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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TL;DR: It is determined for the first time that a sticky end can be made by using the 3′-5′ exonuclease activity of KF, and the cleaved sticky ends were characterized to be mainly 2 nt 5′ overhang.
Abstract: The Klenow fragment (KF) has been used to make the blunt end as a tool enzyme. Its 5'-3' polymerase activity can extend the 5' overhanging sticky end to the blunt end, and 3'-5' exonuclease activity can cleave the 3' overhanging sticky end to the blunt end. The blunt end is useful for cloning. Here, we for the first time determined that a sticky end can be made by using the 3'-5' exonuclease activity of KF. We found that KF can cleave the blunt end into certain sticky ends under controlled conditions. We optimized enzyme cleavage conditions, and characterized the cleaved sticky ends to be mainly 2 nt 5' overhang. By using these sticky ends, we realized ligation reaction in vitro, and accomplished cloning short oligonucleotides directionally with high cloning efficiency. In some cases, this method can provide sticky end fragments in large scale for subsequent convenient cloning at low cost.
7 citations
Cites methods from "Polymerization behavior of Klenow f..."
...The ligation products were analyzed by 18% nondenaturing PAGE and fast silver stain method as described (Zhao and Guan 2010)....
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...The products were analysed by 18% nondenaturing PAGE and fast silver stain method as described (Zhao and Guan 2010)....
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...For sticky-end identification, we used short oligonucleotide substrate and native PAGE as described previously (Zhao and Guan 2010; Zhao 2013)....
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TL;DR: It was found that the Klenow fragment mutants with partial deletions of thumb subdomain were unable to extend primers to the 5′ terminal of templates, thus creating 5′ overhanging sticky ends 2 nt long, which is termed PmTP (premature termination of polymerization).
Abstract: DNA polymerization products by Klenow fragment (KF) are blunt-ended. In the present study, we found that the Klenow fragment mutants with partial deletions of thumb subdomain were unable to extend primers to the 5′ terminal of templates, thus creating 5′ overhanging sticky ends 2 nt long. We termed this phenomenon as PmTP (premature termination of polymerization). The KF mutants produced homogenous sticky-ended products only under mild reaction conditions, whereas under vigorous reaction conditions, the sticky ends were prone to be blunt-ended. It was also identified that deletions of more than four residues of KF thumb subdomain could induce PmTP, and tworesidue deletion of KF thumb subdomain only induced PmTP in a lower-concentration situation. Structure modelling analysis suggested that shortening or destruction of α helix H1 at the tip of the thumb subdomain was crucial to PmTP, while the conserved residues in front of α helix was less important. PmTP might be caused by the reduced DNAbinding affinity of the mutants. The sticky ends made by PmTP have potential applications in gene splicing and molecular cloning techniques.
3 citations
Cites methods from "Polymerization behavior of Klenow f..."
...In polymerase activity assay, after s/s8 was incubated with KF or D424A, respectively, their products demonstrated the same gel shift mobility (figure 3, lanes 5 and 6), indicating that both D424A and KF had polymerase activity (Zhao and Guan 2010)....
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...The ligation products were analysed by 18% nondenaturing PAGE and fast silver stain method as described (Zhao and Guan 2010)....
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...The products were analysed by 18% nondenaturing PAGE and fast silver stain method as described (Zhao and Guan 2010)....
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TL;DR: A novel closed-tube method for amplifying and characterizing heterogeneous target sequences using members of the CTX-M beta-lactamase gene family, which is designed for symmetric PCR or for Linear-After-The-Exponential (LATE)-PCR.
Abstract: The amplification and detection of diverse strains of an infectious virus or bacteria, or variants within a gene family is important for both clinical and basic research but can be difficult using conventional PCR. This report describes and illustrates a novel closed-tube method for amplifying and characterizing heterogeneous target sequences using members of the CTX-M beta-lactamase gene family. Different subgroups of CTX-M genes exhibit low sequence identity, but accurate and efficient detection of these variants is critical because they all confer resistance to penicillin, cefotaxime, and other antibiotics of the beta-lactam class. The method combines a single pair of "thermodynamic consensus primers" (tcPrimers) with one or more "initiator primers" (iPrimers), added at low concentration (5-10 nM). Each iPrimer improves the initial amplification of one or more variants because it has fewer mismatches to its intended target than the more abundant tcPrimers. As a result of initial amplification, each heterogeneous sequence is shifted stepwise toward a better match with the tcPrimers. As soon as the tcPrimer hybridization takes place, amplification proceeds with high efficiency. The tcPrimer pairs can be designed for symmetric PCR or for Linear-After-The-Exponential (LATE)-PCR. LATE-PCR offers the advantage of generating single-stranded DNA that can be characterized for different gene variants in the same closed tube, using low-temperature mismatch-tolerant fluorescent probes.
2 citations
01 Jan 2011
1 citations
TL;DR: A colorimetric assay based on the coupling of gap ligase chain reaction with DNAzyme to detect the target GmSg-1 genes of class A soybean saponins with the naked eye, without the involvement of expensive instruments is proposed.
Abstract: Class A saponins are responsible for the taste of soybean products, and the rapid identification of class A saponins from soybean food is essential for both food safety and cultivar screening. In this study, we propose a colorimetric assay based on the coupling of gap ligase chain reaction (Gap-LCR) with DNAzyme to detect the target GmSg-1 genes of class A soybean saponins with the naked eye, without the involvement of expensive instruments. The limits of detection (LODs) for the GmSg-1a and GmSg-1b genes were determined to be 0.1618 and 0.1625 μM, respectively, with a linear range of 0.2–1.2 μM. The DNAzyme-based Gap LCR assay was successfully employed to identify the target genes from different soybean cultivars, providing a simple means for monitoring the quality of soybean products.
1 citations
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.
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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.
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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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