Design of Primers for Automated Sequencing

One of the most important factors in successful automated DNA sequencing is proper primerdesign. This document describes the steps involved in this process and the major pitfalls to avoid.

**** Use a Computer to Design Primers ****

We highly recommend that a computer be used during primer design in order to check for certain fatal design flaws.Numerous programs are capable of performing this analysis. We generally use 'Oligo' (National Biosciences, Inc,Plymouth MN), a program for the Macintosh that has produced excellent results in our hands. Two other programs you might consider are MacVector (Kodak/IBI) and the GCG suite of sequence analysis programs, but many others are available as well.

The steps in primer design are as follows:

Generally you are starting with some small amount of known sequence that you wish to extend. Here's how to proceed:

I. Design primers only from accurate sequence data.

Automated sequencing (and in fact any sequencing) has a finite probablility of producing errors. Sequence obtained too far away from the primer must be considered questionable. To determine what is 'too far', we strongly suggest that our clients read the memo Interpretation of Sequencing Chromatograms, which describes how to assess the validity of data obtained from the ABI sequencers. Select a region for primer placement where the possibility of sequence error is low.

II. Restrict your search to regions that best reflect your goals.

You may be interested in maximizing the sequence data obtained, or you may only need to examine the sequence at a very specific location in the template. Such needs dictate very different primer placements.

1.Maximize sequence obtained while minimizing the potential for errors:

Generally, you should design the primer as far to the 3' as you can manage as long as you have confidence in the accuracy of the sequence from which the primer is drawn. Primers on opposite strands should be placed in staggered fashion as much as possible.

2.Targetted sequencing of a specific region:

Position the primer so the desired sequence falls in the most accurate region of the chromatogram. Sequence data is often most accurate about 80-150 nucleotides away from the primer. Do not count onseeing good sequence less than 50 nucleotides away from the primer or more than 300 nt away (although we often get sequence starting immediately after the primer, and we often return 700 nt ofaccurate sequence).

III. Locate candidate primers:

Identify potential sequencing primers that produce stable base pairing with the template DNA under conditions appropriate for cycle sequencing. It is strongly suggested that you use a computer at this step.

Suggested primer characteristics:

1.Length should be between 18 and 30 nt, with optimal being 20-25 nt. (Although we have had some successes with primers longer than 30 and shorter than 18).

2.G-C content of 40-60% is desirable.

3.The Tm should be between 55 C and 75 C. Warning: the old "4 degrees for each G-C, 2 degrees for each A-T" rule works poorly, especially for oligos shorter that 20 or longer than 25 nt. Instead, try:

Tm = 81.5 + 16.6* log[Na] + 0.41*(%GC) - 675/length - 0.65*(%formamide) - (%mismatch)

IV. Discard candidate primers that show undesirable self-hybridization.

Primers that can self-hybridize will be unavailable for hybridization to the template. Generally avoid primers that can form 4 or more consecutive bonds with itself, or 8 or more bonds total. Example of a marginally problematic primer:

5'-ACGATTCATCGGACAAAGC-3'

3'-CGAAACAGGCTACTTAGCA-5'

This oligo forms a substantially stable dimer with itself, with four consecutive bonds at two places and a total of eight inter-strand bonds. Primers with 3' ends hybridizing even transiently will become extended due to polymerase action, thus ruining the primer and generating false bands. Be somewhat more stringent in avoiding 3' dimers.

Note that no rule of thumb can accurately predict either success or failure of a primer. A primer that seems marginal may perform exceptionally well, while another that is apparently flawless may not work at all.

V. Verify the site-specificity of the primer.

Perform a sequence homology search (e.g. dot-plot homology comparison) through all known template sequence to check for alternative priming sites. Discard any primers that display 'significant' tendancey to bind to such sites. We can provide only rough guidelines as to what is 'significant'. Avoid primers where alternative sites are present with (1) more than 90% homology to the primary site or (2) more than 7 consecutive homologous nucleotides at the 3' end or (3) abundance greater than 5-fold higher than the intended priming site.

VI. Choosing among candidate primers.

If at this point you have several candidate primers, you might select one or a few that are more A-T rich at the 3' end. These tend to be slightly more specific in action, according to some investigators. You may want to use more than one primer, maximizing the likelihood of sucess.

If you have no candidates that survived the criteria above, then you may be forced to relax the stringency of the selection requirements. Ultimately, the test of a good primer is only in its use, and cannot be accurately predicted by these simplistic rules-of-thumb.