Recipient Organization
DNA Polymerase Technology, Inc.
(N/A)
St. Louis,MO 63104
Performing Department
(N/A)
Non Technical Summary
Detection of microbes directly in soil is difficult. The purpose of this study is to identify enzymes which will enable gene detection direclty in crude soil samples.
Animal Health Component
40%
Research Effort Categories
Basic
20%
Applied
40%
Developmental
40%
Goals / Objectives
The specific technical objectives we set to achieve in developing novel mutant Taq enzymes designed for fast and low-cost PCR analysis of environmental and biochemical soil samples that will also be applicable for high performance in the general PCR research field are: 1. Isolation of humic acid resistant mutant(s) of Taq polymerase: Screen our existing libraries of randomly mutagenized Klentaq enzyme for clones that are resistant to the inhibitory effects of humic substances contained in soil fractions present in the amplification reaction. 2. Determine the DNA sequence of the mutants that meet or exceed the criteria in aim 1 in order to identify candidate amino acid changes responsible for the humic acid resistance. 3. Test the functionality of the mutations responsible for the above phenotypes by re-introducing them in the wild-type Taq and Klentaq enzymes. 4. Extensive functional characterization of the novel mutants: highly purified enzymes from the apparent best
mutant clones will be assayed for their PCR performance with gene targets of environmental and biochemical importance, as well as for their fidelity of DNA synthesis. Successful enzyme mutants will be ones that outperform the wild-type Klentaq, wild-type Taq, and its extant improved versions such as AmpliTaq Gold, MasterTaq, Jump Start Taq, Titanium Taq. For a potential phase two, our objectives would include standardizing the production of high quality enzymes for the market of soil resistant polymerase and develop PCR kits for user-friendly analysis of crude soil samples. We also would attempt to combine the new HA resistance feature with our existing cold-sensitive mutants able to perform hot-start PCR which would improve the specificity of the enzyme. We will attempt to introduce the best mutations characterized in the KlenTaq enzyme into the full length Taq.
Project Methods
Objective 1. Screening of mutagenized Klentaq libraries for humic acid resistant mutant enzyme. Klentaq is now a well-established and widely used enzyme in the PCR practice. Interestingly, we recently found that Klentaq is more resistant to humic acid inhibition than Taq (it can resist at least 5 ng humic acid inhibition in PCR vs. 0.1 ng for Taq). We will adapt the recently described highly effective CSR strategy for directed evolution of enzymes (4, 16) to screen our libraries for humic acid resistant Klentaq mutants. In the case of DNA polymerase, CSR is based on a simple feedback loop consisting of a polymerase clone that replicates only its own encoding gene. Compartmentalization of individual self-replicating reactions is achieved by emulsification of cells expressing enzyme clones with an oil mixture. Objective 2. DNA sequence analysis of the isolated mutants. In order to identify the amino acid changes responsible for the selected phenotype, we shall wholly
sequence across the Klentaq ORF of each isolated clone. The Klentaq gene will be amplified by PCR and cycle-sequenced using dye-ddNTP label. Sequencing service will be purchased from a specialized DNA sequencing facility. The data will then be analyzed in our labs with a software program allowing simultaneous alignment of multiple sequencing files. Sequencing primers to cross the ORF redundantly already exist in our lab. This step will also allow us to design new primers for site-directed mutagenesis of the w.t. enzyme by re-incorporating the functionally significant mutations in the Klentaq gene. Objective 3. Site-directed mutagenesis of Klentaq enzyme. Based on the functional and sequencing data, the best mutations related to the humic acid resistance phenotype will be re-introduced one at a time in the w.t. Klentaq enzyme. The Klentaq gene will be amplified with one end-primer and a second internal primer carrying the amino acid change of interest. The amplified product will serve
next as a primer in a second amplification step in combination with an opposite end-primer of the gene. This second step restores the full-length Klentaq ORF having the desired mutation. Objective 4. Functional testing of the novel mutant enzymes. In order to functionally characterize the new mutants, we shall produce highly purified enzyme of their clones using our standard procedure for preparing commercial quality Klentaq. The procedure, including PEG treatment, BioRex-70 and Heparin-Agarose chromatography, yields DNA-free and nuclease-free Klentaq enzyme purified to homogeneity, as judged by a single band in Coomassie stained protein gel (7). The same purification procedure also works very well for purification of our cold-sensitive Klentaq mutants (7). Should the new mutant proteins show some different features such as changed affinity and elution profile on the chromatography resin, this procedure could be quickly adapted. The efficiency of each step in the purification scheme
can be monitored easily by a standard DNA incorporation assay.