Values in parentheses are after background subtraction. All values were compared to the 2 mM MgCl 2 condition. The 6 mM MnCl 2 and CoCl 2 conditions were excluded from the analysis as their dramatically different magnitudes compared to other values complicates the analysis. An estimate of the base misincorporation frequency can be made from the CMFs in Table 2 and the sequencing results in Figure 3 as described before [ 48 ].
Using a With a CMF of 0. The base region analyzed for mutations is shown. Numbering is as shown in Figure 2 C. Deletions are shown as regular triangles, insertions are shown as downward triangles with the inserted base shown adjacent to the downward triangle, unless it was the same as the base in a nt run, and base substitutions are shown directly above or below the sequence. Substitutions shown correspond to the recovered sequence for the coding strand; however, these mutations could have occurred during synthesis of the non-coding strand as well i.
Individual sequence clones which had multiple mutations more than one mutation event are marked with subscripts adjacent to the mutations.
Several clones with deletions either single or multiple deletions at positions —, just outside of the scored region were also recovered not shown. This was the dominant mutation type recovered in background controls 19 out of 24 total sequences and probably resulted from improper ligation events or damaged plasmid vectors see [ 48 ].
It is also possible to estimate the mutation frequency using the plasmid-based assay results Table 3. The calculations yield a mutation rate of 2. A mutation rate of 2. Kinetic assays have been used by many groups as a reliable way to estimate polymerase fidelity by insertion of specific nt mismatches or extension of specific mismatched primer termini reviewed in [ 54 - 56 ].
Although pre-steady-state assays are more useful for understanding kinetic parameters for misincorporation, steady-state assays are much simpler to perform and typically yield results that are broadly similar to results with pre-steady-state assays [ 54 ]. Mismatched primer extension and running-start assays using the sequences shown in Figure 4 were performed with constant concentrations of free cation of 0.
Note that reactions with each cation were performed using different enzyme concentrations and time points see Materials and Methods. Because of these constraints, a direct comparison of the kinetic and equilibrium constants between the two cations cannot be made.
Experiments were analyzed on denaturing polyacrylamide-urea gels Figure 4 B. The misinsertion ratio for the G. A, B and C. Sequences used in mismatched primer extension and running-start misincorporation assays and examples of analysis. A The sequence of the DNA used in each assay type is shown. The underlined nts show the only differences between the two templates. The four dashes indicate the 4 A nts that must be incorporated before RT incorporates the target nt denoted by X or Y.
B Running-start misincorporation of C. The concentration of the target nt dTTP for C. For other base pair misinsertions noted in the Table 4 , the target nt was changed according to the desired misinsertion. C Extension of a mismatched primer-template with a C. Reactions were performed on the primer-template shown in panel A for the indicated time with the same free cation concentration as above.
See Materials and Methods for a description. G and dATP for C. Higher values indicate greater fidelity. A, and C. C mismatches, however the G. T mismatch showed a statistically insignificant P-value of 0. Consistent with results in the running-start reaction, no extension of the G. In this assay primers with a matched C.
G or a mismatched C. A, or C. A second sequence with a matched G. C or mismatched G. A was also used. C or dGTP for G. T, and G. A extensions. The geometry supported by different cations in the active site of polymerases has been proposed to affect fidelity.
In one report, E. In this regard, E. The effect, if any, of binding at the secondary sites is unknown. Differences between our results and these may stem from intrinsic differences in the enzymes or the different nucleic acid substrate used many of the former experiments used homopolymers.
El-Deiry et al. This indicates that one or more of the steps in catalysis is slow. Conformational transition of the protein after binding the substrate has a significant contribution to the ability of RT to add the correct substrate [ 63 ].
A correctly matched nt then leads to tight binding and alignment of catalytic residues to promote catalysis, whereas a mismatched nt does not induce the tight binding state, thereby facilitating the rapid opening of the specificity domain and release of the misaligned substrate [ 63 ]. Also, it is highly unlikely that these cations could support HIV replication. This suggests that catalysis with these alternative cations is not intrinsically mutagenic and the observed mutagenicity in previous reports, may result from other mechanisms that could occur at high concentrations see Discussion that warrants further investigation.
DNase deoxyribonuclease -free RNase ribonuclease , ribonucleotides, and deoxyribonucleotides were obtained from Roche. Radiolabeled compounds were from PerkinElmer. Pfu DNA polymerase was from Stratagene. G spin columns were from Harvard Apparatus. X-gal was from Denville Scientific, Inc.
Michael Parniak University of Pittsburgh. This enzyme is a non-tagged heterodimer consisting of equal proportions of p66 and p51 subunits. Full extension produced a nt final product see Figure 2 A. The long template was used to make it easier to separate DNA synthesis products from the RNA template on a denaturing polyacrylamide-urea gel see below. Different time points were used to assure that all the reactions were essentially complete with each cation. The final pH of the reactions was 7. Typically two reactions for each condition were combined and material was recovered by standard phenol:chloroform extraction and ethanol precipitation.
After overnight elution, this material was passed through a 0. The gel was run far enough to efficiently separate the nt templates from the nt full extension product of round 2. Dephosphorylated vector was recovered by phenol-chloroform extraction followed by ethanol precipitation and quantified using absorbance at nm.
Ligation and transformation of E. White or faint blue colonies were scored as harboring mutations while blue colonies were non-mutated. Any colonies that were questionable with respect to either being faint blue or blue were picked and replated with an approximately equal amount of blue colony stock. Observing the faint blue colony in a background of blue colonies made it easy to determine if the colony was faint blue rather than blue.
The gapped version of the plasmid pSJ2 was prepared as described [ 53 ]. The reaction pH was 7. The colony mutant frequency CMF was determined using blue-white screening as described above.
The approach used for these assays was based on previous results [ 67 ]. Steady-state kinetic parameters K m , and V max were then calculated as described below. The concentration of free cation was calculated using the formula:.
This assumption leads to an approximate value for the free concentration of these cations in reactions. The approach used for these assays was based on previous results [ 69 ].
The underlined nts in parentheses indicate that templates with either a G or C at this position were used. The amount of free cation in each reaction was adjusted according to the dNTP concentration as described above. Velocity measurement and calculation of V max and K m were performed as described previously for mismatch extension [ 69 ] and running-start assays [ 67 ]. Extension rate determinations for DNA synthesis on the nt RNA template for various cations were performed as described previously [ 20 ].
The average extension rate was calculated by taking into account the length and the relative intensity of all extension products in a time point.
The thermal cycler heats and cools the reaction tubes to achieve the temperatures required at each step of the reaction see below. Many modern thermal cyclers make use of the Peltier effect which permits both heating and cooling of the block holding the PCR tubes simply by reversing the electric current. Thin-walled reaction tubes permit favorable thermal conductivity to allow for rapid thermal equilibration.
Most thermal cyclers have heated lids to prevent condensation at the top of the reaction tube. Older thermocyclers lacking a heated lid require a layer of oil on top of the reaction mixture or a ball of wax inside the tube. When using Pfu DNA polymerase ng of each primer and a hot start for Pfu reaction was carried out. In certain scenarios, chemical additives or co-solvents may be included in the buffer to improve amplification specificity by reducing mispriming and to enhance amplification efficiency by removing secondary structures Table 2.
These reagents are commonly used with difficult samples such as GC-rich templates. Also, they can interfere with certain downstream applications— for example, nonionic detergents in microarray experiments. Hence, it is important to be aware of buffer compositions for successful PCR and downstream usage. Don't have an account? Create Account. Sign in Quick Order. Search Thermo Fisher Scientific. Search All. See Navigation. Table 1.
General recommendations on designing PCR primers. Deoxynucleoside triphosphates dNTPs. Table 2. Common additives or co-solvents used as PCR enhancers, and their recommended final concentrations [6]. Dordrecht: Springer. Gene 93 1 — Anal Biochem 1 — J Biol Chem 30 : — Steitz TA A mechanism for all polymerases. Nature — In: Methods in molecular biology 2nd ed. Totowa: Humana Press. Learn more.
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