Polymerization behavior of Klenow fragment and Taq DNA polymerase in short primer extension reactions.
01 Oct 2010-Acta Biochimica et Biophysica Sinica (Oxford University Press)-Vol. 42, Iss: 10, pp 722-728
TL;DR: It was found that the longer the primer, the more efficient is the primer extension, and these polymerization behavior of short primers lay foundation about DNA polymerization mechanism and development of novel nucleic acid detection assays.
Abstract: DNA polymerases amplify DNA fragments through primer extension reactions. However, polymerization behavior of short primers in the primer extension process has not been systematically explored. In this study, we examined the minimal primer length required for primer extension, and the effect of primer length, mismatches and other conditions on DNA polymerization using a non-radioactive method. Under the condition we conducted, the shortest primers polymerized by Klenow fragment (KF) and Taq DNA polymerase in our experiments were respectively heptamer and octamer. The extension efficiency was also affected by the up-stream overhanging structure of the primer-template complex. We hypothesized a simple model to interpret these observations based on the polymerase structures. Furthermore, it was found that the longer the primer, the more efficient is the primer extension. These polymerization behavior of short primers lay foundation about DNA polymerization mechanism and development of novel nucleic acid detection assays.
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01 Jan 2014
TL;DR: My studies on d(dinitro-tC (O))TP support the existing hypothesis that selection for the correct nucleotide based on size is only true for analogues closely related to the canonical bases, and appear to use both the size and hydrogen bonding patterns of incoming nucleotides to distinguish their correctness.
Abstract: Acknowledgements My most heartfelt thanks goes to my advisor, mentor and friend Dr. Rob Kuchta, for his unending patience and willingness to share his knowledge with me, as well as his continued support for my pursuit of science. I would like to thank my friends in the Kuchta lab, and to some colleagues in particular. Thank you to Dr. Andrew Olsen and Dr. Gudrun Stengel for being the very first overseers of my work, and for not scaring me off. Hougen and Taylor Minckley for the many laughs we've shared benchside, and for answering even the most insignificant of questions. Thank you to my committee members, Dr. Jennifer Martin and Dr. Rob Knight, for their assistance in navigating my thesis work. My work would not have been possible without generous funding from the National Institutes of Health. Thank you to my dear friend Makenna Morck for always being available to discuss biochemistry with me in the wee hours of the morning. I want to extend my profound thanks to my family: my parents, Jean and Michael, for being my first teachers, sparking a passion in me for learning; and my sisters, Sarah and Katelyn, for their unwavering support, and for pretending to be interested when I talk science. Analysis of Tautomeric Equilibrium of the Analogue d(dinitro-tC The studies outlined in this thesis examines the kinetic parameters of incorporation of the novel nucleotide analogue d(dinitro-tC (O))TP by the Klenow fragment of DNA polymerase I, accomplished through a series of polymerization assays. d(dinitro-tC (O))TP contains an additional pair of nitro groups at the top of the cyclic structure, expanding on the structures of previously studied synthetic nucleotides tC and tC (O) (Stengel et al. 2009). Nitro groups have a large electron withdrawing effect due to the highly electron-withdrawing oxygens, resulting in the compound being recognized more as T than as C when compared to the parent tC and tC (O). KF incorporates d(dinitro-tC (O))TP as efficiently as dTTP opposite a templating adenosine base, and about twice as efficiently as dCTP opposite guanosine. My studies on d(dinitro-tC (O))TP support the existing hypothesis that selection for the correct nucleotide based on size is only true for analogues closely related to the canonical bases. KF appears to use both the size and hydrogen bonding patterns of incoming nucleotides to distinguish their correctness. The ability of d(dinitro-tC (O))TP to hydrogen bond in a manner similar to …
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Additional excerpts
...The cleaved enzyme retains 5’—3’ polymerase activity and its 3’—5’ exonuclease domain (Klenow and Overgaard-Hansen 1970; Zhao et al. 2013; Zhao and Guan 2010)....
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TL;DR: A fidelity quantification of Hg2+ based on nucleic acids amplification is developed via a redox reaction or DNA architecture probe that exhibits excellent accuracy and sensitivity with a detection limit down to 300 pM.
Abstract: Mercury(II) ion (Hg2+) is a hazardous pollutant with distinct toxicological profiles that can cause deleterious effects on human health and the environment even at low concentrations, so an accurate detection method with high sensitivity is of great importance. Compared with traditional techniques requiring sophisticated instrumentation and skilled personnel, Hg2+ sensors based on nucleic acids amplification outperform others due to their efficiency, robustness, and ease of operation. Among these nucleic acids amplification-based strategies, a common problem meriting attention is that biothiols such as dithiothreitol (DTT) extensively exist in enzymatic amplification systems as essential components to guarantee tool enzyme activity. However, due to the high affinity of Hg2+ and S-donor atoms, biothiols tend to sequestrate Hg2+ and are probably present to affect the accuracy of detection. Herein, a fidelity quantification of Hg2+ based on nucleic acids amplification is developed via a redox reaction or DNA architecture probe. Upon circumventing a biothiols-induced sequestration in enzymatic amplification system, the proposed strategy exhibits excellent accuracy and sensitivity with a detection limit down to 300 pM.
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References
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TL;DR: A 3.5 angstrom resolution electron density map of the HIV-1 reverse transcriptase heterodimer complexed with nevirapine, a drug with potential for treatment of AIDS, reveals an asymmetric dimer.
Abstract: A 3.5 angstrom resolution electron density map of the HIV-1 reverse transcriptase heterodimer complexed with nevirapine, a drug with potential for treatment of AIDS, reveals an asymmetric dimer. The polymerase (pol) domain of the 66-kilodalton subunit has a large cleft analogous to that of the Klenow fragment of Escherichia coli DNA polymerase I. However, the 51-kilodalton subunit of identical sequence has no such cleft because the four subdomains of the pol domain occupy completely different relative positions. Two of the four pol subdomains appear to be structurally related to subdomains of the Klenow fragment, including one containing the catalytic site. The subdomain that appears likely to bind the template strand at the pol active site has a different structure in the two polymerases. Duplex A-form RNA-DNA hybrid can be model-built into the cleft that runs between the ribonuclease H and pol active sites. Nevirapine is almost completely buried in a pocket near but not overlapping with the pol active site. Residues whose mutation results in drug resistance have been approximately located.
1,902 citations
Journal Article•
TL;DR: The rapid staining protocol significantly decreases the processing time required for silver-stained polyacrylamide gels, which is of particular importance in diagnostic situations, and allows individual bands from complex mixtures to be easily recovered for sequencing or probe preparation.
Abstract: A rapid silver-staining procedure for DNA fragments in polyacrylamide gels is described. The time required for band detection is 15 min and the limit of sensitivity 3 pg/mm2. PCR products subjected to this rapid staining protocol are readily recovered from the gel by excision and elution by incubation at 95 degrees C for 20 min. Bands of up to 3 kb have been recovered and reamplified from either recently prepared or dried gels. The rapid staining protocol significantly decreases the processing time required for silver-stained polyacrylamide gels, which is of particular importance in diagnostic situations. The recovery protocol allows individual bands from complex mixtures to be easily recovered for sequencing or probe preparation.
1,175 citations
TL;DR: The effects of various primer-template mismatches on DNA amplification of an HIV-1 gag region by the polymerase chain reaction (PCR) were investigated, although the G:G mismatches appeared to be more sensitive to sequence context and dNTP concentrations than other mismatches.
Abstract: We investigated the effects of various primer-template mismatches on DNA amplification of an HIV-1 gag region by the polymerase chain reaction (PCR). Single internal mismatches had no significant effect on PCR product yield while those at the 3'-terminal base had varied effects. A:G, G:A, and C:C mismatches reduced overall PCR product yield about 100-fold, A:A mismatches about 20-fold. All other 3'-terminal mismatches were efficiently amplified, although the G:G mismatches appeared to be more sensitive to sequence context and dNTP concentrations than other mismatches. It should be noted that mismatches of T with either G, C, or T had a minimal effect on PCR product yield. Double mismatches within the last four bases of a primer-template duplex where one of the mismatches is at the 3' terminal nucleotide, in general, reduced PCR product yield dramatically. The presence of a mismatched T at the 3'-terminus, however, allowed significant amplification even when coupled with an adjacent mismatch. Furthermore, even two mismatched Ts at the 3'-terminus allowed efficient amplification.
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TL;DR: Of particular interest are the role of editing in the fidelity of copying, the common enzymatic mechanism of polymerases, and the manners in which different domain structures function in the polymerase reaction in analogous ways.
863 citations
TL;DR: Two ternary complexes of rat DNA polymerase beta, a DNA template-primer, and dideoxycytidine triphosphate have been determined at 2.9 A and 3.6 A resolution, suggesting that the polymerase-DNA-ddCTP interactions are not affected by crystal packing forces.
Abstract: Two ternary complexes of rat DNA polymerase beta (pol beta), a DNA template-primer, and dideoxycytidine triphosphate (ddCTP) have been determined at 2.9 A and 3.6 A resolution, respectively. ddCTP is the triphosphate of dideoxycytidine (ddC), a nucleoside analog that targets the reverse transcriptase of human immunodeficiency virus (HIV) and is at present used to treat AIDS. Although crystals of the two complexes belong to different space groups, the structures are similar, suggesting that the polymerase-DNA-ddCTP interactions are not affected by crystal packing forces. In the pol beta active site, the attacking 3'-OH of the elongating primer, the ddCTP phosphates, and two Mg2+ ions are all clustered around Asp190, Asp192, and Asp256. Two of these residues, Asp190 and Asp256, are present in the amino acid sequences of all polymerases so far studied and are also spatially similar in the four polymerases--the Klenow fragment of Escherichia coli DNA polymerase I, HIV-1 reverse transcriptase, T7 RNA polymerase, and rat DNA pol beta--whose crystal structures are now known. A two-metal ion mechanism is described for the nucleotidyl transfer reaction and may apply to all polymerases. In the ternary complex structures analyzed, pol beta binds to the DNA template-primer in a different manner from that recently proposed for other polymerase-DNA models.
728 citations