.78 C  (GC 42.86%)
             Length: 21                              Length: 21
             Tma:    42.45 C                         Tma:    41.82 C


       Product GC: 54.71%
       Product Tm: 57.13 C
       Length:     223


[3]
   477 TGTCTCTGACCAGACACCC       495       728 GGAACGATGCCCTCATTCA       746
       Tm  54.99 C  (GC 52.63%)                Tm  55.20 C  (GC 52.63%)
             Length: 19                              Length: 19
             Tma:    42.28 C                         Tma:    42.34 C


       Product GC: 53.02%
       Product Tm: 58.12 C
       Length:     232


The output file begins with a summary listing all of the constraints used by the program to select appropriate primers or PCR primer pairs. Most of these constraints can be modified by adjusting prompted and optional program parameters.

Following these summaries is an ordered listing of the most appropriate primers or PCR primer pairs selected by prima. The list is ordered by total annealing score so that those primers or PCR primer pairs with the least amount of complementarity to sequences other than the appropriate primer binding sites are listed first. Each output primer or PCR primer pair is designated by a number that corresponds to a line number in the plot of primer sites. While the text output file lists the location of the primer binding site along with each primer sequence, the plot provides a convenient way to review the primer binding sites of many of the selected primers at once.

Data files

None.

Notes

None.

References

None.

Warnings

None.

Diagnostic Error Messages

None.

Exit status

It always exits with a status of 0.

Known bugs

None.

See also

Program nameDescription
primer3Picks PCR primers and hybridization oligos
primersearchSearches DNA sequences for matches with primer pairs
stssearchSearches a DNA database for matches with a set of STS primers

Author(s)

This application was written by Sinead O'Leary (soleary@hgmp.mrc.ac.uk)

History

Finished 2000

Target users

This program is intended to be used by everyone and everything, from naive users to embedded scripts.

Comments