GenomeWalker™ Universal Kit From Clontech

Clontech’s GenomeWalker™ Universal kit offers a very elegant protocol which allows the sequencing of unknown segments of genomic DNA. Based on some very clever modifications of PCR, it allows users to ‘walk’ the genome from pre-selected start points. These start points are known regions of the genome, which can be used as templates for the design of primers.

In our lab, we used this kit, and modified versions of it, to sequence the region of the Arabidopsis genome surrounding an antisense construct that was previously inserted by Agrobacterium transformation. This was a technically challenging experiment and after much consideration, I found the GenomeWalker™ Universal kit to be the most appropriate and efficient approach. We wanted to know exactly where our insert was located and by applying this kit, were able to map the insertion within a few weeks.

This kit relies on the use of very high quality genomic DNA as the starting material. We used the standard CTAB extraction method optimized for plant tissue. Several optimization steps were considered at the stage of tissue selection (young leaves, for example, which contain small amounts of polysaccharides and phenolics) and DNA cleaning by rigorous phenol:choloform extraction.

The GenomeWalker™ Universal kit provides restriction enzymes which are generally suited for the production of blunt ends, enabling efficient adaptor ligation. We have modified this part of the protocol successfully to select our own enzymes based on target sequences to predict the size and frequency of restriction fragments. The restriction digestion protocol itself is easy and explained well in the kit manual. There are several tips provided throughout the protocol to optimize the entire process and continuously evaluate the quality of the various intermediates generated (such as the integrity of the genomic DNA, the quality of the restriction digests etc.).

The next part of the protocol is the really clever bit that has enabled targeted sequencing of fragments containing your gene of interest and its surrounding region. It involves the ligation of the genomic DNA to specific GenomeWalker™ adaptors, which are approximately 30 bp DNA sequences that ligate to blunt end restriction fragments. These new libraries of adaptor ligated DNA are then used as templates for nested PCR, which is designed on two gene specific primers provided by the user and two adaptor specific primers which bind to the adaptor ligated ends of the restriction fragments. The length of the PCR fragments then depends on the frequency of the restriction sites. We were able to generate fragments which were comprised of about 400 to 600 bp of unknown DNA and about 120 bp of our gene specific DNA. These PCR products can then be purified and sequenced. Although we used it for mapping gene insertions, this technique can be effectively applied to identify promotors, intron-exon boundaries and other specific DNA regions of uncharacterized genes.