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EMBOSS: prima |
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.
For efficient priming, you should avoid primers with extensive self-complementarity in order to minimize primer secondary structure and primer dimer formation. Additionally, in PCR experiments, primer pairs with extensive complementarity between the two primers should be avoided in order to minimize primer dimer formation. prima uses the annealing test described in the ALGORITHM topic 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, prima optionally can screen against non-specific primer binding on the template sequence and on any repeated sequences you specify.
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".
T(m)primer) = delta H / (delta S + R x ln(c/4) - 273.15 + 16.6 x log[K(+)]
where delta H is the enthalpy of helix formation, delta S is the entropy of helix formation (including helix initiation), R is the molar gas constant (1.987 cal/degree Celsius/mol), and c is the primer concentration.
prima determines PCR product melting temperatures using the formula of Baldino, et al. (in Methods Enzymol. 168; 761-777 (1989)) as modified slightly by Rychlik, et al. (Nucleic Acids Res. 18; 6409-6412 (1990)).
T(m)product) = 0.41 x (% G+C) + 16.6 x log[K(+)] - 675 / len + 81.5
where len is the length of the product.
If you are selecting PCR primer pairs, the output includes a proposed annealing temperature for each listed primer pair. The annealing temperature is calculated using the formula of Rychlik, et al. (Nucleic Acids Res. 18; 6409-6412 (1990)).
T(a) = 0.3 x T(m)primer) + 0.7 x T(m)product) - 14.9
The same annealing test is used to determine complementarity between the primer and any non-specific binding sites on the template sequences. In this case, the primer in the 5' to 3' orientation is compared over all registers of comparison with the template sequence in the 3' to 5' orientation to determine a total primer-template annealing score. Since complementarity at the 3' end of the primer sequence has a particularly large effect on non-specific primer binding, the 3' primer-template annealing score is also determined. If you screen against non-specific primer binding on any specified repeated sequences, then total primer-repeat and 3' primer-repeat annealing scores, taken over all registers of comparison in all repeated sequences, are also determined.
Total and 3' annealing scores are saved in tests of primer self-complementarity (to check for secondary structure and primer dimer formation) and in tests of complementarity between the two primers in PCR primer pairs (to check for primer dimer formation). Total and 3' annealing scores are also saved when you screen against non-specific primer binding on the template sequence and when you screen against non-specific primer binding on any specified repeated sequences. Primers are rejected that exceed the maximum score you specify for any of these tests. For those primers that are accepted, the program uses the sum of all annealing scores to determine the order of primers or PCR primer pairs in the output list. You can specify weights for each of these scores to adjust their relative contributions in determining the output order. By default, 3' annealing scores have twice the weight of total annealing scores in determining the output order.
% 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]:
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 Minimum 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 |
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 | Minimum 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 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: 232The 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.
Rychlik, W. and Rhoads, R.E. (1990). "Optimization of the Annealing Temperature for DNA Amplification in vitro." Nucleic Acids Research 18, 6409-6412.
Breslauer, K.J., Frank, R., Blocker, H., and Marky, L.A. (1986). "Predicting DNA Duplex Stability from the Base Sequence." Proceedings of the National Academy of Sciences USA 83, 3746-3750.
Baldino, M., Jr. (1989). "High Resolution In Situ Hybridization Histochemistry." In Methods in Enzymology, (P.M. Conn, ed.), 168, 761-777, Academic Press, San Diego, California, USA.
Hillier, L. and Green, P. (1991). "OSP: A Computer Program for Choosing PCR and DNA Sequencing Primers." PCR Methods and Applications 1, 124-128.
Slightom et al. (1994) "Nucleotide sequencing double-stranded plasmids with primers selected from a nonamer library." Biotechniques 17(3), 536-7, 540-4.
When several acceptable PCR primer pairs have the same 3' ends for both primers, prima outputs only the PCR primer pair with the shortest primer sequences. By not allowing duplicate primer endpoints, prima increases the diversity among the PCR primer pairs in the output list.
prima only determines melting temperatures for DNA primers. We do not know of any appropriate nearest-neighbor thermodynamic parameters for RNA-DNA hybrids, so we haven't attempted to calculate melting temperatures for RNA primers. While thermodynamic parameters for RNA duplexes involving mismatches have been described, we do not know of any similar results for DNA duplexes. Therefore, we have not attempted to calculate melting temperatures or other thermodynamic properties for DNA duplexes involving mismatches.
prima does not currently consider formamide concentration in determining primer melting temperatures.
Program name | Description |
---|---|
primersearch | Searches DNA sequences for matches with primer pairs |
stssearch | Searches a DNA database for matches with a set of STS primers |