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. 2012 Aug;40(15):e115.
doi: 10.1093/nar/gks596. Epub 2012 Jun 22.

Primer3--new Capabilities and Interfaces

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Free PMC article

Primer3--new Capabilities and Interfaces

Andreas Untergasser et al. Nucleic Acids Res. .
Free PMC article

Abstract

Polymerase chain reaction (PCR) is a basic molecular biology technique with a multiplicity of uses, including deoxyribonucleic acid cloning and sequencing, functional analysis of genes, diagnosis of diseases, genotyping and discovery of genetic variants. Reliable primer design is crucial for successful PCR, and for over a decade, the open-source Primer3 software has been widely used for primer design, often in high-throughput genomics applications. It has also been incorporated into numerous publicly available software packages and web services. During this period, we have greatly expanded Primer3's functionality. In this article, we describe Primer3's current capabilities, emphasizing recent improvements. The most notable enhancements incorporate more accurate thermodynamic models in the primer design process, both to improve melting temperature prediction and to reduce the likelihood that primers will form hairpins or dimers. Additional enhancements include more precise control of primer placement-a change motivated partly by opportunities to use whole-genome sequences to improve primer specificity. We also added features to increase ease of use, including the ability to save and re-use parameter settings and the ability to require that individual primers not be used in more than one primer pair. We have made the core code more modular and provided cleaner programming interfaces to further ease integration with other software. These improvements position Primer3 for continued use with genome-scale data in the decade ahead.

Figures

Figure 1.
Figure 1.
Organization of Primer3. The web interface communicates with primer3_core using the boulder IO format, as described in the text and Figure 4. The primer3_core main program uses a library for reading boulder-IO arguments from the input stream or from a settings file and then calls choose_primers() in the libprimer3 library. Libprimer3 can also read a repeat library, i.e. a library of highly prevalent repeats that primer pairs should avoid amplifying. For example, for the human genome these repeats include Alus, LINEs, endogenous retroviruses and simple sequence repeats (microsatellites, see RepBase, ftp://ftp.ncbi.nih.gov/repository/repbase/). Libprimer3 calls several other C/C++ libraries that are part of Primer3. The library for thermodynamic alignments for secondary structures, etc. is called thal. The library for the old-style alignments is dpal. The library for calculating oligo melting temperatures is oligotm. The primer3_core main function returns its results using a library to write boulder-IO format.
Figure 2.
Figure 2.
Input web page for Primer3Plus.
Figure 3.
Figure 3.
Example output web page from Primer3Plus. The first major block, labeled ‘Pair 1’, shows the template sequence with locations of the primers highlighted in blue and yellow and key information about the primers and primer pair. Subsequent blocks (‘Pair 2’,…, ‘Pair 5’) show information for alternative primer pairs.
Figure 4.
Figure 4.
Examples of boulder IO (A) input to primer3_core and (B) output from primer3_core. Panel A shows two primer design tasks (SEQUENCE_ID’s example 1 and example 2) separated by a line consisting only of an = character. PRIMER_NUM_RETURN is 1 for both tasks, because the values of tags beginning with PRIMER_… persist between design tasks. However, the value of tags beginning with SEQUENCE_…, such as SEQUENCE_EXCLUDED_REGION = 37,21 in example 1, affect only the current design task. Panel B shows abbreviated output corresponding to panel A.
Figure 5.
Figure 5.
Primer3’s thermodynamic models for predicting the stability of secondary structures. (A–C) Interactions between primers. (D) Hairpin structures. (E) Undesirable binding of primers to template sequence. Short white and black arrows represent forward and reverse primers, respectively. Gray arrows represent hybridization oligos. Long white and black arrows represent forward and reverse template. Curved oligos (including primers) indicate ANY interactions (A, B and D), and straight oligos indicate END (i.e. 3′-anchored) interactions (C and E). Primer3_core and the Primer3 web interfaces now use thermodynamic models by default. The Primer3 manual (http://primer3.sourceforge.net/primer3_manual.htm) and Supplementary Figure S1 document the arguments governing temperature calculations for secondary structures.
Figure 6.
Figure 6.
Constraining the positions of forward and reverse primers using SEQUENCE_PRIMER_PAIR_OK_REGION_LIST. Arrows represent primers. In this example, primer pairs A, B and C are all allowed. In the specification of allowable primer positions, blanks for ‘Start’ and ‘len’ indicate no constraint (example B, reverse primers, example C, forward primers).
Figure 7.
Figure 7.
Unique and non-unique primers. (A) The 3′-end of each forward and reverse primer has a unique position; Primer3 can now ensure this and can ensure a minimum distance between the 3′-ends of all forward and/or all reverse primers. (B) Each primer pair is unique, but the red primers are used more than once.
Figure 8.
Figure 8.
Three types of Primer3 task. Arrows represent PCR primers with 3′-ends at the arrow heads. Thick solid lines represent the template sequence. (A) The task generic directs Primer3 to design primer pairs for PCR when both forward (left) and reverse (right) primers are requested. Thin solid lines represent PCR products. (B) The task pick_sequencing_primers directs Primer3 to pick multiple unpaired primers for Sanger sequencing reads that will cover the template. Thin dashed lines represent Sanger sequencing reads. (C) The task pick_primer_list directs Primer3 to generate lists of legal forward and reverse primers without regard to their positions relative to each other.

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