Polymerase chain reaction (PCR) Subject: Molecular Biology Presented by: yogesh patel. 1st M.Pharmacy Department of pharmacology Oxford college of pharmacy

Contents: History Introduction Principle Procedure Example Verification by gel electrophoresis PCR optimization Application

History: The invention of PCR in 1983is generally credited to ‘Kery Mullis’. He was awarded the Nobel Prize in chemistry in 1993 for this invention. The discovery in 1976 of Taq- Polymerase – a DNA polymerase purified from the thermophilic bacterium, Thermus aquaticus, which naturally lives in hot condition (50- 80 ºC).

Introduction: 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 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 reaction mixtures. For that process thermal cyclers are used.

Continued…(introduction) Thermal cycler

Continued…(introduction) Primers (short DNA fragments) and DNA polymerase are key components to enable selective and repeated amplification. As Polymerase chain reaction progresses, the DNA generated is itself used as a template for replication, setting in a motion a chain reaction in which the DNA template is exponentially amplified. Almost all polymerase chain reaction application employ a heat stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the bacterium Thermus aquaticus. This DNA polymerase enzymatically assembles the nucleotides, single stranded DNA as a template and DNA primers, which are required for initiation of DNA synthesis.

Continued…(introduction)

Continued…(introduction) The majority of polymerase chain reaction methods use ‘thermal cycling’, i.e. alternately heating and cooling the PCR samples 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 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.

Principle: The purpose of PCR is to make a huge number of copies of genes. This is necessary to have enough starting template for sequencing. PCR is used to amplify a specific region of a DNA strand. Most of the PCR methods typically amplify DNA fragments up to 10 kb.

Continued…(principle) A basic set up of PCR requires several components & reagents: DNA template Two primers Taq polymerase or another DNA polymerase Deoxynucleotide triphosphate Buffer solution

Continued…(principle) The PCR is commonly carried out in a reaction volume of 10- 200 µl in small reaction tubes( 0.2- 0.5 ml) in a thermal cycler heats and cools the reaction tubes to achieve the temperature required at each step of the reaction. The cycling reaction There are three major steps in a PCR, which are repeated for 30 or 40 cycles. This is done on an automated cycler, which can heat and cool the tubes with the reaction mixture in a very short time.

Continued…(principle) PCR tubes

Continued…(principle) The cycling process is having following steps: Denaturation Annealing Extension

Denaturation: During the denaturation step the temperature is maintained at 94ºC. Here the double stranded DNA melts open to single stranded DNA, all enzymatic reactions stop during this period.

Annealing: During this step the temperature is maintained at 54ºC. The primers are jiggling around.Ionic bonds are constantly formed and broken between the single stranded primer and the stranded template, the polymerase can attach and starts copying the template. Once there are a few bases built in, the ionic bond is so strong between the template and the primer, that it does not break anymore.

Extension: During this step the temperature is maintained at 72˚C. This is the ideal working temperature for the polymerase. The primers, where there are a few bases built in, already have a stronger ionic attraction to the template than the forces breaking these attractions. Primers that are on positions with no exact match get loose again and do not give an extension of the fragment. The bases are coupled to the primers on the 3ˈ side.

Procedure: Typically, PCR consists of a series of 30- 40 repeated temperature changes called, cycles, with each cycle commonly consists of 3 different temperature steps. The cycling is often preceded by a single temperature step at a high temperature, and followed by one hold at the end for final product extension. The temperatures used and the length of time they are applied in each cycle depend on a variety of parameters like, enzyme used for DNA synthesis, the concentration of dinucleotide triphosphate in the reaction, and the melting temperature of the primers.

Steps of PCR: (procedure) Initialization step Denaturation step Annealing step Extension step Final elongation Final hold

Continued…(procedure) Initialization step: This step consists of heating the reaction to the temperature of 94 - 96˚C. This is held for 1 -9 minutes. It is only required for DNA polymerase that require heat activation. Denaturation step: This step is the first regular cycling event and consists of heating the reaction to 94 – 98˚C for 20- 30 seconds. It causes DNA melting of the DNA template by disrupting the hydrogen bonds between complementary bases, yielding single stranded DNA molecules.

Continued…(procedure) Annealing step: The reaction temperature is lowered to 55- 65˚C for 20 – 40 seconds allowing annealing of the primers to the single stranded DNA template. Stable DNA- DNA hydrogen bonds are only formed when the primer sequences very closely matches the template sequences. The polymerase binds to the primers- template hybrid and begins DNA synthesis using dinucleotide triphosphates.

Continued…(procedure) Extension step: The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 75 - 80˚C, and commonly a temperature of 72˚C is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTP that are complementary to the template in 5ˈ to 3ˈdirection. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified.

Continued…(procedure) Final elongation: This single step is occasionally performed at a temperature of 70-74ºC for 5-15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended. Final hold: This step at 4-15ºC for an indefinite time may be employed for short-term storage of the reaction.

Example: The times and temperatures given in this example are taken from a PCR program that was successfully used on a fragment of insulin-like growth factor (IGF). The reaction mixture consists of • 1.0 µl DNA template (100 ng/µl) • 2.5 µl of primer, 1.25 µl per primer (100 ng/µl) • 1.0 µl Pfu-Polymerase (extracted from pyrococcus furiosus) • 1.0 µl nucleotides • 5.0 µl buffer solution • 89.5 µl water A 200 µl reaction tube containing the 100 µl mixture is inserted into the thermal cycler.

Procedure(example) Denaturation Annealing Elongation Initialization (96ºC, 5 mins) Melting (96ºC,30 sec) Annealing 68ºC, 30 sec Primers+stands attaches Elongation 72ºC, 45 sec, Extension Final elongation & final hold (7ºC)

Verification: Verification of the completion of the process: The PCR product can be identified by its size using Agarose gel electrophoresis. Here, the smaller DNA strands move faster than the larger strands through the gel towards the positive current. The size of the PCR product can be determined by comparing it with a DNA ladder, which contains DNA fragments of known size, also within the gel.

PCR Optimization: Separate area for each step. Laminar flow cabinet for preparation of reaction mixture. Fresh gloves for each step. Separate reagents only for PCR.

Applications: Detecting mutations: The detection of hereditary diseases in a given genome is a long and difficult process, which can be shortened significantly by using PCR. Each gene in question can easily be amplified through PCR by using the appropriate primers and then sequenced to detect mutations. E.g. Retinoblastoma-a childhood cancer of eye. caused by mutation in a gene q14 on chromosome 13. Heredity involvement can be checked by using PCR and sequencing to analyze mutations in tumor tissue and normal tissue.

Continued… (applications) Monitoring cancer therapy: The ability to detect genetic lesions characteristic of tumor cells is a valuable tool to determine a patient being treated for leukemia is free of malignant cells. E.g.in follicular lymphoma there is translocation in chromosomes 14 and 18.

Continued…(applications) To detect bacterial and viral infections: In Mycobacterium tuberculosis, PCR amplification has been performed using primers for a sequence within a gene that is highly conserved in all mycobacterium species.

Continued…(applications) Sex determination of prenatal cells: For inherited X-linked disorder that affects only males, sex determination is the first step in prenatal diagnosis. Male sex determination using DNA is possible because males carry unique sequences on Y chromosome. PCR techniques can be used to amplify a 149-bp fragment on Y chromosome, specific for males.

Continued…(applications) Genetic fingerprinting: It is a forensic technique used to identify a person by comparing his or her DNA with a given sample, such as blood from a crime scene can be genetically compared to blood from a suspect. The sample may contain only a tiny amount of DNA, obtained from a source such as blood, semen, saliva, hair, or other organic material.

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