All sequencing results have some risk of error. The sequence far away from the primer must be considered especially carefully. At MWG, all results are checked with PHRED basecalling and the Fasta data you receive from us meet the PHRED 20 quality standard. This means the probability is 1 in 100 that the base was called wrong. If you take a closer look at the Fasta sequence you will see that part of the sequence has been clipped in comparison to the sequencing chromatograms. (You may be able to read further downstream of the primer and use these results as well. In this case make sure to select a region for primer placement where the possibility of sequence error is low. -don't know about this)
You may be interested in maximizing the sequence data obtained. In this case you should design the primer as far 3' as you can manage as long as you have confidence in the accuracy of the sequence from which the primer is drawn. If you only need to examine the sequence at a very specific location in the template, 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 downstream of the primer. Do not count on seeing good sequence less than 50 nucleotides away from the primer or more than 300 nt away (although MWG often gets sequence starting immediately after the primer, and we often return 700 nt of accurate sequence).
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 stage. Suggested primer characteristics: As a rule of thumb, length should be between 18 and 30 nt, with optimal being 20-24 nt and a G-C content of 40-60% is desirable. The Tm should be between 55 C and 75 C. To calculate the Tm 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:
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:
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.
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' tendancy 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.
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 success.