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PCR Primer Design of Medicilon

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A primer is a short synthetic oligonucleotide which is used in many molecular techniques from PCR to DNA sequencing.  These primers are designed to have a sequence which is the reverse complement of a region of template or target DNA to which we wish the primer to anneal.

pcr primer design


Analysis of Primer Sequences 

When designing primers for PCR, sequencing or mutagenesis it is often necessary to make predictions about these primers, for example melting temperature (Tm) and propensity to form dimers with itself or other primers in the reaction.  The following program will perform these calculations on any primer sequence or pair.

The programs will calculate both the Tm of the primers, as well as any undesireable pairings of primers.  When primers form hairpin loops or dimers less primer is available for the desired reaction.

Primer Design Considerations

One of the single most important factors in successful automated DNA sequencing is proper primer design.  It is important that a primer has the following characteristics:

  • A melting temperature (Tm) in the range of 50 C to 65 C

  • Absence of dimerization capability

  • Absence of significant hairpin formation (>3 bp)

  • Lack of secondary priming sites

  • Low to moderate specific binding at the 3′ end (avoid high GC content to prevent mispriming)

Primers designed according to these criteria will generally be from 18 to 30 bases in length and have %GC of 40 to 60. Try to avoid using primers with Tm’s above 65-70 C, especially on high GC templates, as this can lead to secondary priming artifacts and noisy sequences. We strongly recommend the use of computer software to design primers with these characteristics. Examples of such software are: LaserGene (DNAStar), Oligo (National Biosciences, Inc.), MacVector (Kodak/IBI) and the GCG suite.

If designing a primer based on existing sequencing data, choose a priming site that is greater than 50 nucleotides away from the position where new sequence is needed. Avoid designing primers using regions of poorer quality sequence, such as areas beyond single peak resolution of a chromatogram (typically 600-700 bases). Avoid primers where alternative priming sites are present with more than 90% identity to the primary site or that match at more than seven consecutive nucleotides at the 3′ end.

Finally, be aware that no set of guidelines will always accurately predict the success of a primer. Some primers may fail for no apparent reason, and primers that appear to be poor candidates may work well.

Designing Degenerate Oligonucleotides

A group of degenerate oligonucleotides contain related sequences with differences at specific locations.  These are used simultaneously in the hope that one of the sequences of the oligonucleotides will be perfectly complementary to a target DNA sequence.

One common use of degenerate oligonucleotides is when the amino acid sequence of a protein is known.  One can reverse translate this sequence to determine all of the possible nucleotide sequences that could encode that amino acid sequence.  A set of degenerate oligonucleotides would then be produced matching those DNA sequences.  The following link will take you to a program that will perform a reverse translation.

Also keep in mind that most oligonucleotide synthesis reactions are only 98% efficient.  This means that each time a base is added, only 98% of the oligos will receive the base.  This is not often critical with shorter oligos, but as length increases, so does the probability that a primer will be missing a base.  This is very important in mutagenesis or cloning reactions.  Purification by HPLC or PAGE is recommended in some cases.

PCR Primer Design

PCR (Polymerase Chain Reaction)
Polymerase Chain Reaction is widely held as one of the most important inventions of the 20th century in molecular 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℃, 60℃ and 70℃ 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.

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Tips:  Above is part of PCR Primer Design.  You can also CONTACT US with any question or enquiry you may have. We will be happy to discuss your needs in detail and design an appropriate plan of action.

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