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Can Taq polymerase be used instead of polymerase Vent exo (-)?

Can Taq polymerase be used instead of polymerase Vent exo (-)?


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Instead of using polymerase Vent exo (-), can I go with the usual Taq polymerase?

Do the PCR conditions change (the temperature and master mix concentrations ) in the these two conditions? Do the primers (sequence and concentrations) change if we use vent exo instead of Taq polymerase and vice versa? If the primers are designed specifically for Vent exo (-) how can I modify the PCR conditions so that I can use the same primers (meant for vent exo) using Taq polymerase?


I haven't done ARMS PCR myself, so my answer is based on theory and there might be other practical factors I overlooked.

The characteristic feature of the Vent exo(-) polymerase is the lack of exonuclease activity compared to the regular Vent polymerase. ARMS uses the fact that a mismatch at the 3' position prevents primer extension. For this to work it is important to use a polymerase without exonuclease activity, as such a polymerase could "repair" the mismatch and the whole method would not work. Taq polymerase also doesn't have any exonuclease activity, so in theory you should be able to use it for this experiment.

The primers don't have to be changed for different polymerases, the polymerase buffer though is different and I wouldn't recommend using the wrong one.


DUTPs conjugated with zwitterionic Cy3 or Cy5 fluorophore analogues are effective substrates for DNA amplification and labelling by Taq polymerase

The authors wish it to be known that in their opinion, the first three authors should be regarded as joint First Authors.

Olga A Zasedateleva, Vadim A Vasiliskov, Sergey A Surzhikov, Viktoriya E Kuznetsova, Valeriy E Shershov, Timur O Guseinov, Igor P Smirnov, Roman A Yurasov, Maksim A Spitsyn, Alexander V Chudinov, dUTPs conjugated with zwitterionic Cy3 or Cy5 fluorophore analogues are effective substrates for DNA amplification and labelling by Taq polymerase, Nucleic Acids Research, Volume 46, Issue 12, 6 July 2018, Page e73, https://doi.org/10.1093/nar/gky247


ABSTRACT

We synthesized C5-modified analogs of 2′-deoxyuridine triphosphate and 2′-deoxycytidine triphosphate and investigated them as substrates for PCRs using Taq, Tth, Vent(exo-), KOD Dash and KOD(exo-) polymerases and pUC 18 plasmid DNA as a template. These assays were performed on two different amplifying regions of pUC18 with different T/C contents that are expected to have relatively high barriers for incorporation of either modified dU or dC. On the basis of 260 different assays (26 modified triphosphates × 5 DNA polymerases × 2 amplifying regions), it appears that generation of the full-length PCR product depends not only on the chemical structures of the substitution and the nature of the polymerase but also on whether the substitution is on dU or dC. Furthermore, the template sequence greatly affected generation of the PCR product, depending on the combination of the DNA polymerase and modified triphosphate. By examining primer extension reactions using primers and templates containing C5-modified dUs, we found that a modified dU at the 3′ end of the elongation strand greatly affects the catalytic efficiency of DNA polymerases, whereas a modified dU opposite the elongation site on the template strand has less of an influence on the catalytic efficiency.


RESULTS

The polymerization of a short stretch of DNA (microgene) was first observed when we performed PCR with a primer set of KY-794 and KY-795 (Fig. ​ (Fig.1 1 A) under long PCR conditions (24). These primers form eight complementary base pairs in the 3′ region and have a single extra nucleotide at the 3′-OH end (of KY-795) that does not pair with KY-794 (Fig. ​ (Fig.1 1 A). These primers gave a heterogeneous “smeared” DNA product without any template DNA after 35 cycles of PCR with all four dNTPs and an enzyme mixture of Taq and Pwo polymerases (a similar result is shown in Fig. ​ Fig.2 2 B). The sizes of these heterogeneous DNA products ranged from � bp to 㸒 kb, and increased cycle numbers resulted in the production of a larger population of DNA (data not shown). We were interested in the origin of these heterogeneous DNA molecules and determined the nucleotide sequences after cloning them into a plasmid vector. The cloned products were shown to comprise tandem repeats of a short stretch of DNA (Fig. ​ (Fig.1 1 A). This 36-bp repeating unit is a primer dimer product (25) of KY-794 and KY-795.

Head-to-tail tandem repeats formation in MPR. (A) Primers used in MPR, primer dimer, and schematic representation of head-to-tail tandem polymer of primer dimer. Mismatched nucleotide at 3′-OH end is in green. (B) An example of a microgene polymer (pYT005). Inserted nucleotides or deletion (Δ) at junction points of a microgene unit is in red.

Effects of the 3′-OH mismatch and 3′𠄵′ exonuclease activity of DNA polymerase on MPR. (A) Four sets of primers that have double, single, single, and no 3′-OH mismatch nucleotide(s). Mismatch nucleotides are in green. (B) MPR product (10 μl) was fractionated through a 1.2% agarose gel. Lane M, DNA size marker (1-Kb DNA ladder Life Technologies, Gaithersburg, MD). The sizes of some fragments are indicated on the left (in kilobases). Forty-five cycles of MPR were performed, and the annealing temperature was 69ଌ. (C) Vent (wt) or Vent (exo − ) DNA polymerase was used instead of the Expand Taq polymerase mixture, 45 cycles of MPR were performed, and the annealing temperature was 69ଌ. (D) An example of a microgene polymer (pYT094) created from KY-854 and KY-795 with Vent (exo − ) DNA polymerase. Deletions (Δ) at junctions are in red. Nucleotides originating from the 3′-OH mismatches are in green.

The repeating unit polymerized in a head-to-tail manner without exception as far as examined and was accompanied by insertion or deletion of a few nucleotides at their junctions. In one example (Fig. ​ (Fig.1 1 B) among seven junctions, four had insertions of one or four nucleotides, two had a single nucleotide deletion, and one junction had no insertion⿞letion. Although a tetra-nucleotide CCGG was inserted frequently at junctions from many clones, other inserted nucleotides included a single T or C or the trinucleotides CGG, GCG, and CCA (data not shown). These insertion sequences may not have originated from the primers because the tetranucleotide CCGG (or its complement GGCC) is not contained in the sequences of these primers.

Primers KY-794 and KY-795 were prepared on a DNA synthesizer and were purified by HPLC. To exclude the possibility that the observed polymer formation was due to unexpected modification of the primers during DNA synthesis or HPLC purification, another set of primers with the same sequences as KY-794 and KY-795 was synthesized using another DNA synthesizer and was purified on a NAP-10 gel filtration column (Pharmacia Biotech, Uppsala, Sweden). These newly synthesized primers gave similar polymers of a primer dimer (data not shown), suggesting that the polymerization was not due to aberrant synthesis or unexpected modification of KY-794 and KY-795 primers. Polymers of a primer dimer were obtained reproducibly with these primers under the conditions described, and we named this reaction the microgene polymerization reaction (MPR).

Factors That Affect Polymerization Efficiency in MPR.

To determine factors that gave efficient polymerization of primer dimers in MPR with KY-794 and KY-795, we performed the following experiments. First, we focused on the single extra nucleotide at the 3′-OH end of KY-795 that does not pair with KY-794 (Fig. ​ (Fig.1 1 A). To assess the contribution of the mismatch at 3′-OH ends of primers on MPR, we prepared four pairs of primers that have double, single, and no mismatch pair(s) at their 3′-OH termini (Fig. ​ (Fig.2 2 A). After 45 cycles of MPR with a mixture of Taq and Pwo polymerases, the primer pair with double mismatches (KY-854𯞕) produced the most diffuse smear of DNA whereas primers that lacked mismatches at their 3′-OH ends (KY-803𯞃) did not produce smeared DNA in these conditions (Fig. ​ (Fig.2 2 B). These results indicated that mismatched base pairs at primer 3′-OH termini are a necessary factor in MPR. The effects of a single mismatch were dependent on the nucleotide sequence of the primer. A single base mismatch at the 3′-OH terminus of KY-795 led to the production of smeared DNA whereas the mismatch on KY-854 did not produce a polymer under similar conditions. The factors that caused this difference are not known.

Second, we tested the effect of different polymerases on MPR. The long PCR protocol used in the initial experiments contained a mixture of thermostable Taq and Pwo DNA polymerases. The former enzyme has 5′𠄳′ exonuclease activity in its N-terminal domain (26) but lacks detectable 3′𠄵′ exonuclease activity (27) whereas the latter, archaeal DNA polymerase has 3′𠄵′ exonuclease activity but is believed to lack 5′𠄳′ exonuclease activity (28). Because no heterogeneous “smeared” DNA was observed with the primers when Taq polymerase was used in standard PCR conditions, we reasoned that the 3′𠄵′ exonuclease activity of the Pwo DNA polymerase was critical to MPR. Several 3′ exonuclease plus polymerases and their exonuclease minus derivatives are commercially available. Vent DNA polymerase originates from the hyperthermophilic archaeal bacterium Thermococcus litoralis, and its 3′ exonuclease deficient mutant, Vent exo − , has been constructed (28). We next performed MPRs using these Vent wt and Vent exo − polymerases instead of a mixture of Taq and Pwo polymerases with primer pairs KY-854𯞕 (double mismatches) or KY-803𯞃 (no mismatch). The MPR with these polymerases gave similar results as the MPR with a mixture of Taq and Pwo polymerases (Fig. ​ (Fig.2 2 C). Primers without mismatch pairs at the 3′-OH ends (KY-803𯞃) did not produce polymers in the presence or absence of 3′𠄵′ exonuclease activity, confirming the importance of the 3′-OH end mismatch. The combination of primers with double mismatches (KY-854𯞕) and Vent (exo + ) DNA polymerase resulted in the abundant production of heterogeneous DNA, indicating that a Taq polymerase that has 5′𠄳′ exonuclease activity is not necessarily required for polymer formation. An unexpected result was the production of polymers from the combination of primers with double mismatches (KY-854𯞕) and Vent (exo − ) DNA polymerase although the amount of products was much less compared with that with Vent wt polymerase. Because primer dimers are formed by elongation from one primer using the other primer as template (25), the mismatched 3′ ends of KY-854𯞕 should be removed by the 3′𠄵′ exonuclease activity before chain elongation. We were interested in the structure of heterologous DNA products from MPR with KY-854𯞕 and Vent (exo − ) and so determined the nucleotide sequences of them. The polymers were made essentially from sequences derived from two primers (KY-854𯞕), but additional sequence irregularities were present (one example is shown in Fig. ​ Fig.2 2 D). In these cases, primer dimers were not necessarily repeating units, but, instead, various shorter sequences that were related to the primer dimer were randomly joined. In some cases, nucleotide sequences that originated from the 3′ ends of two primers were observed in the sequence, indicating that the aberrant chain extension had occurred in MRP. In some joining junctions, one primer seemed to connect directly to itself or a primer dimer.

From the above results, we concluded that the factors that enhance the efficiency of MPR are (i) the existence of mismatched nucleotides at 3′-OH ends of primers and (ii) 3′𠄵′ exonuclease activity of DNA polymerase. The role of the mismatched nucleotide at 3′-OH ends on MPR is further discussed below.

MPR with Mixed Primers Increases Molecular Diversity of Polymers.

The aim of our study was to construct a pool of protein-coding DNAs with a high degree of molecular diversity that can serve as a starting point for in vitro protein evolution experiments. Polymerization of microgenes that are deficient in termination codons is one approach to constructing such a library. MPR can be used for this purpose if the primers are designed so that the primer dimers correspond to a stop-codon-free repeating unit. Randomly inserted nucleotides or deletions at junctions alter the reading frame of a microgene in every repeating unit, and the resultant MPR products are a combinatorial library made from 2 × 3 reading frames. In addition to this molecular diversity resulting from frame switching, we tested the possibility that diversity could be further acquired by using a mixture of primers in MPR.

First, we used five primers in MPR instead of two. These included the initial KY-794 and KY-795 and their three derivatives, whose sequences differ from their parent primer at three positions (Fig. ​ (Fig.3 3 A). Sequence analyses of cloned polymers from MPR with these primers revealed that polymers were made from mixtures of the five primers (one example is shown in Fig. ​ Fig.3 3 A). Similarly, a primer that was degenerate at specific positions was used to increase molecular diversity. KY-812, a derivative of KY-794, has two degenerate positions (Fig. ​ (Fig.3 3 B). MPR with KY-812 and KY-795 gave polymers with two positions randomized (one example is shown in Fig. ​ Fig.3 3 B). It should be noted that the first degenerate position (the third nucleotide of KY-794) had a preference for C (Fig. ​ (Fig.3 3 B) whereas the second degenerate position (the 11th nucleotide of KY-794) did not have a preference in this experiment. Thus, usage of mixed primers or degenerated primers in MPR increases the molecular diversity of the resultant polymers.

MPR with mixtures of primers. (A) Five primers used in MPR are shown on the top. Mismatched nucleotides are in green, and sequences that are differ among primers are separately colored. MPR was carried out with an Expand Taq polymerase mixture for 45 cycles at 69ଌ for extension. An example of a microgene polymer (pYT037) is shown on the bottom. Inserted nucleotides or deletion (Δ) at junctions is in red. (B) A degenerated primer was used in MPR. Ns represent a mixture of A, T, G, and C. MPR was carried out with an Expand Taq polymerase mixture for 65 cycles at 69ଌ for extension. An example of a polymers (pYT020) is shown.

Designer Microgenes.

Although a high degree of molecular diversity can be generated from random frame switching at junction points and the use of primer mixtures in MPR, we expected that the nature of the resultant libraries would be greatly influenced by the choice of the initial microgene. To show that the MPR technique is applicable to a wide variety of microgenes, we designed two microgenes starting from polypeptide sequences found in proteins with known three-dimensional structure and constructed libraries through MPR. One microgene had 42 bp and was designed from a 14-amino acid peptide that constituted a part of a coiled coil α-helix in seryl-tRNA synthetase (29) (Fig. ​ (Fig.4 4 A). The microgene was designed so that one frame codes for the 14-amino acid polypeptide and the other five frames do not contain any termination codons. The second 66-bp microgene was designed from one turn of a parallel β-helix protein, pectate lyase from Erwinia chrysanthemi (32). For this microgene, a 22-amino acid consensus sequence was first designed from the repeating units, each of which is comprised of three β-strands (Fig. ​ (Fig.4 4 B). Primer pairs were designed for these two microgenes so that (i) primer pairs could form 10 and 9 base pairs in their 3′ regions (ii) the sequences of primer dimers could recreate microgene sequences and (iii) mismatched single extra nucleotides (A or T) were added at their 3′-OH ends. MPR with these primers and Vent wt DNA polymerase produced heterologous, smeared DNA as expected, and sequence analyses of cloned products revealed that the microgenes were tandemly polymerized with nucleotide insertions or deletions at junctions (Fig. ​ (Fig.4 4 D and H).

Examples of designer microgenes. (A) Sequence of 14 amino acids was extracted from the anti-paralleled coiled coil α-helix region of the N-terminal domain of Escherichia coli seryl-tRNA synthetase (30). The structure of the enzyme from T. thermophilus is shown (29), and the corresponding region in this enzyme is represented in blue ֽrawn using Rasmol (31)]. (B) From the extracted sequence, a microgene was designed so that one of six frames codes for the extracted sequence and the others do not contain any termination codons. The sixth residue (Val) in the extracted sequence had to be changed to Glu in the microgene to avoid the appearance of a termination codon in other frames. (C) MPR primers were designed for the microgene. Mismatched nucleotides were added at 3′-OH ends of both primers (in green). (D) An example of a microgene polymer (pYT071) made from KY-859 and KY-860. MPR was carried out for 65 cycles using Vent (exo + ) DNA polymerase. The annealing and extension temperature was 63ଌ. Inserted nucleotides at junctions are in red. (EH) Design of a microgene from a parallel β-helix protein, E. chrysanthemi pectate lyase E (32), and an example of a microgene polymer. The strategy was same as for AD, except the 22-amino acid sequence was a consensus sequence comprised of three β-strands.

Polymers made from designed microgenes were cloned into an expression vector, and one reading frame was translated in E. coli. Expressed proteins were analyzed by SDS/PAGE. Examples of 10 clones for each microgene are shown (Fig. ​ (Fig.5). 5 ). Three clones from the α-helix microgene and six clones from the β-strand microgene, whose sizes of polymer inserts ranged from 0.25 to 0.8 kb, produced polypeptides with apparent molecular masses of 15� kDa, indicating that the MPR products can serve as a source for in vitro protein evolution experiments. We are now analyzing physical characters of the polypeptides translated from microgene polymers.

SDS/PAGE of proteins from E. coli cells expressing one reading frame of microgenes polymers. (A) α-Helix microgene. (B) β-Strand microgene. Molecular markers were run in lane M, and their sizes are shown on the left in kilodaltons. Visible bands that are expected to be products are indicated by green dots. Some of them (clones 3 and 8 from A and 4, 5, and 8 from B) were confirmed to be products of microgene by purifying them using oligo histidine–tag (data not shown).


Abstract

Replication slippage of DNA polymerases is a potential source of spontaneous genetic rearrangements in prokaryotic and eukaryotic cells. Here we show that different thermostable DNA polymerases undergo replication slippage in vitro, during single-round replication of a single-stranded DNA template carrying a hairpin structure. Low-fidelity polymerases, such as Thermus aquaticus (Taq), high-fidelity polymerases, such as Pyrococcus furiosus (Pfu) and a highly thermostable polymerase from Pyrococcus abyssi (Pyra™ exo − ) undergo slippage. Thermococcus litoralis DNA polymerase (Vent®) is also able to slip however, slippage can be inhibited when its strand-displacement activity is induced. Moreover, DNA polymerases that have a constitutive strand-displacement activity, such as Bacillus stearothermophilus DNA polymerase (Bst), do not slip. Polymerases that slip during single-round replication generate hairpin deletions during PCR amplification, with the exception of Vent® polymerase because its strand-displacement activity is induced under these conditions. We show that these hairpin deletions occurring during PCR are due to replication slippage, and not to a previously proposed process involving polymerization across the hairpin base.


Polymerase Processivity

The importance of proofreading activity to PCR has been widely known for nearly two decades, but another property, processivity, has gained increased attention. &ldquoProcessivity&rdquo is a term that refers to the number of nucleotides incorporated by a polymerase in a single binding event (before dissociation). Taq DNA polymerase adds approximately 50 nucleotides per binding event (8). Why does this matter? A low-processivity or &ldquodistributive&rdquo polymerase extends a population of templates in a noticeably different manner than a processive polymerase. A highly distributive polymerase binds to a template, adds a couple of nucleotides, and dissociates, leaving a population of templates that can be extended equally with time. A highly processive polymerase binds a template and extends with longer binding events.

It would follow that given enough time the outcome of either a processive or distributive polymerase reaction would be a population of copied templates. However, in certain circumstances it is possible that the processive polymerase has superior performance. The E. coli polymerase III &alpha subunit, part of the main replicative polymerase, has a processivity of < 10 base pairs and a speed of < 20 nucleotides/second (nt/s). However, when the subunit associates with the other replisome subunits, particularly the sliding clamp, the effective processivity and replication speed increase to > 50 kb and 1,000 nt/s, respectively (9). The term &ldquoeffective processivity&rdquo is used because there is data indicating the polymerase subunit can exchange in the replisome, but the replisome maintains fast, processive DNA replication (10).

To take advantage of processivity in PCR, researchers have fused a DNA binding domain to an archaeal polymerase (11). This chimeric enzyme has several improved properties, but notably it is able to amplify DNA with shorter extension times and produce longer DNA products more efficiently, thus shortening overall thermocycling times. This fusion idea is the basis of Q5 High-Fidelity DNA Polymerase and Phusion ® High-Fidelity DNA Polymerase, two polymerases available from NEB (Figure 4).

Figure 4: Phire Hot Start DNA Polymerase is constructed by fusing a DNA polymerase (orange) and a small dsDNA-binding protein (yellow). This technology increases the processivity of the polymerase and improves its overall performance.


Routine PCR

40-60% using standard conditions and reagents. Routine PCR is a major component of many molecular biology workflows and is generally fast, robust and reproducible.

Routine PCR is typically performed with Taq or a Taq-based blend of polymerases. that provides enhanced performance. These blends often include a proofreading polymerase that provides enhanced performance and moderately higher fidelity (e.g., 2X) than Taq alone. In cases where true high-fidelity is required, a robust proofreading polymerase (i.e., Q5 ® ) should be used instead of a Taq blend.

For best PCR results, please follow the recommendations that come with each enzyme and calculate an annealing temperature for each primer set using the NEB Tm calculator. For most Routine PCR experiments, limited optimizations are required to achieve reproducible and robust results.

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Feature Articles

Read about the relationship between Polymerase structure and function when copying DNA.


RESULTS

We examined the ability of the DNA polymerases from the thermophilic eubacterium Thermus aquaticus (Taq) and from the thermophilic archaea Pyrococcus furiosus (Pfu) and Pyrococcus woesei (Pwo) to extend a primer across a template containing a single deoxyuridine. The eubacterial Taq DNA polymerase was able to extend the primer to the full length defined by the template. In contrast, the major primer-extension product generated by the archaeal DNA polymerases was significantly shorter (Fig. ​ (Fig.1 1 a). With the equivalent template in which the deoxyuridine was replaced by thymidine, all the DNA polymerases produced full-length products with no premature termination. To determine the position at which primer-extension by the archaeal DNA polymerases had halted on the deoxyuridine template, the products of the reactions were analyzed by gel electrophoresis, alongside sequencing reactions of the same template. The major products generated by the archaeal DNA polymerases corresponded to termination of primer extension around 4𠄶 nucleotides upstream of the position of the uracil in the template strand (Fig. ​ (Fig.1 1 b).

Long-range primer extension reactions on normal and single-uracil templates. (a) Denaturing polyacrylamide gel showing products of reactions in which a 31-mer primer was extended against a 119-nucleotide template (see Methods) containing either a deoxyuridine (U) or a deoxythymidine (T) 23 nucleotides from the 5′ end. Reactions were performed with DNA polymerases from Pyrococcus furiosus (Pfu), Pyrococcus woesei (Pwo), and Thermus aquaticus (Taq). All three polymerases produce full-length products on T templates, but the archaeal enzymes produce smaller major products on U templates. (b) Polyacrylamide gel showing the position of the major premature termination product from the primer extension reaction against the U template with Pyrococcus furiosus DNA polymerase (Pfu exo + ), relative to a sequencing ladder of the segment of pUC19 used in the long-range primer extension reaction (see Methods). The sequence for the template strand is given, reading 3′𡤥′ from the bottom (shorter primer extension products) to the top (longer primer extension products). The position of the deoxyuridine (U) is indicated. The major Pfu products correspond to termination of the polymerase reaction 4𠄶 bases upstream of the template deoxyuridine. The position of the full-length product obtained from primer-extension against the T template (not shown) was consistent with termination at the end of the 119-base template.

The spacing of 4𠄶 nucleotides between the last nucleotide incorporated into the primer and the position of the uracil in the template is suggestive of a uracil-sensor “leading” the polymerase activity in the archaeal enzymes. To further analyze the significance of this spacing, we constructed a series of primer-template duplexes in which a deoxyuridine was placed in the template at between ʱ and ʷ bases downstream of the duplex junction, and we measured the ability of Vent, Pfu, Pwo, Taq, and T4 DNA polymerases to initiate primer extension reactions on these substrates. With deoxyuridine present in the template in the first four positions downstream of the primer-template duplex, minimal primer extension reaction was observed with the archaeal DNA polymerases. At spacings of ʵ, ʶ, and ʷ between the duplex junction and the deoxyuridine, some primer extension products were observed with the archaeal enzymes, corresponding to addition of 1, 2, and 3 nucleotides respectively, with virtually no full-length product produced (Fig. ​ (Fig.2). 2 ). In contrast, Taq and T4 produced a full-length major product with all of these templates. Again these results are consistent with archaeal DNA polymerases stalling DNA synthesis 4𠄶 nucleotides upstream of a template strand deoxyuridine. Polymerase arrest was very persistent incubation of a template with a deoxyuridine in the ⬐ position with Pfu polymerase for up to 1 hr did not result in any significant readthrough (Fig. ​ (Fig.3 3 a). The lack of termination seen with Taq suggests this property is not universally present in thermophilic polymerases. Similarly, the negative results with T4 confirm that deoxyuridine-directed stalling is not a general property of the polymerase α family [the archaeal polymerases and T4 belong to the polymerase α group, whereas Taq is a member of the polymerase I family (14, 15)]. A similar pattern of primer extension was observed with the exo + or exo − versions of the Vent and Pfu polymerases, which contain or lack the 3′-5′ proofreading exonuclease activity. However, products shorter than the original primer were observed with the exo + enzymes and also with Pwo polymerase, for which only the exo + form was available. These observations suggest that, unlike the 5′-3′ polymerase activity, the 3′-5′ exonuclease “proofreading” activity of the archaeal enzymes is insensitive to the presence of deoxyuridine and is not involved in detection and stalling at template deoxyuridine.

Short-range primer extensions. Denaturing polyacrylamide gels showing short-range primer extension products on otherwise identical templates containing either no uracil (no dU) or a single uracil positioned 1𠄷 bases (dU+n) from the end of the primer-template duplex region. The first lane (primer) contains just the primer strand as a marker. Gels are for the following: Vent exo + and exo − (a), Pfu exo + and exo − (b), Pwo exo + (c), Taq (d), and T4 (e). In all cases the “no dU” lane shows the maximum-length 44-mer product. All the archaeal enzymes fail to produce full-length product on templates containing uracil, but they are able to extend the primer slightly when the uracil is 5 or more bases beyond the end of the duplex region. Products shorter than the original primer are present in reactions involving enzymes with functional 3′𡤥′ exonuclease activity (exo + ).

The premature halt generated by the presence of a template-strand uracil was not 100%, and a small proportion of full-length product was sometimes observed with the archaeal enzymes. Scaling-up the reactions produced enough of this full-length product to perform Maxam–Gilbert sequencing, which confirmed the incorporation of adenine opposite the template uracil, as expected from Watson𠄼rick base pairing. Furthermore, analysis of the template strand, after such a treatment, revealed it to be unchanged. Thus, in response to a template deoxyuridine the archaeal polymerases do not nick the template strand, hydrolyze the deoxyuridine glycosidic bond to give an abasic site, or replace the deoxyuridine with another base (data not shown).

Bulky lesions such as pyrimidine photodimers (16) and aminofluorene adducts (17), or abasic sites (18), will stall or at least pause DNA synthesis by a wide variety of polymerases. However, these cause arrest at the site of the lesion, presumably because of poor base pairing interactions available for an incoming nucleoside triphosphate. We observe a similar effect with Pfu, which stalls and misincorporates nucleotides on a template containing a stable abasic site (1′,2′-dideoxyribose) (Fig. ​ (Fig.3 3 b) 10 nucleotide downstream of the primer-template duplex. In marked contrast, the equivalent template with a deoxyuridine at that position results in a shorter product (Fig. ​ (Fig.3 3 b) consistent with arrest 4𠄶 nucleotides upstream of the template strand uracil, and in agreement with the data presented in Figs. ​ Figs.1 1 and ​ and2. 2 . Finally, Pfu polymerase binds tightly and specifically to uracil-DNA, only in a single-stranded context, and not to abasic sites (Fig. ​ (Fig.4). 4 ). Thus the specific arrest of archaeal DNA synthesis elicited by template-strand uracil, characterized by stalling 4𠄶 bases upstream of the uracil and tight binding of single-stranded uracil, is qualitatively different from the nonspecific arrest of DNA polymerases in general at abasic sites. To determine whether upstream stalling was a general response to deaminated bases, a template containing inosine (the deamination product of guanine) in place of uracil was used in similar primer extension reactions it elicited no stalling and produced only full-length product (data not shown).

DNA-binding specificity of Pfu DNA polymerase. (a) Surface plasmon resonance measurements of Pfu DNA polymerase interactions with DNA. The signal in response units (RU) is proportional to the mass of enzyme bound to the oligonucleotides immobilized on the surface of the Biacore chip. The beginning and end of injection of Pfu DNA polymerase over the chip surface are indicated by the arrows. Interactions were measured with single-stranded 35-mer oligonucleotides containing either a single uracil (ssU), a single abasic site (ssab), or no modification (ssC), and with double-stranded 35-mers containing a single U⋅G mispair (U:G) or a guanine opposite and abasic site (ab:G). Pfu polymerase interacts strongly with ssU but no other oligonucleotides. (b) Dose–response curve for binding of Pfu polymerase to ssU. Experimental data (○) were fit to the equation R = RmaxC/(Kd + C), where C is the protein concentration and Rmax is the maximal response, giving an estimated Kd = 0.54 μM.


INTRODUCTION TO BACTERIAL ABC PROTEINS

I. BARRY HOLLAND , in ABC Proteins , 2003

STRUCTURE AND FUNCTION OF THE ABC TRANSPORTERS

Notably, some of the most advanced structural studies of ABC transporters have come from bacterial import and, more recently, bacterial export systems. Thus, we now have high-resolution structures for ABC importers, HisP ( Hung et al., 1998) , a MalK from Thermococcus litoralis ( Diederichs et al., 2000) , one ABC in the family of branched-chain amino acid transporters and one of unknown function ( Karpowich et al., 2001 Yuan et al., 2001) . In this laboratory, we have recently obtained the high-resolution structure of the ABC domain of HlyB (Schmitt et al., in preparation), a member of the large DPL family, which includes the mammalian TAP and Pgp (Mdr1) proteins. The implications of all these structural advances will be considered in other chapters. As discussed in Chapter 7 , a very major and exciting advance in the field was made by the presentation of the first structural data at 4.5Å for the intact bacterial exporter MsbA from E. coli ( Chang and Roth, 2001 ). This provides the first sign of the nature of the membrane domain, and, in particular, that of the membrane-spanning domains. These are finally shown to be helices, settling some previous controversies. Most crucially, of course, this overall structure of MsbA has profound implications for at least a global understanding of how the action of the membrane and ABC domains may be coordinated. Chang and Roth (see also Higgins and Linton, 2001 ) on the basis of this structure have already proposed an exciting solution to a long-standing puzzle – how close are the ABC domains in the transporter? – that most likely they are interfaced at some point in the catalytic cycle (see also Chapter 6 ), but under the influence of the membrane domains they are well separated in the absence of any transport substrate. Unfortunately, mechanistic studies of the nature of the catalytic cycle of ABC proteins in bacteria, and its relationship to the transport function, have lagged relatively far behind those for some of the mammalian proteins. However, recent advances in purifying and reconstituting proteins of the maltose and histidine uptake systems (see Chapter 9 ), combined with the power of microbial genetics, promise much for the future.

Excitingly, as this volume goes to press the high-resolution structure of the bacterial ABC import system for vitamin B12, BtuCD, is reported (Locher et al., Science 296, 1091–1098), providing many new insights into the mechanism of ABC-dependent transport.


Taq and Pfu polymerase give different amplification - (May/04/2006 )

I try to perform get a 500bp-PCR-Product using Pfu-Polymerase. When I do the PCR with Taq-Polymerase everything works out fine. However when I use Pfu under the same condition, no product shows up. I haven't tried out any other polymerase yet.

1. Has anybody had similar problems? Is there an explanation for this?

2. Can you recommend another polymerase with proofreading-ability?

3. Since the fragment is only 500 bp long I thought about gambling a bit: Just do several batches, use Taq and hope that one resulting fragment is a clean match. What do you think?

4. Why is there only one female in smurf village?

1. I've used severall different polymerases and they all have different optimal conditions dependant on your template and primers. The reason is that they are derived from different bacteria/archaea etc. which have different cytoplasmic concentrations of ions etc, so that's why PCR-buffers for different polymerases as well as optimal cycling conditions will be different.

2. Don't give up yet, you have the Pfu now, try playing around with annealing temp, Mg-concentration etc. Polymerases are too expensive to just throw away after one failure. If it keeps occuring: stratagene has PfuTurbo, PfuUltra and PfuUltraII (optimised versions of Pfu), invitrogen has platinum Pfx, accuprime Pfx and Pfx50, Roche has Pwo and Tgo, Finnzyme has Phusion, NEB has Vent and Deep Vent, there's probably countless others and then you still have all the "high fidelity enzyme mixes" (taq + a proofreading enzyme).

3. That might be a good idea. But, this means you would have to clone them and sequence a lot of colonies which will also be expensie (on the other hand: even if you use a proofreading enzyme: there's no guarantee that no error is incorporated, just the chances of errors are lower, but you would still need to do the cloning and sequencing, but most likely you will get a clean match after a smaller number of attempts). If you would just sequence your PCR product right away: you will very likely see a nice match, because for instance taq incorporates ONE mistake in the first cycle of PCR on just ONE of your template (at a specific position) and you started with for instance 10 template molecules, you will only see this mutation in 10% of your PCR products (assuming that this mistake will not be made a again. ), so on the electropherogram you will most likely not notice this one.

4. Smurf's are a bunch of sexists and only need women for procreation? just my 5 cents.

You can use a polymerase cocktail. I have done 10:1 Taq:Pfu. That way, you get the best of both worlds - good amplification from Taq, proof-reading from Pfu.

also, PFU has exonuclease activity. Are you adding the PFU last, on ice and perhaps doing a hostart. This will minimse degradation of your template.

acutally, origninally there were no girls in the smurf village. Gargamel created the smurfette to infiltrate the village, but she felt bad and switched sides to join the smurfs. Does anyone else think that vanity smuf may have been gay?

1. yes. Pfu is pickey. I overcame it by addng more Magnesium, and dmso. Now, with that, i can pfu to amplify things taq won't touch.

2. There are a few out there. The one's i've tried usually flop. Triplemaster seems to work ok.

3. The error rate for Taq is not that high, especially when you consider that you're product isn't that big. You could use Taw, but remember to sequence a number of colonies, just incase.

4. There are 3 female smurfs:
The first was Smurfette, who was created by a magical potion. Her list of ingredients include: "Sugar and spice but nothing nice. A dram of crocodile tears. A peck of bird brain. The tip of an adder's tongue. Half a pack of lies, white, of course. The slyness of a cat. The vanity of a peacock. The chatter of a magpie. The guile of a vixen and the disposition of a shrew. And of course the hardest stone for her heart. "
The second was Sassette. Sassette actually has the same origins as Smurfette (with a minor difference, she wasn't made by Gargamel). The smurflings (Slouchy, Nat, and Snappy) were pitying Smurfette who felt lonely, being the only girl in the village, so they decided to help her. They learned from Papa Smurf that Smurfette was indeed created magically by Gargamel out of clay. They then went to steal Gargamel's formula and magical recipes for creating Smurfette. They successfully created Sassette, who this time didn't need plastic surgery (though her rude behavior and attitude needed a bit of chemical tweaking). As they introduced Sassette to Smurfette, Smurfette was overjoyed at having a female friend, albeit a bit of a tomboy.
The last was Nanny, an old flame of Grandpa Smurf's, Nanny enters the cartoon series after being trapped inside a cursed castle for centuries. Grandpa and the others rescue her, and she makes her home with them in the village.

Wow, you can see where my interests really are.

Ps. vanity gay? never. he was just sensitive, and very handsome.

Check out your extension time and annealing temperature. Pfu polymerase activity is very slow as compared to Taq polymerase and since Pfu prefers sulphate over chloride, you would end up using probably ammonium sulphate in the buffer so you may have to reduce annealing temperature.

That female smurf history is nothing short of amazing. you should publish a review!

Cheers guys.
I never heard about differences in temperature-specifity but I rechecked annealing-temperature and extension time with a gradient-pcr and it worked.