Usefull info

The right primer (how to minimize the risk of choosing wrong primers)

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:

⚫ Choose primers that have approximately 50% GC-content.

⚫ Concentrate more of the GC-rich part of your oligo at the 5'-end.

⚫ DO NOT use GC-rich sequences as the 3'-end of your oligonucleotides. On the other hand, when the last two nucleotides at the 3'-end are G or C (GC clamps) the resulting oligos are generally better than oligos without GC clamps.

⚫ Some data indicate that T at the 3'-end leads more often to mispriming than A, G or C (Kwok, S. et al., Nucl. Acids Res. 1990, 18, 999). When mutations are introduced in the primer, avoid mutating the three bases at the 3'-end (see the above reference).

⚫ The melting temperatures of the two oligos should be similar or identical.

⚫ PCR is more likely to result in non-specific products at lower annealing temperatures, therefore the melting temperatures of your oligos should not be very low.

⚫ Always check your oligos for hairpin, loops, primer dimer etc. formation.

⚫ If your oligos are based on introns or other non-coding sequences, be constantly conscious about the possibility of selecting a primer matching repetitive sequences (Alu- and other repeats, abundant in introns).

⚫ Run a BLAST search with your oligos against the GenBank

"Degenerated" custom oligonucleotides: a mixture of "non-degenerated" DNA molecules

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).

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