Primer Design: A Key Player in Biotechnology

Primer Design affects, if indirectly, almost all areas of biotechnology. This is because PCR technology seems to pervade almost all areas of biotechnology and affects them directly. A good PCR reaction, in turn, is dependent on good primer design. In fact , primer design is so important that it is said to be the rate-limiting step of all PCR based methods.
This makes studying the rules and challenges of primer design very important..

Impact of primer design on other fields
Primer design affects all the fields that employ PCR in some way.
PCR is used in the following fields:
Molecular Diagnostics:
*criminal investigation
* genetic disorder diagnosis
* pathogen detection
* paternity testing
* polymorphism detection
* site directed mutagenesis
* validation of microarray experiments

Rules and Challenges

The job of designing primers would be much easier if we could understand PCR better. The fundamentals of the molecular biology of PCR are not well understood. While all knowledge of what happens is descriptive , the physical chemistry/thermodynamics are not so well understood: this gives us little power to manipulate PCR or design better annealing temeperatures. In other words , if we understand the thermodynamics well , we will be able to understand and thus predict the annealing temperature very well.

The rules for choosing PCR primers are a rough combination of educated guesses and old fashioned trial-and-error. None of the published formulas for calculating annealing temperatures has been proven to give better than a rough estimate -So Far.( see Primer-Bank) The design of PCR (and sequencing) primers is relatively simple from a computational point of view: just search along a sequence and find short sub-sequences that fit certain criteria. However, since the molecular biology of PCR is very complex, the nature of these criteria is not at all obvious. All primers design software uses approximately the same criteria and computing algorithms. Graphical output is not necessary.

Primer Design Rules

* primers should be at least 15 base pairs long
* have at least 50% G/C content
* anneal at a temperature in the range of 50-65 degrees C
* Usually higher annealing temperatures (Tm) are better (i.e. more specific for your desired target)
* Forward and reverse primer should anneal at approximately the same temperature

Primer Problems /Linear constraints : a close look at the challenges

* primers should flank the sequence of interest
* primer sequences should be unique
* primers that match multiple sequences will give multiple products
* repeated sequences can be amplified - but only if unique flanking regions can be found where primers can bind
* primers can have self-annealing regions within each primer (i.e. hairpin and foldback loops)
* pairs of primers can anneal to each other to form the dreaded "primer dimers"

Differential Primers: As if the above mentioned "linear constraints" do not complicate matters enough, the need for information has created some new challenges for primer design.
These new challenges for PCR primer design are:
1. gene-specific primers (for multi-gene families)
2. identify specific species or strains of organisms
3. molecular diagnostics/detectors
Most such studies rely on the polymerase chain reaction to amplify orthologous genes among related organisms, which creates a need for efficient methods to design primers from a nucleotide alignment. For many such studies, the organisms are similar enough that DNA sequences of genes and often even non-coding sequences can be aligned, but dissimilar enough that PCR primers designed from a single species fail to bind efficiently.

Software
Given all the rules and challenges, it only seems logical that we need software to walk us through primer design.

Primer design software is available for some applications. For designing primers for a single nucleotide sequence, web-based and stand-alone software packages are available, such as Primer3 (12) and NetPrimer (Premier Biosoft International). For designing primers from alignments of highly divergent proteins, Codehop (13) uses a mix of consensus bases and degeneracy.

Visual OMP is a software that helps model primers and probes.( http://www.dna-software.com/Products/VisualOMP/IntroVO4.htm)
It uses the industry's most complete database of empirical parameters, combined with theoretical models and advanced computer algorithms to establish a "nearest neighbor model." The model is able to determine the thermodynamics of a multi-state equilibrium given assay experimental conditions and powerful enough to determine secondary structure motifs and suboptimal structures.

Another software is the Expeditor . It is a Pipeline for Designing Primers Using Human Gene Structure and Livestock Animal EST Information.(1) Developed by the Department of Animal Science, Center for Integrated Animal Genomics, Iowa State University, it can be used to combine known gene structure information from human and coding sequence information from farm animal species for a streamlined primer design in target farm animal species. This software has many utilities, which include
1) PCR-based SNP discovery for identification of genes/markers associated with economically important traits in farm animals,
2) comparative mapping analysis, and
3) evolution studies.

Databases
While the software described above is helpful, annealing temperatures are hard to predict.
To be obtain primers that can be handled with a higher level of confidence, and to achieve a higher success rate, looking up primers in a database is very helpful. Some databases for primers are described below.

RTPrimer DB (2)
The real-time polymerase chain reaction (PCR) methodology has become increasingly popular for nucleic acids detection and/or quantification. As primer/probe design and experimental evaluation is time-consuming, we developed a public database application for the storage and retrieval of validated real-time PCR primer and probe sequence records. The integrity and accuracy of the data are maintained by linking to and querying other reference databases. RTPrimerDB provides free public access through the Web to perform queries and submit user based information. Primer/probe records can be searched for by official gene symbol, nucleotide sequence, type of application, detection chemistry, LocusLink or Single Nucleotide Polymorphism (SNP) identifier, and submitter's name. Each record is directly linked to LocusLink, dbSNP and/or PubMed to retrieve additional information on the gene/SNP for which the primers/probes are designed. Currently, the database contains primer/probe records for human, mouse, rat, fruit fly and zebrafish, and all current detection chemistries such as intercalating dyes (SYBR Green I), hydrolysis probes (Taqman), adjacent hybridizations probes and molecular beacons. Real-time PCR primer/probe records are available at http://www.realtimeprimerdatabase.ht.st.

A PCR primer bank for quantitative gene expression
Analysis(3)

The full potential of microarrays as a tool for gene expression profiling can only be realized by eliminating the variability associated with the data sets usually requires that observed differences be validated by some other method, such as real-time quantitative polymerase chain reaction (real-time PCR). However, non-specifc amplifcation of non-target genes is frequently observed in the latter, confounding the analysis in ~40% of real-time PCR attempts when primer-specific labels are not used. Xiaowei Wang and Brian Seed Department of Molecular Biology, Massachusetts General Hospital, present an experimentally validated algorithm for the identification of transcript-specific PCR primers on a genomic scale that can be applied to real-time PCR with sequence-independent detection methods. Also,they provide PrimerBank, an online database, which has been created for researchers to retrieve primer information for their genes of interest. PrimerBank currently contains 147 404 primers encompassing most known human and mouse genes. The primer design algorithm has been tested by conventional and real-time PCR for a subset of 112 primer pairs with a success rate of 98.2%.

In conclusion , it is safe to say that primer design is gaining ground as the the most important aspect of drug discovery and biotechnology. Though fraught with challenges and constraints, software and databases with validated real-time pcr primer and probe sequence records take us closer to a more efficient, cost-effective primer design .

With these primer-banks and databases present, better primer design is now heavily dependent the scientific community submitting results of their experiment, thus saving expenditure of time , effort and of course money. With such an in-silico approach , primer design will no longer be a guessing game, and a series of expensive hits and misses.

References

1)Expeditor: A Pipeline for Designing Primers Using Human Gene Structure and Livestock Animal EST Information
Z.-L. Hu , K. Glenn , A. M. Ramos , C. J. Otieno , J. M. Reecy , and M. F. Rothschild

2)RTPrimerDB: the Real-Time PCR primer and probe database
Filip Pattyn, Frank Speleman, Anne De Paepe and Jo Vandesompele

3) A PCR primer bank for quantitative gene expression analysis Xiaowei Wang and Brian Seed*