Wayne M. Barnes

Mutants of Taq DNA polymerase resistant to PCR inhibitors allow DNA amplification from whole blood and crude soil samples

Potent PCR inhibitors in blood and soil samples can cause false negative results from PCR-based clinical and forensic tests. We show that the effect of these inhibitors is primarily upon Taq DNA polymerase, since mutational alteration of the polymerase can overcome the inhibition to the extent that no DNA purification is now required. An N-terminal deletion (Klentaq1) is some 10–100-fold inhibition resistant to whole blood compared to full-length, wild-type (w.t.) Taq, which is strongly inhibited by 0.1–1% blood. Further mutations at codon 708, both in Klentaq 1 and Taq, confer enhanced resistance to various inhibitors of PCR reactions, including whole blood, plasma, hemoglobin, lactoferrin, serum IgG, soil extracts and humic acid, as well as high concentrations of intercalating dyes. Blood PCR inhibitors can predominantly reduce the DNA extension speed of the w.t. Taq polymerase as compared to the mutant enzymes. Single-copy human genomic targets are readily amplified from whole blood or crude soil extract, without pretreatment to purify the template DNA, and the allowed increase in dye concentration overcomes fluorescence background and quenching in real-time PCR of blood.

Direct DNA amplification from crude clinical samples using a PCR enhancer cocktail and novel mutants of Taq.

PCR-based clinical and forensic tests often have low sensitivity or even false-negative results caused by potent PCR inhibitors found in blood and soil. It is widely accepted that purification of target DNA before PCR is necessary for successful amplification. In an attempt to overcome PCR inhibition, enhance PCR amplification, and simplify the PCR protocol, we demonstrate improved PCR-enhancing cocktails containing nonionic detergent, l-carnitine, d-(+)-trehalose, and heparin. These cocktails, in combination with two inhibitor-resistant Taq mutants, OmniTaq and Omni Klentaq, enabled efficient amplification of exogenous, endogenous, and high-GC content DNA targets directly from crude samples containing human plasma, serum, and whole blood without DNA purification. In the presence of these enhancer cocktails, the mutant enzymes were able to tolerate at least 25% plasma, serum, or whole blood and as high as 80% GC content templates in PCR reactions. These enhancer cocktails also improved the performance of the novel Taq mutants in real-time PCR amplification using crude samples, both in SYBR Green fluorescence detection and TaqMan assays. The novel enhancer mixes also facilitated DNA amplification from crude samples with various commercial Taq DNA polymerases.

Construction of an M13 histidine-transducing phage: a single-stranded cloning vehicle with one EcoRI site.

In order to create a ready source of single-stranded DNA for DNA sequence determination by the dideoxy chain-termination method, the promoter-proximal part of the histidine operon, the hisOGD region of Salmonella typhimurium, was cloned onto the single-stranded phage M13. Both orientations of the his DNA were cloned to supply DNA template for sequencing of each strand. Insertion was achieved at an HaeIII site in the intergenic region (IR) of M13, and a single EcoRI site was purposely regenerated at one boundary of the his DNA insert. Infected colonies, not plaques, were selected using the hisD gene as a selective marker. The single RI site and the hisD marker for auxotrophic selection represent improvements on the wild type M13 as a single-stranded vector for cloning other DNA.

Mammalian microevolution: Rapid change in mouse mitochondrial DNA

We have compared the sequences of mitochondrial DNA extracted from museum skins of white-footed mice caught in the Chicago area since 1855 and from modern mice trapped alive in the same locations. We found a consistently similar directional change of mouse genotype over this period at each of five collection sites that were separated by 10–70 km. The genotype most common 100 years ago is now extremely rare, indicating that the mammalian mitochondrial genome can undergo rapid evolution.

DNA sequence changes of mutations in the histidine operon control region that decrease attenuation

The DNA sequence changes of 18 (9 different) mutations in the control region of the histidine operon of Salmonella typhimurium are presented. All of these mutations increase the level of expression of the operon, presumably by decreasing transcription termination at the attenuator. Five of the mutations were previously isolated hisO mutations, and the other four were isolated here as His+ pseudorevertants of His- stop codon mutations in the leader peptide gene. Only two mutations, O1242 and O3154, directly affect the terminator stem of the leader RNA. One mutation, O1202, creates a strong new stem that would compete with the terminator stem. Most of the other mutations damage other RNA stems. Their effect can best be explained by, and they thus provide supporting evidence for, the prevailing model of attenuator regulation involving alternative, competing RNA stems in the leader RNA. Two mutations that do not appear to significantly affect an RNA stem directly, including a deletion of three of the seven consecutive histidine codons, are best explained as effects of a translating ribosome upon the RNA stem structures, even though the histidine codons are not translated in the pseudorevertants.

Influence of uvrB and pKM101 on the spectrum of spontaneous, UV- and γ-ray-induced base substitutions that revert hisG46 in Salmonella typhimurium

Oligonucleotide probes were used to identify base substitutions in 1089 revertants of hisG46 in Salmonella typhimurium that arose spontaneously or following irradiation with UV- or gamma-rays. The hisG46 allele, carrying a mutant CCC codon (Pro) in place of the wild-type codon CTC (Leu69) reverted via 6 distinguishable mutational events--C to T transitions at codon sites 1 or 2, C to A or C to G transversions at codon site 1, C to A at codon site 2, and an extragenic suppressor mutation. The distribution of hisG46 revertants differed among treatments and was influenced by the DNA-repair capacity of the bacteria. Plasmid pKM101 enhanced the frequencies of both spontaneous and induced mutations; transversion events were enhanced more efficiently by pKM101 than were transition events. Compared to Uvr+ bacteria, Uvr- bacteria had higher frequencies of spontaneous and induced mutations; transition mutations were enhanced more efficiently than were transversion mutations. The influence of DNA-repair activities on the mutational spectra provides some insights on the origins of spontaneous and UV-induced mutations.

Rapid change in mouse mitochondrial DNA

We have compared the sequences of mitochondrial DNA extracted from museum skins of white-footed mice caught in the Chicago area since 1855 and from modern mice trapped alive in the same locations. We found a consistently similar directional change of mouse genotype over this period at each of five collection sites that were separated by 10–70 km. The genotype most common 100 years ago is now extremely rare, indicating that the mammalian mitochondrial genome can undergo rapid evolution.

An analysis of the mutagenicity of 1,2-dibromoethane to Escherichia coli: influence of DNA repair activities and metabolic pathways.

The mutagenicity of 1,2-dibromoethane (EDB) to Escherichia coli was reduced by the UV light-induced excision repair system but unaffected by the loss of a major apurinic/apyrimidinic site repair function. At high doses, 70-90% of the EDB-induced mutations were independent of SOS-mutagenic processing and approximately 50% were independent of glutathione conjugation. The SOS-independent mutations induced by EDB were unaffected by the enzymes that repair alkylation-induced DNA lesions. EDB-induced base substitutions were dominated by GC to AT and AT to GC transitions. These results suggest that EDB-induced premutagenic lesions have some, but not all, of the characteristics of simple alkyl lesions.

A Model System for Gene Transfer in Conifers: European Larch and Agrobacterium

Newly developing forest biotechnologies offer numerous opportunities for rapid and efficient gene transfer. However, conifers remain generally recalcitrant to these techniques, primarily because systems have not yet been developed for recovering whole transformed plants. There are four main components of an effective Aqrobacterium-mediated gene transfer system. First, desirable genes and their promoters must be incorporated within the borders of the plasmid transfer DNA. Second, a suitable Aqrobacterium vector system must be established for delivery of the plasmid into the host. Third, suitable marker genes must be available to allow for in vitro selection of transformed cells, tissues, or organs. Fourth, techniques for regeneration of whole plants are necessary. We have made substantial progress towards these requirements with Aqrobacterium-mediated transfer for European larch (Larix decidua). We have shown several strains of A. tumefaciens and rhizogenes to be virulent to young Larix in vitro. Host tissues were transformed by a binary plasmid vector bearing genetic loci for synthesis of neomycin phosphotransferase (NPT) which conveys resistance to the antibiotic kanamycin. Calli generated either from hairy roots (A. rhizogenes pARC 8) or hypertrophic inoculated hypocotyls (A. tumefaciens pWB101) survived and grew on media supplemented with kanamycin to levels of 30 and 300 mg/1, respectively. These levels of tolerance to kanamycin are consistent with those reported for selection purposes with agronomic species transformed using these vectors. Adventitious shoots of putative larch transformants were isolated after modified leaf disc incubation with A. tumefaciens or after hypocotyl inoculation with A. rhizogenes. These shoots are being grown for verification of transformation via Southern blotting. We believe our larch transformation system will serve as a valuable model system for gene transfer in other coniferous species.

A conservative isoleucine to leucine mutation causes major rearrangements and cold sensitivity in KlenTaq1 DNA polymerase.

Assembly of polymerase chain reactions at room temperature can sometimes lead to low yields or unintentional products due to mispriming. Mutation of isoleucine 707 to leucine in DNA polymerase I from Thermus aquaticus substantially decreases its activity at room temperature without compromising its ability to amplify DNA. To understand why a conservative change to the enzyme over 20 Å from the active site can have a large impact on its activity at low temperature, we solved the X-ray crystal structure of the large (5′-to-3′ exonuclease-deleted) fragment of Taq DNA polymerase containing the cold-sensitive mutation in the ternary (E–DNA–ddNTP) and binary (E–DNA) complexes. The I707L KlenTaq1 ternary complex was identical to the wild-type in the closed conformation except for the mutation and a rotamer change in nearby phenylalanine 749, suggesting that the enzyme should remain active. However, soaking out of the nucleotide substrate at low temperature results in an altered binary complex made possible by the rotamer change at F749 near the tip of the polymerase O-helix. Surprisingly, two adenosines in the 5′-template overhang fill the vacated active site by stacking with the primer strand, thereby blocking the active site at low temperature. Replacement of the two overhanging adenosines with pyrimidines substantially increased activity at room temperature by keeping the template overhang out of the active site, confirming the importance of base stacking. These results explain the cold-sensitive phenotype of the I707L mutation in KlenTaq1 and serve as an example of a large conformational change affected by a conservative mutation.