Lecture 7 Molecular Biology 2 - Polymerase Chain Reaction (PCR) PDF

Molecular Biology Lv2 By Prof. Sami Mohamed Badr University in Cairo, School of Biotechnology Lecture #7 The polymerase chain reaction (PCR) is a la...

Molecular Biology Lv2 By Prof. Sami Mohamed Badr University in Cairo, School of Biotechnology Lecture #7 The polymerase chain reaction (PCR) is a laboratory nucleic acid amplification technique used to denature and renature short segments of DNA using DNA polymerase I enzyme, an isolate from Thermus aquaticus, known as Taq polymerase. In 1985, PCR was introduced by Mullis et al, who were later awarded the Nobel...  - Polymerase DNA Chain Reaction Amplification (PCR) PCR stands for Polymerase Chain Reaction. It is a technique used in molecular biology to amplify a specific segment of DNA. PCR was developed in the 1980s by Kary Mullis, who won the Nobel Prize in Chemistry for his work. PCR is a powerful tool that has revolutionized the field of molecular biology and is used in a wide variety of applications, including: Applications of PCR Identifying and diagnosing infectious diseases Detecting genetic mutations Cloning genes Studying gene expression Forensic analysis Paternity testing Archaeology  The Polymerase Chain Reaction (PCR) is a revolutionary technique in molecular biology that allows for the amplification of specific DNA segments. Kary Mullis, an American biochemist, developed PCR in 1985. His groundbreaking work earned him the Nobel Prize in Chemistry in 1993.  PCR is a repetitive process that involves three main steps: Denaturation: The DNA template is heated to separate the two strands of DNA. Annealing: Primers, which are short pieces of DNA complementary to the target sequence, are added to the reaction. The primers anneal to their complementary sequences on the DNA template. Extension: DNA polymerase, an enzyme that synthesizes DNA, is added to the reaction. DNA polymerase extends the primers, synthesizing new DNA complementary to the target sequence. These three steps are repeated for 20-30 cycles, after each cycle, the amount of target DNA is doubled, this means that after 30 cycles, there will be millions of copies of the target DNA.  This method can generate tens of billions of copies of a particular DNA fragment (the sequence of interest, DNA of interest, or target DNA) from a DNA extract (DNA template). Indeed, if the sequence of interest is present in DNA extract, it is possible to selectively replicate it in very large numbers. Using PCR, copies of very small amounts of DNA sequences are exponentially amplified in a series of cycles of temperature changes. PCR is now often indispensable technique used in medical laboratory research for a broad variety of applications including biomedical research and criminal forensics. Essential Components Of PCR: The following are the essential components of PCR Thermal cyclers (Thermocyclers) Target DNA (DNA template) Two primers(forward and reverse primers) Taq polymerase (Themus aquaticus) Buffers Deoxy nucleotide triphosphates (d NTP’s) Monovalent \bivalent cation Nucleotides (A\T\G\C) Water Principles: PCR amplifies a specific region of a DNA strand (the DNA target). Most PCR methods amplify DNA fragments of between 0.1 and 10 kilo base pairs (kbp) in length, although some techniques allow for amplification of fragments up to 40 kbp. A basic PCR set-up requires several components: 1DNA template that contains the DNA target region to amplify. 2DNA polymerase; an enzyme that polymerizes new DNA strands; heat-resistant Taq polymerase is especially common. PCR Reaction Components Taq polymerase: Taq DNA Polymerase allows replicationis a highly thermostable DNA polymerase from thermophilic bacterium, Thermus aquaticus which lives in hot springs and resists temperatures above 100°C, which are usually sufficient to denature most proteins. Optimum temperature of Taq polymerase activity is at 72°C Pfu polymerase: Today, researchers also use a DNA polymerase from the archaean species Pyrococcus furiosus. This enzyme, called Pfu polymerase, is more accurate and stable but more expensive than Taq polymerase. 3- Two DNA primers, that are complementary to the 3' (three prime) ends of each of the sense and anti-sense strands of the DNA target (DNA polymerase can only bind to and elongate from a double-stranded region of DNA; without primers, there is no double-stranded initiation site at which the polymerase can bind). Forward and reverse primers. Primers are single-stranded DNAs formed of oligonucleotides. The size of the primers is usually between 10 and 30 nucleotides. Specific primers that are complementary to the DNA target region are selected beforehand, and are often synthesized chemically. 4- Deoxynucleoside triphosphates, or dNTPs (sometimes called "deoxynucleotide triphosphates"; nucleotides containing triphosphate groups), the building blocks from which the DNA polymerase synthesizes a new DNA strand 5- a buffer solution providing a suitable chemical environment for optimum activity and stability of the DNA polymerase. 6- Bivalent cations; Typically magnesium (Mg) or manganese (Mn) ions act as co-factors for enzyme activity; Mg2+ is the most common, but Mn2+ can be used for PCRmediated DNA mutagenesis, as a higher Mn2+ concentration increases the error rate during DNA synthesis. The reaction is commonly carried out in a volume of 10 - 200 μL in small reaction tubes (0.2–0.5 mL volumes) in a thermal cycler. History: A very early PCR machine which, rather than cycling through different temperatures, uses three different water baths at constant temperatures between which samples are moved with a robotic arm The thermal cycler (also known as a thermocycler, PCR machine or DNA amplifier) is a laboratory apparatus most commonly used to amplify segments of DNA via the polymerase chain reaction (PCR).  The thermal cycler heats and cools the reaction tubes to achieve the temperatures required at each step of the reaction. Many modern thermal cyclers make use of the Peltier effect, which permits both heating and cooling of the block holding the PCR tubes simply by reversing the electric current. Thin-walled reaction tubes permit favorable thermal conductivity to allow for rapid thermal equilibrium. Most thermal cyclers have heated lids to prevent condensation at the top of the reaction tube. The user interface of a modern thermal cycler Steps of PCR… In the PCR procedure, a three-step cycle causes a chain reaction that produces an exponentially growing population of identical DNA molecules. During each cycle, the reaction mixture is: 1Heated to high temperatures to Denature (Separate) the strands of the double- stranded DNA and then 2Cooled to allow Annealing (Hydrogen bonding) of short, single-stranded DNA primers complementary to sequences on opposite strands at each end of the target sequence; 3Finally, a special DNA polymerase Extends the primers in the 5ʹ → 3ʹ direction. This cycle is then repeated 30-40 times.  If a standard DNA polymerase were used, this enzyme would be denatured along with the DNA during the first heating step and would have to be replaced after Each cycle, The key to automating PCR was the discovery of an unusual heat-stable DNA polymerase enzyme called Taq polymerase Master Mix (PCR): A master mix is a mixture containing precursors and enzymes used as an ingredient PCR techniques. Such mixtures contain a mixture dNTPs, MgCl2, Taq polymerase, a pH buffer and come mixed in nucleasefree water. Master mixes for real-time PCR include a fluorescent compound (frequently SYBR green or flurochrome). ANALYSIS OF PCR PRODUCT There are many ways to analyse PCR products are: 1- Staining of amplified DNA product with a chemical dye like ethidium bromide14. 2- Labelling of PCR primers and nucleotides with fluorophores before PCR amplification14. SYBR Green may also be used instead ethidium bromide. It has the advantage so that it can be used with various pairs of different primers, it also costs less expensive than a probe. 3- Agarose Gel Electrophoresis, Agarose gel consists of 0.9% agarose in 40mM tris- base pH-8.3, 20mM acetic acid, 1mM purified from 0.9% agarose gels using QIA (quick gel extraction kit).  After gel electrophoresis technique, gel is soaked in a buffer containing a die that specifically stains DNA After the compilation of all the 3 process, it is visualized b UV transillumination. If the bands is present, it indicates the target sequence of original DNA sample and absence of any bands indicate the absence of original DNA sample. 29