to minimize the risk of choosing wrong primers)
"Degenerated" custom oligonucleotides: a mixture of non-degenerated DNA molecules
International nomenclature for "degenerated" oligonucleotides
The basics of the oligonucleotide synthesis
A land of plenty: other custom DNA providers
There are numerous software products on the market, that will help you choose the right oligonucleotide sequence. You can find information for some of them (including downloadable "demo" versions) with the help of the Web search engines (Yahoo, Google, Bing and others). When choosing a pair of primers for PCR, the most basic rules are:
In addition to the four widely known "normal" DNA bases, the purines G (Guanine) and A (Adenine), as well as the pyrimidines C (Cytosine) and T (Thymine), the international nomenclature includes abbreviations for the "degenerated" bases. In fact, the bases in any given DNA molecule are not degenerated, most often it is one of the four "normal" nucleotides which is included in the single-stranded oligo that we describe with the help of abbreviated characters. What is in fact degenerated in this case is the position, not the base itself. Therefore, when ordering your custom DNA, do not forget that putting S (meaning G or C) in the sequence will result in roughly 50% of the oligonucleotide molecules having G in that position, and the other 50% harboring C. If your oligonucleotide was ordered with 4 "N", i.e. fully degenerated positions (G or A or T or C = N), the final product will contain 256 distinct types of full-size DNA strands. If you plan to use this oligonucleotide mixture for PCR amplification and subsequent subcloning, do not forget that you may get 256 different clones (in addition, the PCR itself will add mutations, and that will further complicate the picture).
We do synthesize oligonucleotides with "degenerated" positions in any combination at no additional charge, if the mixes are equimolar. Please use the international (IUB-IUPAC) nomenclature for these positions:
R = G or A
K= G or T
S = G or C
W = A or T
M = A or C
Y = T or C
D = G or A or T
V = G or A or C
B = G or T or C
H = A or T or C
N = G or A or T or C
When non-equimolar mixes are needed, for example Y*=95%T+5%C, we need to charge you a small nominal fee reflecting the additional manipulations that are needed to synthesize these mixes.
The basics of the oligonucleotide synthesis. Visit the Web site of Wikipedia for a short overview of the chemical synthesis of DNA.
You may wish to visit the Web site of Dana-Farber Cancer Institute (Harvard University) for a list of custom DNA providers.