Primer

Primers are unmodified oligonucleotides with a typical length of 17 to 40 bases – mainly used in the PCR process. The number of errors increases with the length of the chain. Beyond 60 bases, about 50 % of the oligonucleotides contain the desired sequence (see table below). The maximum viable length of a synthetic DNA chain lies by around 200 nucleotides. Yield of true full-length product assuming a coupling efficiency of 99% per step.

Length in nucleotides

20

30

40

60

100

Yield of desired product (in %)

83%

75%

67%

55%

37%

Common PCR primers can be purified by a regular sephadex gel filtration to remove chemical residues. Side products are mainly 5’- shortened/abbreviated sequences (n-1). These do not critically affect their use in primer extension assays (PCR, sequencing). Incomplete synthesis products (n-1 and deletions) are reduced effectively by reverse phase HPLC chromatography (HPR3), also known as “trityl on” purification. An acidic treatment, which can harm the purine bases (A and G) is necessary to cleave the 5’ protective trityl group. In a final gel filtration step, all HPLC buffer components are removed from the HPR3 quality oligonucleotides..

Oligonucleotide sequences containing degenerated positions (wobble bases) or rare bases (e.g. dUracil, dInosine, LNAs) are not available in the smallest synthesis scale (0.01 umol). In this scale, we deliver exact amounts of 5 nmol or 1 OD (SEQ or PCR) – sufficient for approximately 500 PCR reactions.

Oligonucleotide sequences containing wobble and rare bases (dInosine, dUracil, LNA bases and others) can be ordered in the larger synthesis scales. We deliver these products based on the yield of the synthesis scale purified by GSF or HPR3 purified in defined amounts (OD).

Primers exactly 5 nmol (and aliquots)

Primer custom synthesis

Primer HPLC

SEQ

PCR

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