PCR Strategies

By Michael A. Innis and David H. Gelfand

PCR Strategies expands and updates the landmark volume PCR Protocols. It is a companion laboratory manual that provides a completely new set of up-to-date strategies and protocols for getting the most from PCR.

The editors have organized the book into four sections, focusing on principles, analyses, research applications, and alternative strategies for a wide variety of basic and clinical needs. If you own PCR Protocols, you will want PCR Strategies. If you don't own PCR Protocols, you will want to buy both!

• Concepts explained
• Methods detailed
• Trouble-shooting emphasized
• Novel applications highlighted
• Key concepts for PCR
• Analysis of PCR products
• Research applications
• Alternative amplification strategies

• Language: English
• Publisher: Academic Press
• Release date: Jul 6, 1995
• ISBN: 9780080538549

Book preview

Part One

Key Concepts for PCR

1

The Use of Cosolvents to Enhance Amplification by the Polymerase Chain Reaction

P.A. Landre; D.H. Gelfand; R.M. Watson

The polymerase chain reaction (PCR) (Saiki et al., 1985) uses repeated cycles of template denaturation, primer annealing, and polymerase extension to amplify a specific sequence of DNA determined by oligonucleotide primers. The use of a thermostable DNA polymerase, such as Taq from Thermus aquaticus, permits repeated cycles of heating at high temperatures for reliable template denaturation without the addition of more enzyme at each cycle. It also allows higher temperatures to be used to denature templates that have a high G + C content or stable secondary structures. Taq DNA polymerase also has a relatively high temperature optimum for DNA synthesis (75° – 80 °C). The use of higher annealing/extension and denaturation temperatures increases specificity, yield, and the sensitivity of the PCR reaction.

While the PCR is widely used with great success, certain templates, for example, those with high G + C content or stable secondary structures, may amplify inefficiently, resulting in little or no intended product and often nonspecific products. Thus new applications of the PCR may require some optimization. The PCR parameters most effective in optimizing the reaction are the concentration of enzyme, primers, deoxynucleotide 5′-triphosphates and magnesium; primer annealing/extension temperatures and times; template denaturation temperature and time; and cycle number (Innis and Gelfand, 1990; Saiki, 1989). Use of higher annealing/extension or denaturation temperatures may improve specificity in some cases (Wu et al., 1991). Some investigators have found that incorporation of the nucleotide analog 7-deaza-2′-deoxyguanosine triphosphate (c7dGTP) in addition to deoxyguanosine triphosphate (dGTP) helped destabilize secondary structures of DNA and reduced the formation of nonspecific product (McConlogue et al., 1988).

Various additions or cosolvents have been shown to improve amplification in many applications. Dimethyl sulfoxide (DMSO) increased the amplification efficiency of human leukocyte antigen DQ alpha sequence with the Klenow fragment of Escherichia coli DNA polymerase I (Scharf et al., 1986). DMSO and glycerol were found to improve amplification of G + C rich target DNA from herpesviruses and of long products from ribosomal DNAs containing secondary structure (Smith et al., 1990). Bookstein et al. (1990) found that DMSO was necessary to successfully amplify a region of the human retinoblastoma gene. Sarkar et al. (1990) reported that formamide improved specificity and efficiency of amplification of the G + C rich human dopamine D2 receptor gene, whereas DMSO and dc7GTP did not improve specificity in this application. Hung et al. (1990) found that tetramethylammonium chloride (TMAC), a reagent that improves stringency of hybridization reactions, improved the specificity of PCR, although at concentrations far below those effecting hybridization reactions. DMSO and nonionic detergents have been reported to improve DNA sequencing, presumably by reduction of secondary structures (Winship, 1989; Bachmann et al., 1990).

It is not fully known which PCR parameters are influenced by cosolvents. Some cosolvents, such as formamide, are known to reduce the melting temperature (Tm) of DNA, thus possibly affecting the Tm of the primers and template in PCR. Also, Gelfand and White (1990) reported that agents such as DMSO and formamide inhibit Taq DNA polymerase activity in incorporation assays. In order to understand how cosolvents influence the PCR, their effects on template melting properties and on the activity and thermostability of Taq DNA polymerase were determined.

Materials and Methods

Taq DNA Polymerase Activity Assay

Taq DNA polymerase activity was assayed by determining the level of incorporation of labeled nucleotide monophosphate into an activated salmon sperm DNA template (Lawyer et al., 1989). The assay was performed for 15 min at 74 °C (the optimum temperature for Taq DNA polymerase activity) and at 60 °C (to minimize melting effects on the template). Reaction mixes (50 μl) contained 25 mM TAPS–HCI (pH 9.5), 50 mM KCI, 2 mM MgCI2, 1 mM β-mercaptoethanol, 200 μM each deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate (dTTP), and deoxyguanosine triphosphate, 100 μM α-[³²P]deoxycytidine triphosphate (dCTP) at 100 cpm/picomole; 12.5 μg activated salmon sperm DNA, and co-solvents. Reactions were stopped with 10 μl 60 mM EDTA, precipitated with trichloroacetic acid (TCA), and filtered through Whatman GF/C filters. The filters were counted in a liquid scintillation counter and the picomoles of incorporated label calculated from the CPM and specific activity. Percent control was calculated from the number of picomoles incorporated in the presence of cosolvents divided by the picomoles incorporated in the absence of cosolvents.

Thermal Inactivation Assay

The thermostability of Taq DNA polymerase was determined by steady-state thermal inactivation performed in a constant- temperature waterbath at 95° or 97.5 °C. The enzyme was prepared in a standard GeneAmp PCR (Perkin–Elmer) master mix containing: 10 pmoles each of lambda primers 1 and 2200 μM each dATP, dTTP, and dGTP, 1.25 units Taq DNA polymerase, 0.5 ng lambda DNA template, 1X PCR buffer, and cosolvents. The PCR buffer contained 10 mM Tris–HCI (pH 8.3), 50 mM KCI, and 2 mM MgCI2. Fifty microliters were dispensed to 0.5-ml tubes and overlaid with 50 μl of light mineral oil. Following incubation at high temperature, 5 μl from each tube, in duplicate, were assayed for DNA polymerase activity by measuring the incorporation of labeled nucleoside monophosphate into activated salmon sperm DNA, as described previously. The percent of activity remaining for each time point was determined as the fraction of the initial activity for each enzyme. The half-life, t1/2, was the time at which there was 50% remaining activity.

Determination of DNA Temperature Melting Profiles

DNAs with different G + C contents were denatured in PCR buffer [10 mM Tris–HCI (pH 8.3), 50 mM KCI, and 3 mM MgCI2], and in PCR buffer containing cosolvents to measure their respective melting temperatures (Tm) and strand separation temperatures (Tss). Samples overlaid with mineral oil were heated in a spectrophotometer in stoppered and thermojacketed cells. All samples were heated at an initial concentration of about 1 OD²⁶⁰/ml and the OD²⁶⁰ and temperature were recorded as the temperature was increased. Optical density was plotted as the fractional change in OD²⁶⁰ with the Tm determined as the temperature at the midpoint of the hyperchromic transition, and the Tss as the temperature at the end of the transition.

PCR Reactions

The effects of the cosolvents glycerol and formamide and the denaturation temperature on PCRs were measured in standard 50-μl GeneAmp PCR (Perkin–Elmer) reactions containing 1.25 units Taq DNA polymerase, 0.5 ng DNA, 10 pmole each lambda primers (500 bp product), 200 μM each dNTP, cosolvents, and PCR buffer. PCR buffer contained 10 mM Tris–HCI (pH 8.3), 50 mM KCI, and 2 mM MgCI2. The reactions were thermocycled in a Perkin–Elmer 480 thermocycler programmed for 25 cycles and two-temperature PCR: denaturation temperatures of 90°, 95°, or 98 °C for 1 min and an anneal/ extend temperature of 60 °C for 1 min were used. A 5 min extension at 75 °C was done after thermocycling. The samples were analyzed on 5% polyacrylamide gels by ethidium bromide staining.

For the effect of glycerol and formamide on amplification from a high G + C template, the 50-/μl PCRs contained the following: 1.25 units Taq DNA polymerase (Perkin–Elmer), 0.01 μg plasmid DNA containing the Thermus thermophilus DNA polymerase gene, 50 pmoles each primer (amplified sequence: 2600 bp segment of the cloned gene with 67% G + C), 200 μm each dNTP, cosolvents, and PCR buffer. The thermocycler was programmed for two-temperature PCR: a denaturation temperature of 96 °C for 1 min and an anneal/ extend temperature of 60 °C for 1 min. A 5 min extension of 75 °C was done after thermocycling. Samples were analyzed on a 0.7% agarose gel by ethidium bromide staining.

Results

Effect of Cosolvents on Taq DNA Polymerase Activity

There were two types of effects seen on Taq DNA polymerase activity. A decrease in polymerase activity at both 74° and 60 °C was exhibited in the presence of DMSO and glycerol, which is characteristic of enzyme inhibition (Fig. 1A). In contrast, 1-methyl-2-pyrolidone (NMP) and formamide were inhibitory at 74 °C but stimulatory at 60 °C, presumably as a result of increased template denaturation. Taq DNA polymerase has optimal activity at 74 °C on activated salmon sperm DNA. Cosolvents inhibited activity with this template at this temperature. With 10% glycerol, 79% of the activity remained and with 10% NMP about 20% remained. Ten percent formamide or DMSO produced about 50% of the control activity.

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Figure 1 Effect of cosolvents on taq DNA polymerase activity. Taq DNA polymerase activity was assayed by determining the level of incorporation of labeled nucleotide monophosphate into activated salmon sperm DNA template. The assay was performed for 15 min at 74 °C (the temperature optimum for Taq DNA polymerase activity) and at 60 °C (to determine template melting effects). Reaction mixes (50 μl) contained: 25 mM TAPS-HCl (pH 9.5); 50 mM KCI; 2 mM MgCI2; 1 mM β-mercaptoethanol; 200 μM each dATP, dTTP, and dCTP; 100 μM α-[³²P]dCTP (100 cpm/picomole); 12.5 μg activated salmon sperm DNA and cosolvents. Reactions were stopped with 10 μl 60 mM EDTA, TCA precipitated and filtered through Whatman GF/C filters. The number of picomoles of labeled nucleotide monophosphate were determined. Percent control was calculated from the number of picomoles incorporated in the presence of cosolvents divided by the picomoles incorporated in the absence of cosolvents. Panel A shows the effects of glycerol and dimethylsulfoxide (DMSO) on Taq DNA polymerase activity. Panel B shows the effects of formamide and N-methyl-2-pyrrolidone (NMP) on Taq DNA polymerase activity.

When the activity assay was done at 60 °C, NMP and formamide increased activity compared with the control (Fig. 1B). This is presumably due to melting effects on the activated salmon sperm DNA template used. This template is composed of undefined nicks and gaps, and an increased incorporation of nucleotide monophosphate into this template may occur if pieces of DNA are melted.

The effects of combinations of glycerol and formamide or glycerol and NMP on activity were determined at 74° and 60 °C (data not shown in Fig. 1). Formamide (5 or 10%) and glycerol (10%) are somewhat more than additive in their inhibition of Taq DNA polymerase activity at 74 °C. However at 60 °C, the activity of the combinations is about the same as the activity with formamide alone. Glycerol and NMP in combination did not show an increase in activity at 60 °C, as is seen with NMP alone.

Effect of Cosolvents on the Thermostability of Taq DNA Polymerase

The thermostability of Taq DNA polymerase was determined as described earlier (Fig. 2). At 97.5 °C, Taq DNA polymerase has a half-life (t1/2) after steady-state thermal inactivation of 11 min (Fig. 2A). At 95 °C the t1/2 is 40 min (Fig. 2B). Glycerol improves the thermostability of Taq from 11 min to more than 60 min with 20% glycerol at 97.5 °C. Formamide reduces the thermostability from 40 min at 95.0 °C to 16 min with 5% formamide, and 2 min with 10% formamide. Combining glycerol and formamide improves the thermostability of Taq over formamide alone: 5% formamide and 10% glycerol had a t1/2 of 24 min compared with 16 min for 5% formamide alone.

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Figure 2 Thermostability of Taq DNA polymerase in the presence of cosolvents at 97.5° and 95 °C. The thermostability of Taq DNA polymerase was determined by steady-state thermal inactivation. Thermal inactivation was performed in a constant temperature water-bath at 95° or 97.5 °C. The temperature was monitored using a calibrated thermometer and fluctuated no more than 0.5 °C during the experiment. Enzyme was prepared in a standard GeneAmp PCR master mix containing the following: 10 pmol lambda primers 1 and 2; 200 μM each dATP, dTTP, and dGTP; 1.25 U Taq DNA polymerase; 0.5 ng lambda template; 1 × PCR buffer; and cosolvents. PCR buffer contained: 10 mM Tris–HCI, pH 8.3; 50 mM KCI; and 2 mM MgCI2. Fifty μl were dispensed to 0.5 ml tubes and overlaid with 50 μl of light mineral oil. Five μl from each tube, in duplicate, were assayed for DNA polymerase activity by the incorporation of labeled nucleotide monophosphate into activated salmon sperm DNA as described previously. The percent remaining activity for each timepoint was determined as a fraction of the initial activity for each enzyme. The half-life, t1/2, was the time at which there was 50% remaining activity. Panel A shows the thermal inactivation of Taq DNA polymerase at 97.5 °C in the presence of glycerol. Panel B shows thermal inactivation of Taq at 95 °C with the combination of glycerol and formamide.

Table 1A summarizes the effects of single cosolvents on thermostability. Of the cosolvents tried, only glycerol improved the thermostability of Taq. The other cosolvents reduced thermostability, NMP most dramatically; at 5% NMP the enzyme was immediately inactivated at 97.5 °C. Table 1B shows a summary of combinations of glycerol and NMP or formamide. In both cases, glycerol ameliorated the thermal inactivation of NMP or formamide.

Table 1A

Effects of Single Cosolvents on Thermostability

Table 1B

Combinations of Cosolvents and Thermostability

Effect of Cosolvents on Melting Temperature of DNA

The effect of cosolvents on the melting temperature of DNA depended on template composition. Figure 2A shows that the Tms of various DNAs were lowered using 5% formamide with 10% glycerol, but to different degrees on each template. The templates had different G + C contents and the measured Tms were proportional to the G + C content, as expected. The change in Tm using cosolvents, or delta Tm, was also somewhat dependent on the G + C content of the template and ranged from –6.5° to –4.5 °C for the various templates. Also shown in Table 2A is the effect of this cosolvent on the strand dissociation temperature, or Tss. The Tss is significantly lowered for all the DNAs examined, although the delta Tss shows less-dependence on the G + C content of the template than does the delta Tm.

Table 2A

DNA Melting and Strand Dissociation in Cosolvents

* Estimated.

The effect of different cosolvents on λ DNA was determined and is shown in Table 2B. All the cosolvents studied lowered the Tm, ranging from a reduction of about 1.1° per percent for NMP to 0.3° per percent for glycerol. The effect of formamide on delta Tm was close to reported values of 0.7 °C per percent. The effect of the combination of 5% formamide and 10% glycerol on Tm was additive.

Table 2B

Effects of Cosolvents on λ DNA Denaturation

Cosolvents and PCR

After determining the effects of cosolvents on activity, thermostability, and template melting, a PCR was designed to look at effects of cosolvents on denaturation temperature in a PCR. A standard lambda system (as described earlier) was used with denaturation temperatures (Tden) set at 90°, 95°, and 98 °C (Fig. 3). The reactions were expected not to work with a Tden of 90 °C. At 95 °C the reactions were expected to work and at 98 °C were expected to work but not optimally because of enzyme inactivation. As expected, a Tden of 90 °C did not allow amplification. The addition of 5% and 10% glycerol improved amplification. Glycerol (20%) has slightly less product, probably owing to inhibition of Taq activity. The addition of 5% formamide or 5% formamide plus 10% glycerol also improved amplification at a Tden of 90 °C (Fig. 3A). The effects of cosolvents here are presumably due to a lowering of the Tss of the template and more reliable denaturation of the template. At a higher Tden of 98 °C, the samples without cosolvents amplified well; however, reactions containing 1% or 5% glycerol looked somewhat better owing to their stabilizing effects on the enzyme (Fig. 3C). With glycerol added at 10% or 20%, there was less product because of its inhibition of enzyme activity. Reactions with 5% formamide and 5% formamide plus 10% glycerol had no product, presumably owing to the effect formamide has on thermostability and activity of Taq DNA polymerase.

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Figure 3 Effect of glycerol and formamide and denaturation temperature on PCR. PCR reactions (50  μ l) contained the following: 1.25 U Taq DNA polymerase; 0.5 ng lambda DNA; 10 pmol each lambda primers (lambda 500 bp product); 200  μM each dNTP; cosolvents; and PCR buffer. PCR buffer contained: 2 m M MgCI 2 , 50 m M KCI, and 10 m M Tris–HCI, pH 8.3. The reactions were thermocycled for 25 cycles in a Perkin–Elmer Cetus thermocycler programmed for two temperature PCR: denaturation temperatures of 90°, 95°, or 98 °C for one min and an anneal/extend temperature of 60 °C for one min. A five min extension at 75 °C was done after thermocycling. The samples were analyzed on 5% polyacrylamide gels by ethidium bromide staining. PCR was performed in the absence of cosolvent (lane 1); 1, 5, 10, and 20% glycerol (lanes 2 through 5); 5% formamide (lane 6); or 5% formamide and 10% glycerol (lane 7). Reactions were done in duplicate. Panels A, B, and C show PCR products from reactions performed at denaturation temperatures of 90°, 95°, and 98 °C, respectively.

The effect of cosolvents on amplification of a template with a high G + C content was explored. We attempted to amplify a region of Thermus thermophilus DNA contained on a plasmid (67% G + C in the amplified region) but were not successful (Fig. 4). The addition of small amounts of formamide improved amplification, as did the combinations with 1% glycerol. The absence of product in the 30-/ cycle lane of 1 % glycerol and 1 % formamide was probably a dropout or otherwise unexplained failure to amplify.

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Figure 4 Use of glycerol and formamide to improve PCR amplification from a high G/C content template. PCR reactions (50  μ l) contained the following: 1.25 U Taq DNA polymerase; 0.01  μ g target DNA (plasmid DNA containing Thermus thermophilus DNA polymerase gene, 55% G/C); 50 pmol each primer (amplified sequence: 2600 bp, 67% G/C); 200  μM each dNTP; cosolvents; and PCR buffer. A Perkin–Elmer Cetus thermocycler was programmed for two temperature PCR: a denaturation temperature of 96 °C for one min and an anneal/extend temperature of 60 °C for two min. A five min extension at 75 °C was done after thermocycling. Samples were analyzed on 0.7% agarose gels by ethidium bromide staining. The gel shows PCR products from 20, 25, and 30 cycles performed with: no cosolvent added (set 1); 1% glycerol (set 2); 1% formamide (set 3); 2.5% formamide (set 4); 1% glycerol and 1% formamide (set 5); 1% glycerol and 2.5% formamide (set