Which enzyme of the following is required for a PCR reaction?

Taq DNA Polymerase

Taq DNA polymerase is an 832-amino acid protein with an inferred molecular weight of 93,920 and a specific activity of 292,000 units/ mg; optimal polymerization activity is achieved at 75–80 ° C, with half-maximal activity at 60–70 ° C (Lawyer et al., 1993; see also Table 1). It's thermostability as measured as a half-life of activity is between 45 and 96 min at 95 ° C and 9 min at 97.5 ° C (Lawyer et al., 1993; Kong et al., 1993). Maximal enzymatic activity is achieved in a N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (Taps)-KOH (25 mM), pH 9.4 buffer containing 10–55 mM KCl and 2–3 mM MgCl2 (Lawyer et al., 1993). Under conditions of enzyme excess, the polymerase extends a primer at a maximum rate of 75 nucleotides per second at 70 ° C (Innis et al., 1988; Abramson et al., 1990). The enzyme is moderately processive, extending a primer an average of 50–60 nucleotides before it dissociates (King et al., 1993; Abramson et al., 1990). The fidelity of polymerase base substitution has been estimated to range between 3 × 10–4 and 3 × 10–6 errors per nucleotide polymerized, depending on reaction conditions (Eckert and Kunkel, 1992). In a standard PCR, amplification with Taq DNA polymerase is optimal in a Tris–HCl (10 mM), pH 8.3 buffer containing 50 mM KCl and 1.5 mM MgCl2, although MgCl2 concentration may need to be adjusted for any specific primer pair-template combination (Innis and Gelfand, 1990). Based on sequence homology to Escherichia coli DNA polymerase I, Taq DNA polymerase has been included in a family of DNA polymerases known as Family A (Braithwaite and Ito, 1993). Like E. coli DNA polymerase I, Taq DNA polymerase possesses an 5′ to 3′ exonuclease or "nicktranslation" activity (Longley et al., 1990; Holland et al., 1991; Lawyer et al., 1993). It does not, however, possess a 3′ to 5′ exonuclease or "proofreading" activity (Tindall and Kunkel, 1988; Lawyer et al., 1989).

Similar to the Klenow or large fragment of E. coli DNA polymerase I (Brutlag et al., 1969; Klenow and Henningsen, 1970), amino-terminal truncated forms of the enzyme lacking 5′ to 3′ exonuclease activity have also been described (Lawyer et al., 1989, 1993; Barnes, 1992). One such form, the Stoffel fragment, was constructed by deleting the first 867 bp of the Taq DNA polymerase gene, to yield a 544– amino acid protein with an inferred molecular weight of 61,300 and a specific activity of 369,000 U/mg (Lawyer et al., 1993; see also Table 1). The enzyme has a temperature optimum for activity of 75–80 ° C, with half-maximal activity at 55–65 ° C, and a thermostability half-life of 80 min at 95 ° C and 21 min at 97.5 ° C (Lawyer et al., 1993). Maximal enzymatic activity is achieved in a Tris–HCl (25 mM), pH 8.3 buffer containing 0–10 mM KCl and 3.5–4 mM MgCl2 (Lawyer et al., 1993). Under conditions of moderate enzyme excess, the polymerase extends a primer at a rate of approximately 50 nucleotides per second at 70 ° C; however, rates as high as 180 nucleotides per second are possible with a very large excess of enzyme compared with template molecules (R. D. Abramson, unpublished data). The enzyme possesses low processivity, extending a primer an average of only 5–10 nucleotides before it dissociates (R. D. Abramson, unpublished data). In a standard PCR, amplification with the Stoffel fragment is optimal in a Tris–HCl (10 mM), pH 8.3 buffer containing 10 mM KCl and 3.0 mM MgCl2 (D. H. Gelfand, personal communication). A similarly truncated 67-kDa enzyme has been shown to have a two-fold greater fidelity than the full-length protein (Barnes, 1992), suggesting that the Stoffel fragment may also have improved fidelity, possibly as a result of its lower processivity (see fidelity discussion).

Subtle differences exist between the polymerase activity of the Stoffel fragment and full-length Taq DNA polymerase (Lawyer et al., 1993). Whereas the Stoffel fragment is more thermostable than Taq DNA polymerase, it is not as active at temperatures greater than 80 ° C. This may be due to a difference in the ability of the enzyme to bind to DNA at high temperatures. That a difference in DNA binding exists between the two enzymes is reflected in the lower processivity of the Stoffel fragment. This difference in processivity may also explain the observed difference in sensitivity to ionic strength between the enzymes, as well as possible differences in polymerase fidelity. Differences are also apparent in the response of the two enzymes to divalent cations. The Stoffel fragment is optimally active over a broader range of Mg2 + concentrations, but is less active when either Mn2 + or Co2 + is substituted for Mg2 + .

Reagents

Taq polymerase and oligonucleotide primers. These can be purchased from a variety of different companies, such as Applied Biosystems (Warrington), Thermo-Fisher (Runcorn) and Sigma-Genosys (Poole). The oligos are usually 17–22 bases in length.

PCR buffers. These are usually supplied with the Taq polymerase. Three different buffers can be prepared as follows:

×10 PCR buffer I: 100 mmol/l Tris-HCl, pH 8.3, 500 mmol/l KCl, 15 mmol/l MgCl2, 0.1% (w/v) gelatin, 0.5% (v/v) NP40 and 0.5% (v/v) Tween 20

×10 PCR buffer II: 670 mmol/l Tris, pH 8.8, 166 mmol/l (NH4)2SO4, 25 mmol/l MgCl2, 670 μmol/l Na2EDTA, 1.6 mg/ml bovine serum albumin (BSA) and 100 mmol/l β-mercaptoethanol. This buffer is used in conjunction with 10% dimethyl sulphoxide (DMSO) in the final reaction mixture

×10 PCR buffer III: 750 mmol/l Tris, pH 8.8, 200 mmol/l (NH4)2SO4, 0.1% (v/v) Tween 20. A solution of 25 mmol/l MgCl2 is also prepared and added separately to the PCR reaction.

dNTP, 10 mmol/l. Take 10 μl of 100 mmol/l dATP, 10 μl of 100 mmol/l dTTP, 10 μl of 100 mmol/l dCTP, 10 μl of 100 mmol/l dGTP and 60 μl of water to make a 100 μl of 10 mmol/l dNTP.

DMSO

Agarose, Type II medium electroendosmosis. ×10 Tris-borate-EDTA (TBE) buffer. Add 216 g of Trizma base, 18.6 g of EDTA and 110 g of orthoboric acid to 1600 ml water. Dissolve and top up to 21; dilute 1 in 20 for use as ×0.5 TBE buffer.

SYBR Safe DNA Stain, Invitrogen, Cat no S33102 (×10 000 concentrated). Add 5 μl every 50 ml of agarose gel preparation.

Tracking dye. Weigh 15 g of Ficoll (type 400), 0.25 g of bromophenol blue and 0.25 g of xylene cyanol. Make up to 100 ml with water, cover and mix by inversion; it will take a considerable amount of mixing to get the solution homogeneous. Dispense into aliquots.

9.2 TaqMan probes

Taq polymerase, frequently used for standard PCR applications, exhibits 5′-exonuclease activity: if the enzyme encounters a double-stranded region during extension, the polymerase will nick the nontemplate strand, freeing its constituent nucleotides. This property is exploited by the TaqMan probe (Lee et al., 1993; Fig. 23.3(b)). A pair of PCR primers is designed, and an additional primer is designed within the amplicon. This internal primer is the foundation of the TaqMan probe and is labeled at the 5′ end with one fluorescent reporter dye, typically FAM, TET, VIC, or HEX, and the 3′ end is labeled with a quencher dye, often TAMRA, DABCYL, or a black hole quencher (BHQ). As this technology relies upon FRET (fluorescence resonance energy transfer) between the reporter and quencher dyes, the choice of reporter and quencher dye is not arbitrary; the two dyes must have overlapping excitation/emission spectra. When the reporter and quencher are in close proximity (labeling the intact primer), FRET occurs and there is no fluorescence observable. As Taq polymerase extends through this region and encounters the probe, the enzyme's exonuclease activity nicks the 5′ end of the probe, liberating the reporter dye from the primer anchoring the quencher. As the reporter and quencher are no longer in close proximity, FRET does not occur and fluorescence is observed. A major advantage of TaqMan probes is the added specificity conferred by the probe's hybridization between the two PCR primers. One drawback is that a separate probe must be designed for each gene of interest; however, the multiple available combinations of reporters and quenchers in concert with sequence specificity conferred by the probe itself enable multiplexing, that is, the combination of several probes in one reaction (Gibson et al., 1996).

When designing TaqMan probes, several guidelines must be kept in mind: the probe should be 15–30 nucleotides long, be ~ 30–80% GC, have a Tm 10 °C higher than the amplification primers, and be free of nucleotide runs. It is also desirable to have fewer than 2 GCs at the 3′ end to introduce a small degree of instability to prevent mispriming. Additionally, a G in the 5′ position should be avoided as it may quench fluorescence (Arya et al., 2005). If maximal specificity is desired, such as for allelic discrimination, a nonfluorescent quencher (such as a BHQ) may be coupled with a minor groove binder (MGB). MGBs increase Tm, allowing shorter probes, and show higher specificity for single base mismatches (Kutyavin et al., 2000).

i. Overall features.

Taq DNA Pol I consists of 832 amino acids (Mr 94,000) (1). The N- and C-terminal residues are predicted to be Met and Glu, respectively. The enzyme contains no Cys.

Taq DNA Pol shows ∼50% sequence homology with E. coli DNA Pol I within the C-terminal ∼400 residues that constitute the polymerase domain. Taq Pol has an independent 5 ‘-nuclease activity in the N-terminal domain (residues 1–290) but has no 3′ → 5′-exonuclease activity.

The crystal structures of Taq Pol (69) and Klentaql (75) reveal that the C-terminal polymerase domain is identical in fold to that of Klenow Pol. Regarding the 3′ → 5′-exonuclease domain (residues 324–515) of Klenow Pol, Taq Pol is markedly different by having deletions of four loops of lengths 8 to 27 residues. All four of the acidic residues (D424, D501, D355, E357) known to be essential for divalent metal binding and exonucleolytic catalysis in Klenow Pol are replaced by residues incapable of binding metal ions (L356, R405, G308, V310) in the vestigial 3′ → 5′-exonuclease domain of Taq Pol.

The N-terminal 5′-nuclease domain of Taq Pol has a deep cleft that contains at its bottom a cluster of acidic residues highly conserved in 5′-nuclease domains of the Pol I family. Seven of the carboxylate groups are presumed to form three divalent metal-binding sites (a triangle of ∼5 Å × −10 Å × ∼10 Å). Two residues (R25 and R74) are essential for 5′-nuclease activity.

Among other notable differences from Klenow Pol, Klentaql has 19 opposite-charge substitutions, suggesting a global charge redistribution which is likely to play a role in the thermostability.

6.2 Enzymes for Cloning

Taq polymerase remains to be the most ubiquitously used polymerase for PCR. This enzyme has a high rate of dNTP incorporation but does not have any proof-reading activity. This means that Taq is prone to introducing base-pair errors during PCR amplification. If the PCR product is to be used for functional protein analysis, then this can be problematic. Single base pair changes have the potential to change the amino acid sequence which could in turn impact upon the protein product and its functionality. However, Taq does have a very useful characteristic that is exploited for cloning. Namely that it incorporates a single adenosine base at either end of the PCR product. This in essence creates “overhangs” that will readily bond with thymidine nucleotides by complementary base pairing—how this can be used for cloning will be described later in this chapter.

To overcome the problem associated with Taq-introduced errors during PCR, alternative polymerase must be used. Pfu is a polymerase with 3′-5′ exonuclease activity meaning that as the PCR product is synthesized it checks which nucleotides have been incorporated and can work backwards to remove incorrect bases and replace them with the correct ones. Almost all suppliers of polymerase enzymes for PCR will produce a version of a so-called “proof-reading” enzyme, check the product descriptions before you buy. The drawbacks of using “pure” preparations of proofreading polymerase are that they do not incorporate an adenosine base at the ends of the PCR product. This can be overcome by using a Taq/Pfu mixture which many companies produce—the merits of using these enzymes mixtures will be described later on in this chapter.

So, it is highly likely that you will need to use a high-fidelity enzyme, and it does not really matter which one you choose. The method of cloning you wish to utilize will ultimately dictate this decision.

Intracellular Localization of Sep15

As noted above, Sep15 occurs as a complex with UGTR, an ER-resident protein,19 which is involved in the recognition of misfolded proteins. UGTR glycosylates misfolded proteins for proper folding20,21 or for degradation. To determine the intracellular localization of Sep15, several constructs have been made with Sep15 gene fused to the green ftorescent protein (GFP) gene. generating the fusion protein facilitates tracking the expressed protein by monitoring green fluorescence in transfected cells, using confocal microscopy. The technique described below is designed to detect the cellular location of proteins in either the ER or the golgi. However, because Sep15 is complexed with UGTR, which is known to reside in the ER, this technique can likely be used to demonstrate that Sep15 is located in the ER.

3.2 Third exponential PCR