New real-time PCR primer and probe databases:

• RTPrimerDB  => http://medgen.ugent.be/rtprimerdb/                                                

RTPrimerDB: the Real-Time PCR primer and probe database. Nucleic Acids Research, 31(1): 122-123)
PATTYN, F., SPELEMAN, F., DE PAEPE A. & VANDESOMPELE, J. (2003)

• PCR PRIMER DESIGN AND REACTION OPTIMISATION    by    Ed Rybicki, Dep. Molecular and Cell Biology

• The BiSearch web server.
  
  Aranyi T, Varadi A, Simon I, Tusnady GE.
  BMC Bioinformatics. 2006 ;7: 431.
  lnstitute of Enzymology, BRC, HAS, H-1113 Karolina ut 29, Budapest, Hungary.
  
  BACKGROUND: A large number of PCR primer-design softwares are available online. However, only very few of them can be used for the design of primers to amplify bisulfite-treated DNA templates, necessary to determine genomic DNA methylation profiles. Indeed, the number of studies on bisulfite-treated templates exponentially increases as determining DNA methylation becomes more important in the diagnosis of cancers. Bisulfite-treated DNA is difficult to amplify since undesired PCR products are often amplified due to the increased sequence redundancy after the chemical conversion. In order to increase the efficiency of PCR primer-design, we have developed BiSearch web server, an online primer-design tool for both bisulfite-treated and native DNA templates.  RESULTS: The web tool is composed of a primer-design and an electronic PCR (ePCR) algorithm. The completely reformulated ePCR module detects potential mispriming sites as well as undesired PCR products on both cDNA and native or bisulfite-treated genomic DNA libraries. Due to the new algorithm of the current version, the ePCR module became approximately hundred times faster than the previous one and gave the best performance when compared to other web based tools. This high-speed ePCR analysis made possible the development of the new option of high-throughput primer screening. BiSearch web server can be used for academic researchers at the http://bisearch.enzim.hu site.  CONCLUSION: BiSearch web server is a useful tool for primer-design for any DNA template and especially for bisulfite-treated genomes. The ePCR tool for fast detection of mispriming sites and alternative PCR products in cDNA libraries and native or bisulfite-treated genomes are the unique features of the new version of BiSearch software.

• BiSearch: primer-design and search tool for PCR on bisulfite-treated genomes.
  
  Tusnady GE, Simon I, Varadi A, Aranyi T.
  Nucleic Acids Res. 2005 Jan 13;33(1):e9.
  Institute of Enzymology, BRC, Hungarian Academy of Sciences H-1113 Budapest, Karolina ut 29, Hungary.
  
  Bisulfite genomic sequencing is the most widely used technique to analyze the 5-methylation of cytosines, the prevalent covalent DNA modification in mammals. The process is based on the selective transformation of unmethylated cytosines to uridines. Then, the investigated genomic regions are PCR amplified, subcloned and sequenced. During sequencing, the initially unmethylated cytosines are detected as thymines. The efficacy of bisulfite PCR is generally low; mispriming and non-specific amplification often occurs due to the T richness of the target sequences. In order to ameliorate the efficiency of PCR, we developed a new primer-design software called BiSearch, available on the World Wide Web. It has the unique property of analyzing the primer pairs for mispriming sites on the bisulfite-treated genome and determines potential non-specific amplification products with a new search algorithm. The options of primer-design and analysis for mispriming sites can be used sequentially or separately, both on bisulfite-treated and untreated sequences. In silico and in vitro tests of the software suggest that new PCR strategies may increase the efficiency of the amplification.
  

• Design of Primers for Automated Sequencing
  

• Advanced qPCR design software  by BioSearch-Technologies

• http://www.realtimeprimers.org/

• PrimerBank - PCR Primers For Gene Expression Detection or Quantification

Publication: Xiaowei Wang and Brian Seed (2003) A PCR primer bank for quantitative gene expression analysis. 
Nucleic Acids Research 31(24): e154; pp.1-8.

• The Quantitative PCR Primer Database (QPPD) provides information about primers and probes that can be used to quantitate human and mouse mRNA by reverse transcription polymerase chain reaction (RT–PCR) assays. All data has been gathered from published articles, cited in PubMed.

• Human Endogenous Control Gene Panel  (TATAA Biocenter AB)
  

  For all gene expression studies using quantitative PCR it is necessary to compensate for differences between samples due to material losses, differences in RT yields and PCR inhibition. Normalization should include an endogenous control gene, but can also be complemented by identical sample input amounts. The endogenous control gene should have constant expression in all the samples compared. There is no universal control gene, expressed at a constant level under all conditions and in all tissues.
  The best way to choose the proper reference gene is by running a panel of potential genes on a number of representative test samples. The gene(s) most appropriate for normalization are chosen in each case.
  The Human Endogenous Control Panel consists of 12 validated qPCR assays for the most common endogenous control genes for gene expression studies, and provides a rapid and cost efficient way to identify your control genes. The panel is compatible with most commercial mastermixes containg SYBR Green I    =>  short manual
  

PCR Primer Design Guidelines                                                                                

by Premier Biosoft
http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html

Polymerase Chain Reaction is widely held as one of the most important inventions of the 20th century in molecular biology. Small amounts of the genetic material can now be amplified to be able to a identify, manipulate DNA, detect infectious organisms, including the viruses that cause AIDS, hepatitis, tuberculosis, detect genetic variations, including mutations, in human genes and numerous other tasks.

PCR involves the following three steps: denaturation, annealing and extension. First, the genetic material is denatured, converting the double stranded DNA molecules to single strands. The primers are then annealed to the complementary regions of the single stranded molecules. In the third step, they are extended by the action of the DNA polymerase. All these steps are temperature sensitive and the common choice of temperatures is 94^oC, 60^oC and 70^oC respectively. Good primer design is essential for successful reactions. The important design considerations described below are a key to specific amplification with high yield. The preferred values indicated are built into all our products by default.

1. Primer Length: It is generally accepted that the optimal length of PCR primers is 18-22 bp. This length is long enough for adequate specificity, and short enough for primers to bind easily to the template at the annealing temperature.

2. Primer Melting Temperature: Primer Melting Temperature (T_m) by definition is the temperature at which one half of the DNA duplex will dissociate to become single stranded and indicates the duplex stability. Primers with melting temperatures in the range of 52-58 ^oC generally produce the best results. Primers with melting temperatures above 65^oC have a tendency for secondary annealing. The GC content of the sequence gives a fair indication of the primer T_m. All our products calculate it using the nearest neighbor thermodynamic theory, accepted as a much superior method for estimating it, which is considered the most recent and best available.

Formula for primer T_m calculation:

Melting Temperature T_m(^oK)={ΔH/ ΔS + R ln(C)}, Or Melting Temperature T_m(^oC) = {ΔH/ ΔS + R ln(C)} - 273.15 where

ΔH (kcal/mole) : H is the Enthalpy. Enthalpy is the amount of heat energy possessed by substances. ΔH is the change in Enthalpy. In the above formula the ΔH is obtained by adding up all the di-nucleotide pairs enthalpy values of each nearest neighbor base pair.

ΔS (kcal/mole) : S is the amount of disorder a system exhibits is called entropy. ΔS is change in Entropy. Here it is obtained by adding up all the di-nucleotide pairs entropy values of each nearest neighbor base pair. An additional salt correction is added as the Nearest Neighbor parameters were obtained from DNA melting studies conducted in 1M Na+ buffer and this is the default condition used for all calculations.

ΔS (salt correction) = ΔS (1M NaCl )+ 0.368 x N x ln([Na+])

Where
N is the number of nucleotide pairs in the primer ( primer length -1).
[Na+] is salt equivalent in mM.

[Na+] calculation:

[Na+] = Monovalent ion concentration +4 x free Mg2+.

3.Primer annealing temperature : The primer melting temperature is the estimate of the DNA-DNA hybrid stability and critical in determining the annealing temperature. Too high T_a will produce insufficient primer-template hybridization resulting in low PCR product yield. Too low T_a may possibly lead to non-specific products caused by a high number of base pair mismatches,. Mismatch tolerance is found to have the strongest influence on PCR specificity.

T_a = 0.3 x T_m(primer) + 0.7 T_m (product) – 14.9

where,

T_m(primer) = Melting Temperature of the primers

T_m(product) = Melting temperature of the product

4. GC Content : The GC content (the number of G's and C's in the primer as a percentage of the total bases) of primer should be 40-60%.

5. GC Clamp : The presence of G or C bases within the lat five bases from the 3' end of primers (GC clamp) helps promote specific binding at the 3' end due to the stronger bonding of G and C bases. More than 3 G's or C's should be avoided in the last 5 bases at the 3' end of the primer.

6. Primer Secondary Structures : Presence of the primer secondary structures produced by intermolecular or intramolecular interactions can lead to poor or no yield of the product. They adversely affect primer template annealing and thus the amplification. They greatly reduce the availability of primers to the reaction.

i) Hairpins : It is formed by intramolecular interaction within the primer and should be avoided. Optimally a 3' end hairpin with a ΔG of -2 kcal/mol and an internal hairpin with a ΔG of -3 kcal/mol is tolerated generally.

ΔG definition : The Gibbs Free Energy G is the measure of the amount of work that can be extracted from a process operating at a constant pressure. It is the measure of the spontaneity of the reaction. The stability of hairpin is commonly represented by its ΔG value, the energy required to break the secondary structure. Larger negative value for ΔG indicates stable, undesirable hairpins. Presence of hairpins at the 3' end most adversely affects the reaction.

ΔG = ΔH – TΔS

7. Repeats : A repeat is a di-nucleotide occurring many times consecutively and should be avoided because they can misprime. For example: ATATATAT. A maximum number of di-nucleotide repeats acceptable in an oligo is 4 di-nucleotides.

8. Runs : Primers with long runs of a single base should generally be avoided as they can misprime. For example, AGCGGGGGATGGGG has runs of base 'G' of value 5 and 4. A maximum number of runs accepted is 4bp.

9. 3' End Stability : It is the maximum ΔG value of the five bases from the 3' end. An unstable 3' end (less negative ΔG) will result in less false priming.

10. Avoid Template secondary structure : A single stranded Nucleic acid sequences is highly unstable and fold into conformations (secondary structures). The stability of these template secondary structures depends largely on their free energy and melting temperatures(T_m). Consideration of template secondary structures is important in designing primers, especially in qPCR. If primers are designed on a secondary structures which is stable even above the annealing temperatures, the primers are unable to bind to the template and the yield of PCR product is significantly affected. Hence, it is important to design primers in the regions of the templates that do not form stable secondary structures during the PCR reaction. Our products determine the secondary structures of the template using the Mfold algorithm and design primers avoiding them.

11. Avoid Cross homology : To improve specificity of the primers it is necessary to avoid regions of homology. Primers designed for a sequence must not amplify other genes in the mixture. Commonly, primers are designed and then BLASTed to test the specificity. Our products offer a better alternative. You can avoid regions of cross homology while designing primers. You can BLAST the templates against the appropriate non-redundant database and the software will interpret the results. It will identify regions significant cross homologies in each template and avoid them during primer search.

Parameters for Primer Pair Design:

1. Amplicon Length : The amplicon length is dictated by the experimental goals. For qPCR, the target length is closer to 100 bp and for standard PCR, it is near 500 bp. If you know the positions of each primer with respect to the template, the product is calculated as: Product length = (Position of antisense primer-Position of sense primer) + 1.

2. Product position : Primer can be located near the 5' end, the 3' end or any where within specified length. Generally, the sequence close to the 3' end is known with greater confidence and hence preferred most frequently.

3. Tm of Product : Melting Temperature (T_m) is the temperature at which one half of the DNA duplex will dissociate and become single stranded. The stability of the primer-template DNA duplex can be measured by the melting temperature (T_m).

4.Optimum Annealing temperature (T_a Opt): The formula of Rychlik is most respected. Our products use this formula to calculate it and thousands of our customers have reported good results using it for the annealing step of the PCR cycle. It usually results in good PCR product yield with minimum false product production.

T_a Opt = 0.3 x(T_m of primer) + 0.7 x(T_m of product) - 25

where
T_m of primer is the melting temperature of the less stable primer-template pair
T_m of product is the melting temperature of the PCR product.

5. Primer Pair Tm Mismatch Calculation : The two primers of a primer pair should have closely matched melting temperatures for maximizing PCR product yield. The difference of 5^oC or more can lead no amplification.

Primer Design Using Software

A number of primer design tools are available that can assist in PCR primer design for new and experienced users alike. These tools may reduce the cost and time involved in experimentation by lowering the chances of failed experimentation.

Primer Premier follows all the guidelines specified for PCR primer design. Primer Premier can be used to design primers for single templates, alignments, degenerate primer design, restriction enzyme analysis. contig analysis and design of sequencing primers.

The guidelines for qPCR primer design vary slightly. Software such as AlleleID and Beacon Designer can design primers and oligonucleotide probes for complex detection assays such as multiplex assays, cross species primer design, species specific primer design and primer design to reduce the cost of experimentation.

PrimerPlex is a software that can design ASPE (Allele specific Primer Extension) primers and capture probes for multiplex SNP genotyping using suspension array systems such as Luminex xMAP® and BioRad Bioplex.

References :

1. “A critical review of PCR primer design algorithms and cross-hybridization case study” By F.John Burpo.
2. “Optimization of the annealing temperature for DNA amplification in vitro” By W.Rychlik, W.J.Spencer
and R.E.Rhoads.
3. “A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics” By John SantaLucia.
4. “A computer program for selection of oligonucleotide primers for polymerase chain reactions” Lowe T, Sharefkin J, Yang SQ, Dieffenbach CW.
5. “Optimization strategies for the polymerase chain reaction” Williams JF.Perkin-Elmer Corporation, Norwalk, CT 06859-0251.
6. “Algorithms and thermodynamics for RNA secondary structure prediction. A Practical guide.” Zuker.m.athews, D.Turner, D.

Summary of Primer Design Softwares:

Biosearch Technologies introduces advanced qPCR design software Biosearch Technologies, Inc. (BTI), innovative developer of the patented Black Hole Quencher®, CAL Fluor®, Quasar® and Pulsar® series of quenchers and dyes for multiplex qPCR, recently released the first in a series of qPCR assay probe and primer design modules for its new web-based software engine, RealTimeDesign™. Hosted on BTI's website, www.biosearchtech.com, RealTimeDesign software is available free of charge and can be used on any desktop computer having internet access. This first module for TaqMan® assay design (TaqMan is a registered trademark of Roche Molecular Systems, Alameda, CA) significantly refines and enhances existing TaqMan-proven assay design algorithms with the latest insights into rules governing primer-dimer formation, amplification efficiency, secondary structure and mis-hybridizations. Whether the user is a novice or seasoned expert in assay design, RealTimeDesign ensures that TaqMan assays will routinely demonstrate detection and amplification efficiencies averaging 99%. For the novice user, RealTimeDesign™ Express Mode takes all the guess work out of assay design and fully automates all steps of the assay design process. For more experienced users, Custom Mode allows user-defined design parameter modifications at every step of the assay design process. In both Express and Custom Mode, assays can be designed against 1 to 10 different targets simultaneously with results archived for future review.  Visit www.qpcrdesign.com to learn more.

PrimeSyn lab provides custom DNA synthesis services ranging from custom oligos and simple primers to labeled probes (FRET,Scorpion, probes for RTPCR).  Other services include protein analysis, protein purification, and analytical method development. http://www.primesyn.com               e-mail: info@primesyn.com

PCR Primer Design Guidelines Polymerase Chain Reaction is widely held as one of the most important inventions of the 20th century in molecular biology. Small amounts of the genetic material can now be amplified to be able to a identify, manipulate DNA, detect infectious organisms, including the viruses that cause AIDS, hepatitis, tuberculosis, detect genetic variations, including mutations, in human genes and numerous other tasks. http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html

Beacon Designer designs molecular beacons and TaqMan® probes for robust amplification and fluorescence in real time qPCR. Beacon Designer finds the best possible primer pair, TaqMan® and molecular beacon for single or multiplex real time QPCR assays. For designing primers, Beacon Designer avoids cross homologies identified by automatically interpreting BLAST search results and template secondary structures identified by connecting to the Mfold server. The resultant primers are highly specific and efficient. Beacon Designer can be used to design primers and probes for allele discrimination in multiplex experiments and to evaluate pre-designed assays as well. The highlights of the Beacon Designer 2.0 are as follows:   beacon-designer2.pdf PREMIER Biosoft International - Intuitive software for molecular biologist. PCR primer design, DNA microarray, molecular beacon, and plasmid vector drawing software. Array Designer | Beacon Designer | Primer Premier | SimVector | Xpression Primer | Netprimer Beacon Designer 4.0 Design SYBR Green primers, TaqMan® probes, FRET probes and molecular beacons for robust amplification and fluorescence in real time qPCR. http://www.premierbiosoft.com/DATAFILES/quick_preview/BD_quick_preview/html/1_introduction.htm

Alkami Biosystems Quick Guide for PCR This 158 page PCR manual covers the following topics: PCR Primer design, PCR Methods, PCR Polymerases, PCR Variables, PCR Troubleshooting, Special PCR Topics, Appendixes and a comprehensive Index. This FREE online guide is in PDF format.

http://ihg.gsf.de/ihg/ExonPrimer.html ExonPrimer is a Perl script that helps to design intronic primers for the PCR amplification of exons. The script needs a cDNA and the corresponding genomic sequence as input. It aligns these sequences using Blat and designs PCR primers to amplify each exon using Primer3. The positions of the exons are deduced from the alignment of the genomic and the cDNA sequences. Insertions/deletions up to 6 base pairs are bridged by postprocessing. Exons with small introns in-between are combined. Exons smaller than 20-25 bp will not be recognized. The user can define the maximum exon size. Exons larger than this size will be divided into several parts. The poly-A tail of the cDNA should be clipped to allow the alignment of the cDNA and the genomic DNA sequence. The genomic sequence must be longer than the cDNA sequence. Otherwise the design of primers for the first and/or last exon is not possible. Download of the human genome sequence with all SNPs masked by N's. Using this sequence, one can avoid primers to be positioned across SNPs. ExonPrimer is also available in the UCSC Genome Browser for the human genome assemblies hg16 (July 2003) and hg17 (May 2004). One can find a link to ExonPrimer in the 'Quick Links to Tools and Databases' section of the known genes details page.

Design of Primers for Automated Sequencing  A detailed guide for designing primers for automated sequencing

Primer3 pick primers from a DNA sequence (Whitehead Institute/MIT Center for Genome Research)

Web Primer DNA and Purpose (PCR or Sequence Primer) Entry. Sequencing primers will be evenly spaced along the DNA. PCR primers will be at the ends of the DNA selected in a region of DNA the length of which is user defined. (Saccharomyces Genome Database-Stanford University)

DoPrimer for design of PCR and sequence Primers (INTERACTIVA The Virtual Laboratory)

PCR primer selection was designed for selection of PCR primers to amplify precise regions from relatively low complexity genomes. (Virtual Genome Center)

Xprimer Primer Selection provides an interface to a PCR primer selection program based on xprimer. (Virtual Genome Center)

Primerfinder (Basic Mode & Advanced Mode) is a tool to design oligonucleotides suitable for PCR within any sequence you specify. (Tim Niacaris)

OLIGOCALC  Oligo On-line Calculator (Molecular Biology Shortcuts MBS)

GenePrimer (help) Prediction of PCR Primers for Experimental Gene Identification. This software implements an algorithm for experimental gene identification by multiple PCR amplifications. (USC Computational Biology Software Packages)

GeneFisher (manual pages) Interactive Primer Design ( Folker Meyer & Chris Schleiermacher)

GeneWalker (manual) helps you designing your primers. (CyberGene AB)

Primer Design is designed to make simple the work of selecting primer pairs for PCR.This is a Java version of the algorithms used in PCR primer designed as implemented by the software primer 0.5.(Whitehead Institute/MIT Center for Genome Research)

CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primers) (help) PCR primers designed from protein multiple sequence alignments. (Blocks WWW Server)

Cassandra (help) Primers Prediction Software (USC Computational Biology Software Packages)

Oligonucleotide Tm determination (Virtual Genome Center)

Oligo Calculator

Oligonucleotide Properties Calculator

Oligonucleotide Analyzer