Friday, September 2, 2011

Cleanup… more than meets the Eye

The beautiful state of Vermont is a state of disrepair following Hurricane Irene last weekend. Our sales meeting was postponed due to the storm, and BioTek fellow Vermonters hit by the worst of Irene are without power, roadways, and basic services. The damage toll made national news. Blizzards, ice storms, and severe winter weather are what we do best in Vermont, the rest of the year is (supposed to be) our reprieve and reward. The irony of facing the wrath of hurricanes on the cusp of our coveted autumn foliage months is not lost on those of us who live in this little piece of landlocked paradise. Who doesn’t both love and hate the weather? Tilt your head back and soak in a postcard perfect sunny, breezy day (usually the kind that follow a hurricane).Throw your arms up and curse at a dark, menacing, flood threatening sky on a day off to go kayaking – it is to no avail when nature will prevail. Aside from defensive preparation for weather disasters, there is the gesture of necessary and heartfelt emergency response that can get us through - helping others in need, giving where one can, supporting those who didn’t fair weather as well ( or It is time to heal, repair, cleanup.

Scientifically speaking of cleanup, segue to my blog posting this week…DNA sequencing cleanup. Although Next Generation Sequencing garners a lot of attention, traditional sequencing based on the Sanger method is still an indispensable part of contemporary molecular biology. Over the past 10-12 years the evolution of market leading dye terminator methods and automated capillary electrophoresis instrumentation has largely standardized traditional sequencing. A critical component of this workflow is the cleanup procedure, where contaminating artifacts from the sequencing reaction are removed prior to capillary electrophoresis. There are currently a number of viable sequencing cleanup methods available using either filtration, precipitation, or sequestering as a process of choice.

BioTek is making a foray into the cleanup procedures of the genomic workflow, demonstrating first a filtration method for sequencing cleanup. The BioTek ELx405 vacuum filtration module was utilized with a Millipore membrane based size-exclusion kit to compare performance to a matrix gel filtration method used by the Vermont Cancer Center DNA Analysis Facility affiliated with the University of Vermont and Fletcher Allen Healthcare. We were fortunate enough to work with very experienced staff using state of the art technology for this application. A poster presentation at European Lab Automation in June 2011 highlighted the results of this collaboration. An Application Note will also be published soon offering additional information for optimizing vacuum performance among other lessons learned. A Quality Matrix and chromatogram of comparative results are shown below.

The promising outcomes of this collaboration have led to additional research investigating the potential viability of our ELx405 and EL406 for performing magnetic bead based sequencing cleanup, a nice companion piece to the filtration methods already demonstrated. To round out this niche market application analysis stay appraised for updates on a potential mag bead pcr cleanup demonstration.

quality matrix
Quality Matrix comparing Vacuum (n=16, n=15) and Gel Matrix (n=8) filtration methods for all samples in the group (350 ng plasmid pGEM®-32f(+) samples). Refer to the poster for definitions of sequencing quality indicators. Results show high correlation between methods.
Representative chromatograms captured through Finch TV of results achieved during Run #2 of the sequencing cleanup experiment. A typical sample result utilizing ELx405vacuum filtration is shown left, and one using SDS/gel matrix filtration right. Samples are the derived control DNA sequence of pGEM-3Zf(+) with a 5 pmol concentration of M13 reverse primer.

By, BioTek Instruments

1 comment:

  1. Well getting cDNA Library Construction can make you more easy to find the correct dna for your research... A cDNA library is made using mRNA instead of DNA as the starting material. The mRNA can be extracted from cells of specific tissues from the organism of interest. The “c” in cDNA stands for copy because a double stranded DNA copy is made from a mRNA . Because of the different starting material, cDNA libraries will have some key distinctions from a genomic library. The reasons for the differences above are because of the nature of gene expression. Learn more and know how DNA Sequencing is done. Thanks.