PCR Strategies

Michael A. Innis, David H. Gelfand, John J. Sninsky

• Publisher: Academic Press
• Year: 1995
• Print ISBN: 9780123721822
• eBook ISBN: 9780080538549
• Topic: Biological Sciences
• Subtopic: Biotechnology
• Index: Biological Sciences

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

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 (T_m) of DNA, thus possibly affecting the T_m 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 thermostabili...

Table of contents

• Cover image
• Title page
• Table of Contents
• Copyright page
• Contributors
• Foreword
• Preface
• Part One: Key Concepts for PCR
• Part Two: Analysis of PCR Products
• Part Three: Research Applications
• Part Four: Alternative Amplification Strategies
• Index