Biocurious is a weblog about biology, quantified.

Hot off the Press: Single molecule DNA sequencing

by PhilipJ on 12 August 2006

The recent achievement of single-basepair (bp) resolution measurements of RNAp transcription in an optical trapping system (which I talked about here) was remarkable not only from a technical point of view, but also because of the new sequencing possibilities opened up by this assay. With single-basepair resolution, if the dynamics of the polymerase were in some way dependent on the underlying sequence, it should be possible to sequence individual strands of DNA.

Building on the work in the single-basepair resolution paper, the Block lab has a new result in this week’s Science, where they are (mostly) able to sequence a 32 bp section of DNA upon which an RNA polymerase is stepping.

By keeping the concentration of one of the four nucleotides required for transcription extremely low, pausing events will occur as the RNAp attempts to add this nucleotide to the growing chain. Holding each of the four nucleotides at the rate-limiting concentrations in four otherwise identical assays, the paper shows that it is possible to sequence the underlying DNA by analyzing the ordered sequence of pausing in each of the four cases. An example of their transcription data is shown below (A), with the inferred sequence shown in (B).

These data, acquired over a span of three minutes and using only four DNA molecules, are capable of determining 30 of the 32 bases in the sequence. To increase the accuracy of the assay, one need only combine the statistics from a number of identical transcription assays.

This doesn’t mean, however, that we’ll get ultrafast sequencing from now on. RNAp is quite processive (traveling up to ~2000 bp), but other elements of the dynamics make long sequencing difficult. This assay uses the pausing induced by low concentrations of a nucleotide required for transcription, but RNAp is known to have random pausing events due to misincorportation of a nucleotide as well, which complicates the analysis.

Still, these are very exciting results, and here is a direct link to the paper (PDF), courtesy the Block lab website!



  1. chad    4023 days ago    #

    man, thats hot.


  2. TheBrummell    4014 days ago    #

    Cool! The Pocket Sequencer is one small step closer to reality! Yay Biophysics!


  3. 16s rdna sequencing    605 days ago    #

    Now the sequencing method has been developed to the third generation, which can achieve high throughput sequencing.


Name
Email
http://
Message
  Textile help