Polymerase Chain Reaction

12 Key excerpts on "Polymerase Chain Reaction"

• 

  eBook - PDF

  Genetic Engineering

  A Glimpse of Techniques and Applications

  • Farrukh Jamal(Author)
  • 2020(Publication Date)
  • IntechOpen

    (Publisher)

  13 Chapter 2 Polymerase Chain Reaction Shaheen Shahzad, Mohammad Afzal, Shomaila Sikandar and Imran Afzal Abstract Polymerase Chain Reaction (PCR) is an efficient and one of the most common methods used in biological sciences for in vitro multiplication of a target DNA molecule. The technique has significantly contributed in changing and developing different fields of biological sciences since 1980s. PCR has a vital role in support-ing the processes involved in genetic engineering, particularly the cloning of DNA fragments used to modify the genomes of microorganisms, animals, and plants. Consequently, the technique has numerous applications in fundamental and applied research in medicine agriculture, environment, and bio-industry. The main focus of this chapter is to describe briefly the principles, methodology, various types, and applications of PCR in different fields. Besides, different components of PCR, trouble shooting during the execution, and limitations of the techniques are also outlined. Keywords: PCR, primer, DNA template, nucleotides, sequence, polymerase 1. Introduction Polymerase Chain Reaction (PCR) is one of the most commonly used method in modern molecular biology. The technique was developed by the Nobel laureate, Kary Mullis, in 1984. It is an in vitro process to multiply a target molecule of DNA with extreme precision, making it easy to be handled and examined by routine molecular biological methods [1–4]. Since its inception, PCR has significantly con-tributed in changing and developing biological sciences. The first PCR machine was introduced in market in 1988. The Human Genome Project has been result of PCR based approaches [5, 6]. Owing to its wide range of applications, numerous variants of PCR techniques have emerged over the past few decades [2–4].

  Sign up to read

  Learn more about book
• 

  eBook - ePub

  Genetic Engineering

  Volume 1: Principles Mechanism, and Expression

  • Tariq Ahmad Bhat, Jameel M. Al-Khayri, Tariq Ahmad Bhat, Jameel M. Al-Khayri(Authors)
  • 2023(Publication Date)
  • Apple Academic Press

    (Publisher)

  Chapter 6 Polymerase Chain Reaction ASIMA TAYYEB and ZHUHA BASIT School of Biological Sciences, University of the Punjab, Lahore, Pakistan

  ABSTRACT

  Polymerase Chain Reaction (PCR) is a remarkable technique which can be used for amplification of DNA at exponential rate. It was invented in 1983 by Mullis. In the history of biological and medical sciences, the development of the PCR has been a turning point. PCR with its ability to amplify even a single molecule makes it a highly sensitive and qualitative technique. PCR has wide range of applications in the fields of molecular genetics, systematics, epidemiology, archaeology, anthropology, evolutionary genetics, and other branches of forensic sciences. Moreover, DNA amplified through PCR can be employed in number of processes like cloning, sequencing, sizing and analytical detection (Arnheim & Erlich, 1992 ).

  6.1 History

  The credit of invention of remarkable PCR in method 1983 goes to Kary Mullis, who was later on awarded with Nobel Prize in Chemistry for his invention in 1985 which he shared with Michael Smith. Techniques which were quite similar to PCR were in use in past, but a major breakthrough in this field occurred with the discovery of Taq polymerase in 1976, which opened new avenues and provided opportunity for utilization of thermocycler which could be programmed (Mullis and Faloona, 1987 ). Taq polymerase has been derived from bacterium Thermus aquaticus, which are thermophilic and live at extreme hot temperature. One of main characteristic of this enzyme is that is remains active even beyond the denaturation temperature which makes it much effective and ends up the need to add enzyme at every new cycle (Chamberlain et al., 1994 ).

  ________________________________ Genetic Engineering, Volume 1: Principles, Mechanism, and Expression. Tariq Ahmad Bhat & Jameel M. Al-Khayri (Eds.) © 2023 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)

  Sign up to read

  Learn more about book
• 

  eBook - PDF

  Molecular research in Aquaculture

  • Bruno Augusto Amato Borges(Author)
  • 2019(Publication Date)
  • Delve Publishing

    (Publisher)

  Finally, this chapter discusses this PCR application in detail. 4.1. INTRODUCTION Polymerase Chain Reaction (PCR) is defined as a technique which is greatly used in the field of molecular biology. It is generally used to make or produce several copies of a particular segment of deoxyribonucleic acid (DNA). The technique of PCR makes investigators capable of producing large quantities of DNA. These produced DNA will further be used in numerous procedures and experiments in molecular biology, medical diagnostics, evolutionary biology and forensic analysis. The revolutionary in vitro PCR technique was initially developed by Kary B. Mullis, an American biochemist in year 1983 to reach huge concentration of specific fragments of DNA. He won the Nobel Prize in 1993 for his contributions in the field of chemistry. Prior to the development of PCR, the techniques used to amplify or produce numerous copies of rDNA fragments were labor-intensive as well as time-consuming. A machine designed to perform reactions of PCR can easily complete several rounds of replication which ultimately produces billion copies of a specific DNA fragment in just few hours. The technique of PCR is based on the natural process by which a cell used to replicate a DNA strand. Some biological ingredients are required in the PCR reaction. The integral component of PCR reaction is the template DNA. This is the DNA which has the region to be copied, for example, gene. The only information required for this DNA fragment to be replicated is the sequence of two short regions of nucleotides at either end of the region. Nucleotides are known as the subunits of DNA. Two short template sequences Polymerase Chain Reaction: Principles and Components 77 should be known so that two primers can be produced. Primers are known as the short stretches of the nucleotides that resemble the template sequences. The primers produced then bind to the template at their complementary sites.

  Sign up to read

  Learn more about book
• 

  No longer available |Learn more

  Introduction to DNA Profiling

  • (Author)
  • 2014(Publication Date)
  • Orange Apple

    (Publisher)

  ________________________ WORLD TECHNOLOGIES ________________________ Chapter- 5 Polymerase Chain Reaction A strip of eight PCR tubes, each containing a 100 μL reaction mixture The Polymerase Chain Reaction ( PCR ) is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the ________________________ WORLD TECHNOLOGIES ________________________ reaction for DNA melting and enzymatic replication of the DNA. Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase (after which the method is named) are key components to enable selective and repeated amplification. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. PCR can be extensively modified to perform a wide array of genetic manipulations. Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the bacterium Thermus aquaticus . This DNA polymerase enzymatically assembles a new DNA strand from DNA building blocks, the nucleotides, by using single-stranded DNA as a template and DNA oligonucleotides (also called DNA primers), which are required for initiation of DNA synthesis. The vast majority of PCR methods use thermal cycling, i.e., alternately heating and cooling the PCR sample to a defined series of temperature steps. These thermal cycling steps are necessary first to physically separate the two strands in a DNA double helix at a high temperature in a process called DNA melting. At a lower temperature, each strand is then used as the template in DNA synthesis by the DNA polymerase to selectively amplify the target DNA.

  Sign up to read

  Learn more about book
• 

  eBook - PDF

  Insect Molecular Genetics

  An Introduction to Principles and Applications

  • Marjorie A. Hoy(Author)
  • 2013(Publication Date)
  • Academic Press

    (Publisher)

  203 204 Chapter 9 DNA Amplification by the Polymerase Chain Reaction Recently, other methods, including the ligase chain reaction (LCR) and several other amplification techniques, have become available to amplify and/or detect specific nucleic acids. Introduction The Polymerase Chain Reaction (PCR) is an in vitro or cell-free method for synthesizing specific DNA or RNA sequences in nearly any amount required. It is one of the most accessible and versatile techniques available to entomologists interested in both basic and applied problems. The PCR is recognized to be one of the most powerful new techniques in biology. It is used to isolate specific DNA fragments, end-label DNA, mutagenize specific DNA fragments, clone cDNA and genomic DNA, sequence DNA, quantitate RNA and DNA, and alter a variety of sequences to study gene expression. DNA polymerase and the PCR technique were designated the Molecule of the Year by Science magazine in 1989 (Guyer and Koshland 1989). The PCR has become a standard laboratory meth-od in an extraordinarily short time since it was invented in 1985 (K. Mullis, U.S. patent 4,683,195, July 1987; U.S. patent 4,683,202, July 1987). Between 1985 and 1989, the PCR was cited in more than 1000 publications and in 1993 Kary Mullis received the Nobel Prize in chemistry for his work on the PCR. Improvements in and optimization of the PCR have led to its use by numer-ous scientists. The PCR has rapidly become a common procedure in forensics and diagnostics, and is revolutionizing studies of basic biology, ecology, and evolution. This relatively simple technique has given scientists who have lim-ited experience in molecular biology the opportunity to apply molecular genetic techniques to diverse problems (Arnheim et al. 1990). While the PCR technique is relatively simple, the process is, in fact, not completely understood. A number of parameters can be modified to optimize the PCR (Carbonari et al. 1993).

  Sign up to read

  Learn more about book
• 

  No longer available |Learn more

  Forensic Toxicology and DNA Profiling

  • (Author)
  • 2014(Publication Date)
  • College Publishing House

    (Publisher)

  ________________________ WORLD TECHNOLOGIES ________________________ Chapter 10 Polymerase Chain Reaction A strip of eight PCR tubes, each containing a 100 μL reaction mixture The Polymerase Chain Reaction ( PCR ) is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase (after which the method is named) are key components to enable selective ________________________ WORLD TECHNOLOGIES ________________________ and repeated amplification. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. PCR can be extensively modified to perform a wide array of genetic manipulations. Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the bacterium Thermus aquaticus . This DNA polymerase enzymatically assembles a new DNA strand from DNA building blocks, the nucleotides, by using single-stranded DNA as a template and DNA oligonucleotides (also called DNA primers), which are required for initiation of DNA synthesis. The vast majority of PCR methods use thermal cycling, i.e., alternately heating and cooling the PCR sample to a defined series of temperature steps. These thermal cycling steps are necessary first to physically separate the two strands in a DNA double helix at a high temperature in a process called DNA melting. At a lower temperature, each strand is then used as the template in DNA synthesis by the DNA polymerase to selectively amplify the target DNA.

  Sign up to read

  Learn more about book
• 

  eBook - ePub

  An Introduction to Biotechnology

  The Science, Technology and Medical Applications

  • W.T. Godbey(Author)
  • 2014(Publication Date)
  • Academic Press

    (Publisher)

  Chapter 11

  The Polymerase Chain Reaction

  Abstract

  At this point in the text, we are headed toward producing our own gene sequences. However, a single copy of a DNA sequence is seldom (if ever) enough for the biotechnologist to work with. PCR is a means by which exponential numbers of a given DNA sequence are produced from minimal quantities of starting material. PCR is used to indicate the transcriptional activity of a specific gene (which can, for example, imply drug bioactivity), to help identify cell type or human identity, or to generate sufficient starting material for molecular cloning.

  The rudiments of the PCR process will be described, and then different uses of the chain reaction will be presented to compare and contrast traditional and real-time PCR. Mechanisms of qPCR that use SYBR green and primer/probe sets will be discussed.

  Keywords Amplicon Melt Anneal Extend PCR loops Cycle number Transcriptome Internal control Primer dimer Background fluorescence qPCR

  Cycle threshold (

  Ct

  )

  Delta-delta

  Ct

  (2− ΔΔ )

  Traditional and real-time PCR SYBR green Primer Primer efficiency Probe

  DNA polymerase, which we have already discussed in terms of cellular replication, can be used in the laboratory to make multiple copies of a DNA fragment. While the cell will use DNA polymerase to make a single copy of its genome before division, in the laboratory, we can use the polymerase repeatedly to amplify a region of DNA (called an amplicon ) at an exponential rate. In the first round of amplification, the amplicon will be replicated once. In the second round, the amplicons of the original DNA and the copies made during the first round will be replicated. In the third round, the amplicon and all of the previously constructed copies (a total of four dsDNA molecules) will be replicated, leaving us with a total of eight dsDNA fragments at the end of the round. These rounds of replication will continue, ultimately yielding (by one theory) 2n

  Sign up to read

  Learn more about book
• 

  eBook - PDF

  Recombinant DNA Laboratory Manual, Revised Edition

  • Judith W. Zyskind, Sanford I. Bernstein(Authors)
  • 2012(Publication Date)
  • Academic Press

    (Publisher)

  LabX THE Polymerase Chain Reaction TECHNIQUE The Polymerase Chain Reaction or PCR has caused a minirevolution in molecular biology. PCR is an in vitro method of DNA synthesis by which a particular segment of DNA can be specifically amplified. Using PCR, molecular biologists can produce microgram quantities of DNA from just picogram amounts of starting material. To perform PCR, two oligonucleotide primers that flank the DNA fragment to be amplified are annealed to their complementary sequences. These primers hybridize to opposite strands of the target sequence and are oriented so that DNA synthesis occurs in the region between the two primers. Following primer extension, the strands are denatured and new primers are annealed. DNA synthesis is permitted to occur again. These cycles of denaturation, annealing, and polymerization are repeated a number of times. Y DNA sequence to be amplified (-100-3000 bp) PCR was invented by Kary Mullis at Cetus Corporation in 1985, and Cetus holds the patents on this procedure (Barinaga, 1991). Dr. Mullis originally used DNA polymerase I Klenow fragment for the primer exten-sion step in his first PCR reactions. Because Klenow fragment is unstable at high temperatures, new enzyme had to be added each PCR cycle. He and David Gelfand, also at Cetus, replaced the Klenow enzyme with Tag DNA Lab X. The Polymerase Chain Reaction Technique polymerase, which has an optimum temperature of 75°C. Tag DNA polymerase, produced by the thermophilic bacterium, Thermus aquaticus, can withstand high temperatures without significant loss of activity and remains active throughout the PCR reaction cycles.

  Sign up to read

  Learn more about book
• 

  eBook - PDF

  Ion Channels in the Pulmonary Vasculature

  • Jason X. J. Yuan(Author)
  • 2005(Publication Date)
  • CRC Press

    (Publisher)

  35 Screening for mRNA Expression Using the Polymerase Chain Reaction Ivana Fantozzi, Carmelle V. Remillard, Mehran Mandegar, and Jason X.-J. Yuan Department of Medicine, University of California, San Diego, California, U.S.A. I. INTRODUCTION Polymerase Chain Reaction (PCR) is an in vitro technique for amplifying a target DNA sequence in a short time. This is because both strands of DNA are copied simultaneously during PCR; therefore, there is an exponen-tial increase in the number of copies of the target DNA fragment or gene with each cycle (1,2). Three fundamental aspects make PCR such a powerful tool in determining the qualitative expression of target genes: 1. PCR is less expensive, less time-consuming, and simpler than pre-vious DNA (and RNA) duplication techniques, making it possi-ble for anyone, even one with no training in molecular biology, to do genetic and molecular biological research. 2. PCR has an enormous amplification capability. Therefore, start-ing from an extremely tiny biological sample, it is possible to obtain a nearly unlimited supply of genetic material to manipulate. 3. Perhaps the most important aspect is the specificity of PCR. The specificity comes from the specific hybridization of the primers with DNA target regions. The length of the target sequence, if limited to less than a few kilobases, has a beneficial effect on specificity (3). 711 One of the strengths of PCR is its versatility. Modifications in the tech-nique can change its application such that it can be used to analyze and amplify cellular RNA. This particular application, referred to as ‘‘reverse transcription-PCR’’ (RT-PCR), highly increases the sensitivity of detection and enables sequence analysis of RNA using the low amount of RNA templates available within the cells. This technique can also be used to screen for the intensity of mRNA expression of known genes.

  Sign up to read

  Learn more about book
• 

  eBook - PDF

  PCR/RT- PCR in situ

  Light and Electron Microscopy

  • Gerard Morel, Mireille Raccurt(Authors)
  • 2002(Publication Date)
  • CRC Press

    (Publisher)

  Polymerase Chain Reaction (PCR) 94 5.1.3 Exponential Amplification With each cycle the number of DNA strands doubles, so that the repetition of the cycles leads to the exponential amplification of a specific nucleotide sequence according to the theoretical formula: number of copies = 2 n where n represents the number of cycles. ➫ In other words, 20 amplification cycles pro-duce 2 20 copies. ➫ The yield is never 100%; for liquid-phase PCR it is closer to 70%. It is difficult to define in situ . ➫ Target DNA, with the sequence of interest First cycle: No fragment of the desired size has been synthesized. Second cycle: By the end of this cycle, two fragments of the required size have been obtained. Third cycle: Two double strands of the required size have now been synthesized. Fourth cycle: Eight double strands have now been synthesized. Figure 5.3 Exponential amplification of a sequence of interest during the first four PCR cycles. 5 ′ 3 ′ 3 ′ 5 ′ 5 ′ 3 ′ 5 ′ 3 ′ 3 ′ 5 ′ 5 ′ 3 ′ 3 ′ 5 ′ 3 ′ 5 ′ 5 ′ 3 ′ 3 ′ 5 ′ 3 ′ 5 ′ 5 ′ 3 ′ 5 ′ 3 ′ 5 ′ 3 ′ 3 ′ 5 ′ 3 ′ 5 ′ 5 ′ 3 ′ 5 ′ 3 ′ 5 ′ 3 ′ 3 ′ 5 ′ 3 ′ 5 ′ 3 ′ 5 ′ 5.2 Diagram of the Different Steps 95 5.2 DIAGRAM OF THE DIFFERENT STEPS Frozen sections Cell smears Cell suspensions Paraffin sections Pretreatments RNA DNA Amplification Washing Reverse transcription Washing Amplification Washing Fixation of amplified product Fixation of amplified product Polymerase Chain Reaction (PCR) 96 5.3 TOOLS 5.3.1 Deoxynucleotide Triphosphates (dNTP) 5.3.1.1 Characteristics These are generally supplied either: • In liquid form, at pH 7, in ultrapure water or in lyophilized form or ➫ Their degree of purity, as given by the manu-facturers, is generally > 98%. • Separately (dATP, dCTP, dGTP, dTTP), or in equimolar mixtures ➫ In liquid form, dNTPs are supplied at a con-centration of 100 m M . 5.3.1.2 Working concentration The usual concentration is between 50 and 250 µ M ; an equimolar concentration ensures the fidelity of the enzyme.

  Sign up to read

  Learn more about book
• 

  Available until 25 Jan |Learn more

  Forensic Biology

  • Richard Li(Author)
  • 2015(Publication Date)
  • CRC Press

    (Publisher)

  Finally, the length of the molecule also affects the T m simply because a longer mol- ecule of DNA requires more energy to break more bonds than a shorter molecule. The single strands in a solution of denatured DNA can, under certain conditions, reanneal into double-stranded DNA. The process is called renaturation and two requirements must be met for it to occur. First, sufficient amounts of charged molecules, such as salts, must be present in the solu- tion to neutralize the negative charges of the phosphate groups in DNA. This prevents the comple- mentary strands from repelling each other. Additionally, the temperature must be high enough to disrupt hydrogen bonds that formed randomly between the bases of DNA strands. However, exces- sively elevated temperatures can disrupt the base pairs between the complementary DNA strands. 7.2 Basic Principles of Polymerase Chain Reaction The Polymerase Chain Reaction (PCR) allows the exponential amplification of specific sequences of DNA to yield sufficient amplified products, also known as amplicons, for various downstream Forensic Biology, Second Edition 144 applications. The technique is highly sensitive and can amplify very small quantities of DNA. Therefore, it can be utilized for the analysis of samples of limited quantity. PCR-based assays are rapid and robust. Thus, PCR forms the basis of many forensic DNA assays such as DNA quan- titation (Chapter 6), short tandem repeat (STR) profiling (Chapter 20), and mitochondrial DNA (mtDNA) sequencing (Chapter 23). The concept of synthesizing DNA by a cycling process was first proposed in the early 1970s. In the mid-1980s, PCR technology was finally developed by Kary Mullis and his coworkers (Cetus Corporation) to amplify the β-globin gene for the diagnosis of sickle-cell anemia.

  Sign up to read

  Learn more about book
• 

  eBook - PDF

  Fundamental Laboratory Approaches for Biochemistry and Biotechnology

  • Alexander J. Ninfa, David P. Ballou, Marilee Benore(Authors)
  • 2015(Publication Date)
  • Wiley

    (Publisher)

  Thus, the PCR reactions are carried out simultaneously in a single tube, and the electrophoretic bands corresponding to length of DNA are detected and identified by color of fluorescence with a laser- Fundamental Laboratory Approaches for Biochemistry and Biotechnology 406 excited fluorimeter. An excellent review of the process can be found at the Web site for the University of Michigan core sequencing facility. (http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/sequencing.html). Another way PCR is used in sequencing produces separate single strands from double-stranded DNA (asymmetric PCR). The individual single strands can then be isolated, for example, by cutting the band out of an agarose gel, and sequencing it by the Sanger method. In Experiment 4 of our laboratory exercises, you will selectively amplify single strands of the E. coli glnL gene by asymmetric PCR, using genomic DNA as the starting material. This technique has been used to rapidly sequence many different mutant versions of the glnL gene from genomic DNA, without first cloning each mutant gene (Atkinson and Ninfa 1992). GENE TYPING Because the PCR technique permits the rapid examination of a target DNA sequence, it is useful for applications in which one needs to verify the genotype of a sample. The basic principle of DNA typing is as follows. All organisms have a set of genes that make up part of its genome. However, the DNA sequences of those genes are not identical in all individuals. The differences in DNA sequence of our genes partially accounts for why individuals within a species are recognizable from one another. For example, among humans, hair color, eye color, height, and weight are influenced by our genetic make-up. The latter two characteristics are, of course, also influenced by environmental factors. When the same gene is found to have different versions, with slightly different DNA sequences, the different versions are called alleles.

  Sign up to read

  Learn more about book