Improvement of chromosome walking technology
Genome walking is a cloning method that can obtain regions of unknown genome adjacent to known sequences. In the past 20 years, researchers have developed a variety of chromosome walking methods, which can be roughly divided into three categories: reverse PCR, ligation-mediated PCR, and random primer PCR. KRIBB's chief scientist Jung-Hoon Sohn believes that chromosome walking is a very effective method of gene cloning. "Once you get some genomic fragments, you can continue to obtain flanking fragments through PCR. If PCR is not used, cloning from a DNA library is not only monotonous, but also difficult." However, PCR-based chromosome walking methods face many problems: The specificity and efficiency are low, the walking distance is short, and the method is complicated. Dr. Song explained that although PCR generally requires two start sites, ligation-mediated PCR chromosome walking requires only a single start site. By connecting to the cassette lacking the 5 'phosphate group, a second starting site was created. The problem with this method is that many DNA fragments are often amplified non-specifically. The "template-blocking" method developed by Dr. Song and colleagues reduced this non-specific amplification.
In this new method, the researchers filled the 3'-OH of the restriction enzyme digested genomic fragment with dideoxy NTP (ddNTP) and connected it to a properly designed box. In this way, the genomic DNA fragments flanked by frames serve as templates for target gene amplification, and the amplification primers are gene-specific primers and frame primers, respectively. The ends of the genomic DNA are locked by ddNTP, which greatly reduces non-specific amplification and produces simple and rapid chromosome walking. Dr. Song said: "Template-locked PCR shows high specificity. It requires neither specially designed primers nor nested PCR."
The researchers tested this template-locked PCR by cloning a new PGK1 promoter from Pichia pastoris and two new cellulase genes from Penicillium. The complete genome sequences of these two organisms are not yet available.
Dr. Song explained: "Fungal cellulase is very important for degrading cellulosic biomass to produce sugar and bioenergy. We isolated this fungus from rotten wood for the cloning of new cellulase. It is just template-locked PCR An example of how to clone genes from microorganisms without knowledge of genomic information. "
For a few species whose genome sequencing has been completed (such as humans, mice, nematodes, rice, Arabidopsis, etc.), flanking sequences of known sequences can be easily found from the database. However, for most organisms, before knowing their genomic sequence, if they want to know the DNA sequence on both sides of a known region, they can only use the chromosome walking technique.
Although the whole genome sequencing technology is developing rapidly, it is easier to obtain the whole genome sequence of multiple species, but Song believes that the improvement of chromosome walking is still critical to the daily work of the laboratory, because many interested organisms have not completed the genome Sequencing, or only completed part.
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