Basic Science Methods for Clinical Researchers

Basic Science Methods for Clinical Researchers addresses the specific challenges faced by clinicians without a conventional science background. The aim of the book is to introduce the reader to core experimental methods commonly used to answer questions in basic science research and to outline their relative strengths and limitations in generating conclusive data.

This book will be a vital companion for clinicians undertaking laboratory-based science. It will support clinicians in the pursuit of their academic interests and in making an original contribution to their chosen field. In doing so, it will facilitate the development of tomorrow’s clinician scientists and future leaders in discovery science.

• Serves as a helpful guide for clinical researchers who lack a conventional science background
• Organized around research themes pertaining to key biological molecules, from genes, to proteins, cells, and model organisms
• Features protocols, techniques for troubleshooting common problems, and an explanation of the advantages and limitations of a technique in generating conclusive data
• Appendices provide resources for practical research methodology, including legal frameworks for using stem cells and animals in the laboratory, ethical considerations, and good laboratory practice (GLP)

• Language: English
• Publisher: Academic Press
• Release date: Mar 31, 2017
• ISBN: 9780128030783

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science.

Chapter 1

The Polymerase Chain Reaction

PCR, qPCR, and RT-PCR

Mehdi Jalali¹, Justyna Zaborowska² and Morteza Jalali²,    ¹University of Liverpool, Liverpool, United Kingdom,    ²University of Oxford, Oxford, United Kingdom

Abstract

The polymerase chain reaction (PCR) is a laboratory technique used for the amplification of a specific DNA fragment in a simple enzyme reaction. The basic PCR method has been modified to expand its application. Development of quantitative PCR (qPCR) has enabled detection and quantification of the target sequence in real time, while it is being synthesized. Another popular variation is reverse transcription polymerase chain reaction (RT-PCR), a technique used to detect and measure RNA. PCR technology has revolutionized the field of molecular biology and medical research. Because of its widespread use, it is important to understand the scientific principles of PCR. The aim of this chapter is to explain the concepts underlying this method and to explore the clinical usefulness and potential of this technique. The chapter also provides detailed protocols on how to undertake PCR in the laboratory, including techniques for RNA isolation, cDNA synthesis, and data analysis. A scenario in which PCR is utilized to answer a research question is also described, as well as guidance on how to troubleshoot experimental problems.

Keywords

cDNA; primer design; PCR; qPCR; RT-PCR; Taq polymerase

Chapter Outline

Introduction 2

In Principle 2

Analysis of the PCR Product 3

Phases of PCR 5

Variations of PCR Method: Quantitative PCR 5

Variations of PCR Method: Reverse Transcription PCR 8

In Practice 9

Extraction of Total RNA 9

Synthesis of cDNA by Reverse Transcription 11

Real-Time PCR Amplification 11

Data Analysis: Generating a Standard Curve 12

Relative Quantification (Pfaffl Method) 13

Applications of PCR 14

Scenario 15

Key Limitations 15

Troubleshooting 15

Little or No PCR Product 15

Efficiency of Reaction Is Too Low 15

Efficiency of Reaction Is Too High 16

Multiple Bands On Gel, Or Multiple Peaks On Melting Curve 16

No RT Control Produces a Band 16

Negative PCR Control Produces a Band 16

Conclusion 16

References 17

Suggested Further Reading 18

Glossary 18

List of Acronyms and Abbreviations 18

Objectives

 Describe the scientific principles behind PCR

 Provide PCR laboratory protocols

 List PCR applications

 Describe a typical scenario in which qRT-PCR might be used

 Discuss key limitations and troubleshooting

Introduction

The polymerase chain reaction (PCR) was developed in the 1980s by Dr. Kary Mullis. The technique has been compared to a molecular photocopier owing to its ability to recognize a specific sequence of DNA, and rapidly and accurately synthesize a high number of copies [1]. It has revolutionized molecular biology, and in particular genetic manipulations, the diagnosis of genetic and infectious diseases, genotyping and DNA forensics. It is considered one of the greatest scientific discoveries of the 20th century. To date, a variety of spin-off techniques based on the original PCR method have been developed. Real-time PCR, also known as quantitative PCR (qPCR), combines PCR amplification and detection in a single step. Another technique known as reverse transcription polymerase chain reaction (RT-PCR) uses RNA as the nucleic acid starting template.

In Principle

PCR resembles an in vitro and elementary form of DNA replication, a physiological process used by all living cells to duplicate their genetic material prior to cell division [2]. It involves repeated cycles of heating and cooling of a reaction mixture containing DNA template, DNA polymerase, primers, and nucleotides (Table 1.1). DNA template is the DNA containing the target sequence. Primers are short chains of nucleotides which locate the specific target DNA of interest and bind to it upon cooling, through complementary base pairing. They act as a starting point for DNA polymerase to create the new complementary strand. DNA polymerase is an enzyme that synthesizes new strands of DNA complementary to the target sequence.

Table 1.1

Components of PCR and reaction guidelines for reaction optimization

Template DNA contains the DNA sequence that will be amplified by PCR. DNA polymerases copy DNA molecules during the PCR reaction. Primers are short single-stranded DNA molecules that bind by complementary base pairing to opposite DNA strands. The melting temperature (Tm) of the primer is the temperature at which one-half of the double-stranded DNA dissociates to become single stranded [3].

Each cycle of PCR consists of three steps (Fig. 1.1):

 Denaturation—reaction mixture heated to over 90°C to unwind double helix of DNA by breaking apart hydrogen bonds.

 Primer annealing—reaction mixture cooled to 45–65°C to allow for primer annealing. Forward and reverse primers hybridize through complementary base pairing to opposite strands of the DNA. They must be complementary to the 3ʹ ends of the antiparallel strand of template DNA.

 Extension—the reaction mixture is heated to 72°C toward the optimal temperature for DNA polymerase enzyme activity. Polymerase binds to the primer-template hybrid complex and then assembles a new complementary strand using the free nucleotides in the reaction mixture.

Figure 1.1 First cycle of PCR. During the denaturation step, DNA is heated to above 90°C and the two strands of the DNA target sequence separate. The temperature of the reaction is then cooled to 45–65°C and primers anneal to their complementary sequence in the template DNA. In the extension step, the reaction is heated to 72°C to allow the DNA polymerase to synthesize a new DNA strand complementary to the template strand.

Following extension, the reaction is returned to the denaturation step and PCR continues. Each cycle approximately doubles the amount of DNA, as a new strand of DNA subsequently acts as a template for replication in the following cycle. This results in an exponential increase in quantity of DNA. For example, after 6 cycles there are 2⁶ copies. A total of 25–40 PCR cycles is carried out depending on the expected yield of the PCR product [2].

Analysis of the PCR Product

The PCR product or amplicon can be visualized and analyzed with the use of agarose gel electrophoresis, which separates DNA products on the basis of size and charge (Fig. 1.2). qPCR, which will be discussed later in this chapter, does not require such postamplification analyses and instead the product is analyzed throughout the reaction in real-time. Gel electrophoresis involves the separation of charged molecules in an electrical field on an agarosegel, followed by staining with ethidium bromide. PCR by-products such as primer dimers appear as diffuse, smudgy bands near the bottom of the gel. The other way to validate a PCR reaction is to directly sequence the amplicon.

Figure 1.2 PCR experimental design. (A) The polymerase chain reaction (PCR) requires several components including DNA template, DNA polymerase, deoxynucleotide triphosphates (dNTPs) and oligonucleotide primers. (B) The prepared PCR reactions are placed in the thermal cycler. (C) The size of a DNA fragment can be estimated by gel electrophoresis. In an electric field, negatively charged DNA molecules will migrate toward the positive electrode.

Phases of PCR

The PCR reaction can be split into three phases—exponential, linear, and plateau (Fig. 1.3). During the exponential phase, reaction components are in excess and there is exact doubling of product at each cycle. During the linear stage, the reaction components start to run out and consequently the reaction slows down. In the plateau phase, the reaction stops and no more product is generated.

Figure 1.3 Phases of the PCR amplification curve. The exact doubling of product accumulates at every cycle during the exponential phase. In the linear phase the reaction is slowing as components are being consumed. In the plateau phase the reaction stops and no more products are being made.

The depletion in reagents will occur at varying rates due to the different reaction kinetics in each PCR tube. Rates of depletion will start to vary in the linear phase, and each sample will plateau at a different point. Therefore, even replicate DNA template samples can end up with different copy numbers in the plateau phase, despite starting with the same quantity [4]. The exponential phase is optimal for data analysis, as it yields high-quality quantitative data. Conventional PCR quantifies data from the linear and plateau phases. Therefore, data from conventional PCR can only be considered semi-quantitative at best, and allows detection of only a tenfold change in gene expression. The PCR variant named real-time PCR measures at the exponential phase of the PCR reaction, allowing detection of twofold change in gene expression [5].

Variations of PCR Method: Quantitative PCR

In recent years, a technological innovation of PCR, qPCR, has become increasingly important in clinical diagnostics. qPCR allows for detection and quantification of the target DNA, as the reaction progresses. A variety of fluorescent chemistries correlate PCR product concentration to fluorescence intensity [6]. The most commonly used fluorescent DNA binding dye is SYBR Green I. It emits fluorescence when bound to double-stranded DNA and the intensity of fluorescence increases proportionally to the concentration of PCR product (Fig. 1.4).

Figure 1.4 Action of SYBR Green I Dye. (A) When DNA is denatured, SYBR Green I Dye floats free and emits low fluorescence. (B) SYBR Green Dye binds to the double-stranded product and fluoresces.

SYBR Green I can bind to any double-stranded DNA, including nonspecific products such as primer dimers [7]. Therefore, it is important to carefully design primers that only bind to the selected target sequence. In addition, a melt curve is required when using dye-based methods to validate the results and ensure specific product. At the end of the PCR run, the reaction mixture is exposed to a temperature gradient from around 60°C to 95°C and fluorescence readings are continually collected. At a certain temperature, the amplified product will fully dissociate. This results in a rapid fall in fluorescence emission, as SYBR Green I dissociates.

Double-stranded DNA melts at different temperatures according to its length, GC content, and the presence of base pair mismatches and secondary structures (Fig. 1.5). Therefore, it is possible to validate how many products of amplification are present in a sample. If binding is specific, a single tight peak should appear on the graph representing the specific amplicon of interest. Primer dimers instead appear as shorter broader waves at lower temperatures [8] (Fig. 1.6).

Figure 1.5 Hairpin structure. Owing to the presence of complementary sequences within the length of single-stranded nucleic acid sequences, a secondary hairpin structure might form.

Figure 1.6 Melt curve analysis from a qPCR assay. A uniform melt curve with a single tight peak means that only the target DNA of interest has been generated. A smaller peak to the left may correspond to the dissociation curve for primer dimers.

Obtained qPCR fluorescence data can be plotted on an amplification graph with cycle number on the X-axis and fluorescence on the Y-axis (Fig. 1.7). Reactions are characterized by the PCR cycle at which amplification of a PCR product is first detected [9]. The baseline in the amplification plot is the average background. The threshold is the level of fluorescence above the baseline. It is usually calculated automatically and corresponds to the three standard deviations above the mean baseline values, within the exponential phase. The threshold cycle (CT value) is defined as the cycle in which the fluorescent signal crosses the threshold, exceeding background level. Accordingly, the greater the quantity of target DNA in the sample, the sooner the fluorescence crosses the threshold, yielding a lower CT [9].

Figure 1.7 qPCR amplification plot. Baseline-subtracted fluorescence versus number of PCR cycles. The threshold cycle (CT) is the cycle number at which the fluorescent signal of the reaction crosses the established threshold line.

There are two major methods for normalization of qPCR assays: absolute quantification and relative quantification [10]. Whether absolute or relative quantification is required depends on the experimental question that needs answering. For instance, absolute quantification is used to measure the exact number of target molecules in a sample, for example the number of viral particles in a given amount of blood. Alternatively, relative quantification might be used to compare gene expression between samples, and to calculate the fold difference in expression.

Variations of PCR Method: Reverse Transcription PCR

Reverse transcription (RT)-PCR is used to amplify RNA targets. The RNA template is converted into complementary (c)DNA by the enzyme reverse transcriptase. The cDNA serves later as a template for exponential amplification using PCR. RT-PCR can be undertaken in one or two steps. One-step RT-PCR combines the RT reaction and PCR reaction in the same tube. Only sequence-specific primers may be used. During two-step RT-PCR, the synthesized cDNA is transferred into a second tube for PCR. Oligo (dT), random hexamer or gene-specific primers can be used. Oligo (dT) primers are generally preferred as they hybridize to the 3ʹ poly (A) tails in mRNAs (transcribed gene sequences), whereas random primers prime anything including ribosomal RNA [11]. There are advantages and disadvantages to both methods. One-step reactions are easier to set up and ideal for high throughput screening. Two-step reactions are ideal for detection of several messages from a single RNA sample.

In Practice

This section provides detailed experimental protocols for qRT-PCR using SYBR Green I. We begin with total RNA extraction, followed by RT-PCR. cDNA is then used as a template for qPCR with gene-specific primers. When working with RNA, the researcher must maintain an RNase-free environment, which means wearing gloves, keeping tube lids covered, using RNase-free pipette tips and microcentrifuge tubes, and RNase-free water. Control samples should be used for both RT and PCR. The no- template control helps to detect contamination with template or amplicons from previous reaction steps. In addition, a no-reverse-transcriptase control detects contaminating DNA within the RNA sample. The positive control consists of a source of quantified DNA/RNA with the target sequence and provides assurance that the reaction is working if there is absence of gene expression in the target cells.

Extraction of Total RNA

RNA can be extracted in 30–60 minutes using the method presented below, which has been adapted from Chomczyński and Sacchi 1987 [12]. This protocol utilizes TRIzol Reagent, which is designed to isolate high-integrity total RNA.

Homogenization

1. For tissues, add 1 mL of TRIzol reagent per 50–100 mg of tissue sample. Homogenize sample using, for example, a power homogenizer.

For cells grown in suspension, centrifuge at 300×g for 5 minutes to pellet the cells and pour off supernatant. Add 0.75 mL of TRIzol Reagent per 0.25 mL of sample.

For cells grown in a monolayer, remove growth media from culture dish and rinse cells with ice-cold phosphate-buffered saline. Add 1 mL of TRIzol Reagent per 10 cm² of culture dish surface area.

TIP: Thoroughly homogenize your samples. Fragments left undisrupted during homogenization step represent RNA lost. Also, rinse your homogenizer in-between samples to prevent cross-contamination.

2. Re-suspend the lysate using a 1 mL pipette tip.

3. Incubate mixture at room temperature for 5 minutes.

Phase separation

4. While working in the fume hood, add 200 μL of chloroform per 2 mL of TRIzol Reagent.

5. Vortex samples for 15 seconds and incubate them at room temperature for 3 minutes.

6. Centrifuge the samples at 12,000 g for 15 minutes at 4°C. The mixture separates into a lower red phenol-chloroform phase, an interphase, and a colorless upper aqueous phase where RNA remains.

7. Transfer the upper aqueous phase to a fresh tube. Make sure you do not draw any of the interphase or organic layer into the pipette.

RNA precipitation

8. Add 500 μL of 100% isopropanol and incubate samples at room temperature for 10 minutes.

9. Centrifuge at 12,000×g for 10 minutes at 4°C. The RNA will form a gel-like precipitate at the bottom and side of the tube.

RNA wash and resuspension

10. Remove supernatant and wash precipitate by adding 1 mL of 75% ethanol. Mix the samples by vortexing.

TIP: Don’t forget to vortex! This allows ethanol to penetrate the nucleic acid pellet to dissolve any residual salt.

11. Centrifuge at 7500×g for 5 minutes at 4°C.

12. Remove the supernatant. Allow a few minutes for any remaining ethanol to dry.

13. Re-suspend pellet in RNase-free water (20–50 μL).

At this point, the extracted RNA may be treated with DNase enzyme to prevent genomic DNA contamination. In particular, for downstream RT-PCR, co-amplification of genomic DNA can lead to nonspecific results. For DNase I digestion, use 1 U of DNase I per 1 µg–5 µg of total RNA. Keep total volume of 50 µL. Incubate for 10 minutes at +37°C. Finally, the RNA sample must be purified of DNases, to avoid any destruction of cDNA generated subsequently in the RT-PCR step.

Assessing RNA quantity, purity and integrity

Nucleic acids can be quantified by UV absorption using a spectrophotometer such as the Nanodrop. Ultraviolet absorbance at 260 nm is used to measure the amount of nucleic acid in the sample. An A260 reading of 1.0 is equivalent to around 40 μg/mL of RNA. The RNA purity is determined from the relative absorbance at 260 and 280 nm. A ratio A260/280 greater than 1.8 is satisfactory.

To determine RNA integrity or quality one can look at the intensity of rRNA bands on denaturing agarose gels. Alternatively, the use of a bioanalyzer automatically measures the sizes of the rRNA bands using laser and fluorescent technology, and attributes an RNA integrity number (RIN) to the sample. The RIN is a standardized measure that may be compared between different RNA samples [13].

For short-term storage RNA can be kept at −20°C; for long-term storage at −80°C.

Synthesis of cDNA by Reverse Transcription

The protocol below has been adapted from Invitrogen [11,14]. RT can be undertaken in 90–120 minutes depending on the number of samples. For cDNA synthesis use high-integrity