Kabeya J.

Muamba Department of Crop Physiology, University of Agricultural Sciences, Bangalore Tips on choosing or designing primers for sequencing
Choosing stock primers - Be sure to check the map of your vector for available priming sites. Do not guess! If possible, select U, R-1 or R-2 stock primers over the other stock primers. These primers will often give superior results and longer reads over other choices, even if the priming site is located slightly farther from your insert. We offer several stock primers free of charge because their priming sites are commonly present on many vectors but it is not an endorsement of their quality as sequencing primers. Tips for Designing Custom Primers -Your decisions concerning primer sequence selection, method of primer synthesis and approach to primer purification can have a significant effect on the quality of sequence data. Try to follow the guidelines below when designing your primers.

Primers should be at least 18-20 nucleotides in length to minimize the chance of encountering problems with a secondary hybridization site on the vector or insert. Primers with long runs of a single base should generally be avoided. It is especially important to avoid 3 or more G's or C's in a row. For cycle sequencing, primers with melting temperatures above 50 C (calculated by the formula Tm = 2(A+T) + 4(G+C)) generally produce better results than primers with lower melting temperatures. Primers should have a G/C content between 40 and 60 percent. For primers with a G/C content of less than 50%, it may be necessary to extend the primer sequence beyond 18 bases to keep the melting temperature above the recommended lower limit of 50 C. Primers should be "stickier" at their 5' ends than at their 3' ends. A "sticky" 3' end, as indicated by a high G/C content, could potentially anneal at multiple sites on the template DNA. A "G" or "C" is desirable at the 3' end but the first part of this rule should apply. Primers should not contain complementary sequences (palindromes) within themselves. This prevents hairpins. A hairpin is a portion of the primer folding back on itself and results in a poor priming event decreasing signal strength. Primers should not contain sequences of nucleotides that would allow one primer molecule to anneal to itself (hairpin) or to the other primer if it's being used in a PCR reaction (primer dimer). If possible, run a computer search against the vector and insert DNA sequences to verify that the primer and especially the 8-10 bases of its 3' end are unique.

Do not design primers with degenerate bases. Do not request inosine in sequencing primers. They either do not work or give poor cycle sequencing results though there have been exceptions.

The design of PCR and DNA sequencing primers follow very similar guidelines. Even though primer characteristics can be visually inspected for the presence of the elements listed above, a number of computer programs have been developed which use several of these guidelines for primer selection. Ordering primers - For PCR and sequencing applications, the 50-nmol scale of crude primer is adequate. Purification is not usually necessary. The 50-nmol scale will yield enough primer for at