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| Technical bulletin - Primer Design |
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Our professionally designed real-time PCR assays have the following attributes:
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Guaranteed priming specificity (single product by melt curve analysis)
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Guaranteed priming efficiency (>90% priming efficiency)
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No SNPs in our primers (On request our primers can be screened for SNPs)
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Flexible design (we can design to your individual requirements)
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Optimal Design for Real time PCR :-
Short amplicons
No template secondary structure
Minimise primer dimers
Minimise primer hairpins
Optimal Tm
Primer Design Flexibility
One of the greatest benefits you will find in using our services is the flexibly we can offer at the level of assay design. We appreciate that many of our customers no long wish to just measure global expression levels, but to elucidate which splice forms are abundant and biologically relevant.
We are are well equipped with our expert team to assist these types of projects. Specific design requests are usually included in the standard cost of the assay. here is a list of request that you can make that are free of charge.
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Design my assay to span at least one intron.
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Design my assay to detect this specific exon.
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Design my assay to detect gene x and make sure it wont detect gene y which is closely related.
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Design a single exon assay so that I can detect both cDNA and genomic DNA.
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Design my assay close to the 3'end.
Splice variants
Designing assays for specific splice variant detection has no additional cost. However, splice variant detection assays are supplied with a positive control which caries a small extra charge. This is for your benefit. Many splice variants that are sited in papers or submitted to the main sequence databases are expressed at very low levels or not at all in most tissues and circumstances. Our positive control enables you to prepare a copy number standard curve and also to quickly eliminate from your study splice variants that are not relevant. From our point of view, it is very hard for us to find the correct cDNA from our BioBank to validate splice variant specific assays. A synthetic positive control template enables us to properly validate your assay whilst also providing you the positive control you will need should to get conclusive results fast.
How do we design the very best primers?
Our primer design process is first and foremost an expert driven process. Our team of scientists and advisors includes some of the World's foremost authorities in the field of real-time PCR and DNA chemistry. Every single assay is designed by a genuine expert rather than a computer algorithm.
Of course computers are a powerful tool and software is integral to our design process. We use an especially modified version of Beacon Designer in our computer aided design model . This software is the best in its class for real time PCR primer design. In addition to Beacon Designer we use the industries finest DNA analysis software (visual OMP) to model in silico the thermodynamic properties of each real-time PCR probe we design. There is no better or more detailed way to ensure that each probe has the ideal attributes before synthesis.
For more information on Beacon Designer please visit the Beacon Designer homepage (here)
Our professional approach to primer design prioritises many key attributes:
1) Primer Specificity.
It is utterly fundamental to a quality real-time PCR assay to have primers that detect only the product of interest. During the design process primers are cross checked with the NCBI database to ensure only the specific target of interest will be amplified in the real-time PCR reaction. All primers are then tested on real biological cDNA to prove the point:
Post run melt-curve analysis: Two exemplary real-time PCR assays detecting products of different sizes.
2) Minimal template secondary structure.
Efficient primer binding requires a linear template. If your template contains secondary structure that effects primer binding this can have a massive impact on the accuracy of your data. Reference in Nature.
We check all amplicons to ensure they have minimal secondary structure during the design process.
3) Minimal potential for primer interactions.
Primer dimers occur when amplifying low copy number transcripts. They reduce both the sensitivity of the assay at the limits of detection and the reproducibility of any results obtained. Only primers with minimal propensity to form primer dimers are designed by our expert team.
Getting these characteristics right is critical to sensitivity, specificity and achieving reproducible results.
In addition, the following attributes are also included in the design parameters:
Target sequence Amplicon length
Cross homology with related genes and pseudo-genes (if present)
Amplicon location (distance from 3'end)
Primer Tm
Primer length
Primer DG
3' end
Intron spanning (if requested)
Primer hair-pin structure
PerfectProbe /template annealing Tm
Probe 5' and 3' extensions
Probe melting point
Probe length
Probe G/C content
Design real time PCR assays or microarrays for bacterial identification and pathogen detection using AlleleID from PREMIER Biosoft International.
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ORDER ONLINE, E: ORDERS@PRIMERDESIGN.CO.UK, T: +44 (0)2380 473274, F: +44 (0)8708 362 155 |
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