If my insert is longer than your read length, how can I sequence it?

Basic Primer Walking

Our sequencers will show you 800-1000 nt of unknown sequence, starting from a known primer site. If your unknown region extends beyond this, you have a couple of options. The discussion here is intended for situations where the unknown sequence is just a few kb long. If your unknown is longer - say, 10 kb or more - then please go to the page on shotgun sequencing.

**** Note that we do not perform primer-walking or shotgun sequencing. ****

Primer walking is the consecutive design of new primers to extend into unknown sequence. It takes ~4 days for each primer "step", assuming you submit the samples to the UM Sequencing Core for sequencing (~2 days) and you order primers from typical oligo vendors (~2 days). The longer you need to "walk", obviously the longer this process will take. For just a few kb, this is the best method. Longer sequences may require random-fragment approaches ("shotgun" sequencing).

  • You MUST know at least one region of sequence from which to start. You use that region (typically a vector sequence) to get the first increment of unknown sequence:

  • Now you have more 'known' sequence, you can use that to design a new primer to 'walk' further into the unknown region. Don't use questionable regions of sequence for designing the next primer!! See our web page on Design of Sequencing Primers.

  • Continue that pattern, sequencing with each primer, then designing the next, until you have stepped through the entire unknown region:

  • With that first-pass determination of your unknown sequence, you should now sequence the opposite strand, in order to confirm the sequence and correct any residual errors. Note that the opposite-strand primers are placed in a staggered fashion, in order to maximize your ability to get good sequence at any given nucleotide:

  • A few comments about that final step:
    • Since you know the entire sequence now, you can design and use all the remaining primers in one sequencing batch, for best speed and efficiency.

    • Don't count on getting ideal read lengths throughout this process. If your DNA turns out to be contaminated, you'll be safer to have designed the primers only 500 nt apart. If you want to gamble, extend the spacing to 700 nt.

    • Please refer to the Design of Primers for Automated Sequencing guide for information about how to design succesful sequencing primers.

    • You MAY know some vector sequence for starting the opposite strand (starting at the right end of the insert, progressing towards the left). If so, consider "walking" from that side at the same time as you walk from the left, and you'll cut the walking time in half.

Contact Dr. Robert Lyons, the Core Director, with questions or suggestions.