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|• Standard Service:||Free Design||Super Service:||Proportional Control|
Degenerate Primers DNA Oligo Synthesis,
Degenerate Oligo Pool DNA Synthesis,
Proportional Control Oligo Synthesis
Degenerate Primers Custom Degenerate Oligo Pool Synthesis DNA Oligo Synthesis
DNA oligos are chemically synthesized with four deoxyribonucleotides of A, T, C and G. These sequences are widely used in various ways in field of biology. CELLFREE is confident in our ability to provide high-quality DNA oligo products to scientific researchers and industrial customers around the world. With the combination of our state-of-the-art synthesizer, advanced production technology and professional technical support team we have consistently achieved low mutation rates, short turnaround times, high cost performance.
Genetic codes have the tendency to degenerate, which lead to the use of degenerate primers in PCR experiments to exhibit specific genes. CELLFREE provides both the design and synthesis of these degenerate primers, as well as custom degenerate oligo pool synthesis upon request.
A degenerate primer is defined as: “A mix of oligonucleotide sequences in which some positions contain a number of possible bases, giving a population of primers with similar sequences that cover all possible nucleotide combinations for a given protein sequence” (Iserte 2013). For example:
refers to a series of primers in which the seventh and twelfth nucleotides are degenerate. The amount of degeneracy is defined by the number of different primer combinations in the mix. The above example has a degeneracy value of six.
|Symbol||Base Type||Standard Ratio||Price|
|Standard Level||Superior Level|
|R||A,G||50% : 50%||Free||Ask for quotation|
|Y||C,T||50% : 50%|
|M||A,C||50% : 50%|
|K||G,T||50% : 50%|
|S||C,G||50% : 50%|
|W||A,T||50% : 50%|
|H||A,C,T||33% : 33% : 33%|
|B||C,G,T||33% : 33% : 33%|
|V||A,C,G||33% : 33% : 33%|
|D||A,G,T||33% : 33% : 33%|
|N||A,C,G,T||25% : 25% : 25% : 25%|
* Non-standard base proportion should be declared clearly, for example, N(20%A:30%C:40%G:10%T).
Designing degenerate primers
1) Align multiple amino acid sequences using free online software.
2) Target an area approximately 200-500 base pairs in length for optimal PCR amplification.
3) Position forward and reverse primers in more conserved regions – the less degenerate, the further apart these can be.
4) Include between 6 and 7 amino acids in the primers, equating to ~15-20 base pairs.
5) Try to include amino acids methionine and tryptophan, which are coded by a single codon (three-letter nucleotide code), and avoid amino acids leucine, serine and arginine, which can each be coded by six codon combinations.
6) For subsequent cloning procedures and to increase primer length (and therefore annealing temperature) add a 5’ tail (6-9 base pairs) containing a restriction enzyme site.
7) If there is complete degeneracy (no matches among any given species), consider using the base inosine (structurally similar to guanine) as it can pair with any of the four bases, although it will bind to cytosine preferentially. Alternatively, insert N for aNy base to ensure equimolar concentrations of each base at that position in your primer mix.
8) Avoid degeneracy at the 3’ terminus (this would not be a good place to insert inosine).