An introduction to PCR

An introduction to PCR 1.1 Introduction: PCR, a ‘DNA photocopier’ Does it really work? It is so simple! Why did I not think of it? These thoughts were probably typical of most molecular biologists on reading early reports of the polymerase chain reaction or PCR as it is more commonly called. PCR uses a few basic everyday molecular biology reagents to make large numbers of copies of a specific DNA fragment in a test-tube. PCR has been called a ‘DNA photocopier’. While the concept is simple, PCR is a complicated process with many reactants. The concentration of template DNA is initially very low but its concentration increases dramatic- ally as the reaction proceeds and the product molecules become new templates. Other reactants, such as dNTPs and primers, are at concentra- tions that hardly change during the reaction, while some reactants, such as DNA polymerase, can become limiting. There are significant changes in temperature and pH and therefore dramatic fluctuations in the dynamics of a range of molecular interactions. So, PCR is really a very complex process, but one with tremendous power and versatility for DNA manipu- lation and analysis. In the relatively short time since its invention by Kary Mullis, PCR has revolutionized our approach to molecular biology. The impact of PCR on biological and medical research has been like a supercharger in an engine, dramatically speeding the rate of progress of the study of genes and genomes. Using PCR we can now isolate essentially any gene from any organism. It has become a cornerstone of genome sequencing projects, used both for determining DNA sequence data and for the subsequent study of putative genes and their products by high throughput screening method- ologies. Having isolated a target gene we can use PCR to tailor its sequence to allow cloning or mutagenesis or we can establish diagnostic tests to detect mutant forms of the gene. PCR has become a routine laboratory tech- nique whose apparent simplicity and ease of use has allowed nonmolecular biology labs to access the power of molecular biology. There are many scientific papers describing new applications or new methods of PCR. Many commercial products and kits have been launched for PCR applications in research and for PCR-based diagnostics and some of these will be discussed in later chapters. 1.2 PCR involves DNA synthesis PCR copies DNA in the test-tube and uses the basic elements of the natural DNA synthesis and replication processes. In a living cell a highly complex system involving many different proteins is necessary to replicate the complete genome. In simplistic terms, the DNA is unwound and each strand of the parent molecule is used as a template to produce a comple- 1 mentary ‘daughter’ strand. This copying relies on the ability of nucleotides to base pair according to the well-known Watson and Crick rules; A always pairs with T and G always pairs with C. The template strand therefore specifies the base sequence of the new complementary DNA strand. A large number of proteins and other molecules, such as RNA primers, are required to ensure that the process of DNA replication occurs efficiently with high fidelity, which means with few mistakes, and in a tightly regulated manner. DNA synthesis by a DNA polymerase must be ‘primed’, meaning we need to supply a short DNA sequence called a primer that is complementary to a template sequence. Primers are synthetically produced DNA sequences usually around 20 nucleotides long. The DNA polymerase will add nucleotides to the free 3′-OH of this primer according to the normal base pairing rules (Figure 1.1). 2 PCR Primer DNA polymerase Template Synthesis of new DNA strand 5' 3' 3' 5' 5' dNTPs T G T T C C C A A G G A T T T G G GGA A A C C CC 3' 3' 5' 5' 3' AAA AA GG G TT TTTCC C Figure 1.1 Primer extension by a DNA polymerase. The primer anneals to a complementary sequence on the template strand and the DNA polymerase uses the template sequence to extend the primer by incorporation of the correct deoxynucleotide (dNTP) according to base pairing rules. PCR requires only some of the components of the complex replication machinery to copy short fragments of DNA in a simple buffer system in a test tube. Unwinding of the DNA in the cell uses a multi-component complex involving a variety of enzymes and proteins, but in PCR this is replaced simply by a heating step to break the hydrogen bonds between the base pairs of the DNA duplex, a process called denaturation. Following template denaturation two sequence-specific oligonucleotide primers bind to their complementary sequences on the template DNA strands according to normal base pairing rules (Figure 1.2). These primers define the region of template to be copied. DNA polymerase then begins to add deoxynucleotides to the 3′-OH group of both primers producing new duplex DNA molecules (Figure 1.2). This requirement of DNA polymerases to use primers to initiate DNA synthesis is critical for the PCR process since it means we can control where the primers bind, and therefore which region of DNA will be replicated and amplified. If the DNA polymerase was like an RNA polymerase that does not require a primer then we would have no way of defining what segment of DNA we wanted to be copied. At the next heating step the double-stranded molecules, which are heteroduplexes containing an original template DNA strand and a newly synthesized DNA strand produced during the first DNA synthesis reaction, are now denatured. Each DNA single strand can now act as a template for the next round of DNA synthesis. As discussed in detail in Chapter 2, it is during this second cycle of PCR that the first DNA single strand of a length defined by the positions of the primers can be formed. In cycle 3 the first correct length double-stranded PCR products are formed. In subsequent cycles there is then an exponential increase in the number of copies of the ‘target’ DNA sequence; theoretically, the number of copies of the target sequence will be doubled at each PCR cycle. This means that at 100% efficiency, each template present at the start of the reaction would give rise to 10 6 new strands after only 20 cycles of PCR. Of course the process is not 100% efficient, and it is usually necessary to carry out more reaction cycles, often 25 to 40 depending upon the concentration of the initial template DNA, its purity, the precise conditions and the application for which you require the product. The specificity and efficiency of PCR, however, means that very low numbers of template molecules present at the start of the PCR can be amplified into a large amount of product DNA, often a microgram or more, which is plenty for a range of detailed analyses. Of course, this ability to amplify also means that if you happen to contaminate your reaction with a few molecules of product DNA from a previous reaction, you may get a false result. This is why performing control reactions is so impor- tant and we will deal with such contamination problems in Chapter 4. 1.3 PCR is controlled by heating and cooling PCR relies on the use of different temperatures for the three steps of the reaction, denaturation, annealing and extension. A high temperature, usually 94–95°C, is used to denature (separate) the strands of the DNA template. The temperature is then lowered to allow the primers to anneal by base pairing to their complementary sequences on the template strands; this temperature varies depending on the primers (see details in Chapter 3). An introduction to PCR 3 The annealing temperature is important to ensure high specificity in the reaction; generally the higher the annealing temperature the more specific will be the reaction. A temperature of 55°C is commonly used, but in many cases a higher temperature is better and this can even be as high as 72°C for some experiments, leading to a two-temperature PCR cycle. Finally, for 4 PCR G 5' 5' 5' 3' 3' 3' 3' 3' 5' 5' 5' 3' 3' 5' 3' Primer 2 dNTPs Primer 1 Synthesis of new DNA strands defined by primers T T T G G G A AA A A CC C C C CC CC CC CC CC CC CC CC CC AA AAA AA A A AAAA AAA G GG GG GG GG G GG GG GG GG TT T TT T TTT TT TTTT 5' TTG A C 5' 3' 3' 5' DNA denatured and primers annealed TT TTTT GG GG GG C CCCAA AC TGGCCCAA AA Figure 1.2 The first cycle of a PCR. A double-stranded template molecule is denatured. Primers anneal to their complementary sequences on the single-stranded template. DNA synthesis is catalyzed by a thermostable DNA polymerase. The result of this PCR cycle is that two copies of the target sequence have been generated for each original copy. efficient DNA synthesis, the temperature is adjusted to be optimal for the DNA polymerase activity, normally 72°C (see Chapter 3). To amplify the target DNA it is necessary to cycle through these temperatures several times (25 to 40 depending on the application). Conveniently, this temperature cycling is accomplished by using a thermal cycler, a programmable instru- ment that can rapidly alter temperature and hold samples at the desired temperature for a set time. This automation is one of the important advances that led to PCR becoming widely accessible to many scientists and is covered in more detail in Chapter 3. Before thermal cyclers became avail- able, PCR was performed by using three water baths set to temperatures of typically 95°C, 55°C and 72°C, and reaction tubes in racks were moved manually between the baths. The other major technological advance that preceded the development of thermal cyclers was the replacement of DNA polymerase I Klenow fragment with thermostable DNA polymerases, such as Taq DNA poly- merase, which are not inactivated at the high denaturation temperatures used during PCR. The ability to carry out the reaction at high temperatures enhances the specificity of the reaction (Chapter 4). At 37°C, where Klenow works best, primers can bind to nontarget sequences with weak sequence similarity, because mismatches between the two strands can be tolerated. This leads to poor specificity of primer annealing and the amplifi- cation of many nontarget products. The introduction of thermostable DNA polymerases also reduced the cost of a reaction by reducing the amount of polymerase required. With Klenow, at each denaturing step the enzyme was also denatured and therefore a fresh aliquot had to be added at each cycle. Thermostable polymerases retain their activity at the denaturation temperatures and therefore only need to be added at the start of the reaction. 1.4 PCR applications and gene cloning PCR has revolutionized our approach to basic scientific and medical research, to medical, forensic and environmental testing. It provides an extremely flexible tool for the research scientist, and every molecular biology research laboratory now uses PCR routinely; often adapting and tailoring the basic procedures to meet their own special needs. It has become an indispensable tool for routine and repetitive DNA analyses such as diagnosis of certain genetic diseases within clinical screening laboratories where speed and accuracy are important factors, and also for sample identification in forensic and environmental testing. In particular PCR has become a central tool in the analysis and exploitation of genome sequence information, for example in gene knockout through RNA interference where PCR allows the rapid generation of appropriate constructs. It also facilitates measurement of levels of gene expression by ‘real-time’ PCR that monitors the level of product amplification at each cycle of the PCR (Chapter 9), providing information on the relative concentrations of template cDNA. In some cases PCR provides an alternative to gene cloning, but in other cases it provides a complementary tool. In gene cloning a fragment of DNA is joined by ligation to a cloning vector which is able to replicate within a An introduction to PCR 5 host cell such as the bacterium Escherichia coli. As the bacterium grows, the new recombinant DNA molecule is copied by DNA replication, and as the cell divides the number of cells carrying the recombinant molecule increases. Finally, when there are enough cells you can isolate the recombinant DNA molecules to provide sufficient DNA for analysis or further manipulation of the cloned DNA fragment. This type of cloning experiment takes about 2–3 days. PCR also amplifies your target DNA fragment so that you have enough to analyze or manipulate, but in this case the DNA replication occurs in a test-tube and usually takes no more than 1–3 hours. In many cases, for example in diagnostic tests for cancers or genetic diseases, including ante- natal screening, or in forensic testing, PCR provides the most sensitive and appropriate approach to analyze DNA within a day. For studying new genes and genetic diseases it is often necessary to create gene libraries and this may involve PCR followed by cloning into a suitable vector. Also many experiments to produce proteins to study their structure and function require expression in host cells and this requires the cloning of the gene perhaps as a PCR product, into a suitable expression vector. So in many cases PCR and gene cloning represent complementary techniques. It is important to consider carefully the most appropriate strategy for the experiments you wish to undertake. Integrating PCR and cloning will be covered further in Chapter 6 while diagnostic applications of PCR are covered in Chapter 11. 1.5 History of PCR As long ago as 1971, Khorana and colleagues described an approach for replicating a region of duplex DNA by using two DNA synthesis primers designed so that their 3′-ends pointed towards each other (1). However, the concept of using such an approach repeatedly in an amplification format was not conceived for another 12 years. ‘Sometimes a good idea comes to you when you are not looking for it.’ With these words, Kary Mullis, the inventor of PCR, starts an account in Scientific American of how, during a night drive through the mountains of Northern California in Spring 1983, he had a revelation that led him to develop PCR (Mullis, 1990). Mullis was awarded the 1993 Nobel Prize for Chemistry for his achievement. The practical aspects of the PCR process were then developed by scientists at Cetus Corporation, the company for which Mullis worked at that time. They demonstrated the feasibility of the concept that Mullis had provided, and PCR became a major part of the business of Cetus, before they finally sold the rights to PCR in 1991 for $300m to Roche Molecular Systems. PCR and the thermostable polymerase responsible for the process were named as the first ‘Molecule of the Year’ in 1989 by the international journal Science. Since the myriad of applications of PCR were recognized it has become rather entangled in commercialism, due to the large amounts of money to be made from licensing the technology. PCR is covered by patents, granted to Hoffman La-Roche and Roche Molecular Systems, and these have been vigorously enforced to prevent unlicensed use of the method. Some of these patents terminated on 28 March 2005. From this date it has been possible to perform basic PCR in the US without a license, although some other 6 PCR patents still apply to instruments and specific applications. Outside the US in countries covered by the equivalent patents, there is a further year of patent protection to run. Key milestones in the development of PCR 1983 Kary Mullis of Cetus Corp. invents PCR. 1985 First paper describing PCR using Klenow fragment of DNA polymerase I (2). 1986 Cetus Corp. and Perkin Elmer Corp. establish a joint venture company (Perkin Elmer Cetus) to develop both instruments and reagents for the biotechnology research market. 1987 Cetus develop a partnership with Kodak for PCR-based diagnostics, but Kodak terminate this agreement and Hoffman-La Roche become the new partner. 1988 First paper describing the use of Taq DNA polymerase in PCR (3). 1990 Cetus licence certain reagents companies, namely Promega, Stratagene, USB, Pharmacia, Gibco-BRL and Boehringer, to sell native Taq DNA polymerase for non–PCR applications. 1991 Cetus wins court case against DuPont who challenged the Cetus PCR patents. 1991 Perkin Elmer Cetus joint venture dissolved as the PCR rights are acquired by Hoffman LaRoche. 1991 Perkin Elmer form a ‘strategic alliance’ with Roche to sell PCR reagents in the research market. Roche continue to develop the diagnostics reagents business. Perkin Elmer assume total respon- sibility for the thermal cycler business. 1991 Cetus is acquired by Chiron Corp. for non-PCR business aspects, in particular interleukin-2-based pharmaceuticals. 1993 Roche file a lawsuit against Promega for alleged infringement of their license to sell native Taq polymerase for non-PCR applications. Action also taken against several smaller companies for selling Taq DNA polymerase without license agreements. These disputes between Roche and Promega are still proceeding through the courts in 2005, and do not look like they will be resolved quickly or easily. 1993 Perkin Elmer merges with Applied Biosystems. The Applied Biosystems Division of Perkin Elmer assumes responsibility for all DNA products such as DNA synthesis, sequencing and the PCR in addition to the other products associated with protein sequencing and analysis. 1993 License granted to Boehringer-Mannheim to supply reagents, including Taq polymerase for use in PCR. 1993 Kary Mullis, inventor of PCR, wins a Nobel Prize for Chemistry. 1993 + Widespread licensing of PCR technology and Taq DNA polymerases to a large number of Biological Supplies Companies. 1998 Promega Corporation challenges the original patents on native Taq DNA polymerase and court proceedings continue. 2005 March 28 2005 is the date on which several US PCR patents expire: ● 4 683 195 Process for amplifying, detecting and/or cloning nucleic acid sequences; An introduction to PCR 7 ● 4 683 202 Process for amplifying nucleic acid sequences; ● 4 965 188 Process for amplifying, detecting and/or cloning nucleic acid sequences using a thermostable enzyme; ● 6 040 166 Kits for amplifying and detecting nucleic acid sequences including a probe; ● 6 197 563 Kits for amplifying and detecting nucleic acid sequences; ● 4 800 159 Process for amplifying, detecting and/or cloning nucleic acid sequences; ● 5 008 182 Detection of AIDS-associated virus by PCR; ● 5 176 995 Detection of viruses by amplification and hybridization. The speed and simplicity of PCR technology accompanied by an increased range of high quality products has led to a more rational approach to PCR experimentation. We understand better the molecular processes underlying PCR (see Chapter 2) so that it is seen less as a ‘witches’ brew’. It is impor- tant to highlight good practices that increase confidence in results by reducing the likelihood of artefactual results. The importance of good PCR technique, particularly with regard to proper controls and the prevention of contamination (Chapter 4) cannot be overemphasized. Remember, if you work in a research laboratory a wrong result may be inconvenient leading to a waste of time, effort and money and so should be avoided. But, if you work in a diagnostic laboratory, a wrong result could mean the difference between life and death. It is a good idea to start with the highest standards and expectations so that you can be confident in your results no matter where you work. PCR has now been adapted to serve a variety of applications and some of these will be described in this book (Chapters 5 to 11). Further reading Mullis KB (1990) The unusual origins of the polymerase chain reaction. Sci Am 262: 56–65. White TJ (1996) The future of PCR technology: diversification of technologies and applications. Trends Biotechnol 14: 478–483. References 1. Kleppe K, Ohtsuka E, Kleppe R, Molineux R, Khorana HG (1971) Studies on polynucleotides. XCVI. Repair replication of short synthetic DNA’s as catalysed by DNA polymerases. J Mol Biol 56: 341–346. 2. Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N (1985) Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230: 1350–1354. 3. Saiki RK, Gelfand DH, Stoffel S, Scharf S, Higuchi R, Horn GT, Mullis KB, Erlich HA (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487–491. 8 PCR . sequencing and the PCR in addition to the other products associated with protein sequencing and analysis. 1993 License granted to Boehringer-Mannheim to supply. complementary tool. In gene cloning a fragment of DNA is joined by ligation to a cloning vector which is able to replicate within a An introduction to PCR 5