MOLECULAR BIOLOGY AND DIAGNOSTICS LABORATORY POLYMERASE CHAIN REACTION OUTLINE • Polymerase Chain Reaction o Introduction o Principle of the test o Materials o Procedure • PowerPoint Notes o Polymerase Chain Reaction (PCR) o Components of PCR o PCR Steps/Procedure o Product of PCR (Amplicon POLYMERASE CHAIN REACTION (PCR) INTRODUCTION • Polymerase Chain Reaction is a technique used in the lab to make millions of copies of a particular section of DNA. Using PCR it is possible to generate thousands to millions of copies of a particular section of DNA from a very small amount of DNA. Polymerase Chain Reaction are used to the possible detection of pathogenic virus or bacteria, identification of individual DNA fingerprints and for purpose in research. PRINCIPLE OF THE TEST • Typically, the goal of PCR is to make enough of the target DNA region that it can be analyzed or used in some other way. The technique is used to amplify specific, target DNA fragments from low quantities of source DNA or RNA after a reverse transcription step to produce complementary DNA (cDNA). The three major steps in PCR are Denaturation, Annealing and Extension. MATERIALS • DNA Sample • Taq polymerase - aquaticus, Like DNA replication in an organism, PCR requires a DNA polymerase enzyme that makes new strands of DNA, using existing strands as templates. The DNA polymerase typically used in PCR is called Taq polymerase, after the heat-tolerant bacterium from which it was isolated (Thermus aquaticus) • PCR primers - Taq polymerase can only make DNA if it's given a primer, a short sequence of nucleotides that provides a starting point for DNA synthesis. In a PCR reaction, the experimenter determines the region of DNA that will be copied, or amplified, by the primers she or he chooses. PCR primers are short pieces of single-stranded DNA, usually around 202020 nucleotides in length. Two primers are used in each PCR reaction, and they are designed so that they flank the target region (region that should be copied). That is, they are given sequences that will make them bind to opposite strands of the template DNA, just at the edges of the region to be copied. o The primers bind to the template by complementary base pairing. • Template – for RNA and DNA Purification. • Nucleotide bases - (also known as dNTPs). DNA bases (A, C, G and T) are the building blocks of DNA and are needed to construct the new strand of DNA. PROCEDURE 1. Denaturation (96 °C): Heat the reaction strongly to separate, or denature, the DNA strands. This provides single-stranded template for the next step. 2. Annealing (55-65 °C) Cool the reaction so the primers can bind to their complementary sequences on the single-stranded template DNA. 3. Extension (72 °C): Raise the reaction temperatures so Taq polymerase extends the primers, synthesizing new strands of DNA. POLYMERASE CHAIN REACTION (PPT) POLYMERASE CHAIN REACTION (PCR) • Introduced by Kary Mullis • Target amplification method • Procedure/Steps: o Denaturation o Annealing o Extension COMPONENTS OF PCR • Templates to be copied • Primers • Nucleotides • Polymerase enzyme • Buffer components COMPONENTS OF A TYPICAL PCR REACTION Component Purpose 0.5 mM each primer (oligodeoxynucleotides) Directs DNA synthesis to the desired region 0.2 nM each dATP, dCTP, dGTP, dTTP Building blocks that extend the primers 50 mM KCl Monovalent cation (salt), for optimal hybridization of primers to template 10 mM Tris, pH 8.4 Buffer to maintain optimal pH for the enzyme reaction 1.5 mM MgCl 2 Divalent reaction, required by the enzyme 1.5 units polymerase The polymerase enzyme that extends the primers (adds dNTPs) 10 2 -10 5 copies of template Sample DNA that is being tested
PRIMERS • Critical component • Determine the specificity • Analogous to the probes • Contain sequences homologous to sites flanking the region • Forward primer must bind to the target DNA; Reverse primer must bind just 5′ to the sequence to be amplified on the opposite strand of the DNA • Dictate the size of the amplified product • The primer Tm can serve as a starting point for setting the optimal annealing temperature • aberrant primer binding, or mispriming, can occur in PCR. o Mispriming of one primer creates an unintended product that could interfere with subsequent interpretation. Mispriming can also occur in regions unrelated to the intended target sequence. • Primer dimers - result from the binding of primers onto each other through short (2 –3 base) homologies at their 3′ ends and the copying of each primer sequence o Formation of primer dimers occurs when there are three or more complementary bases at the 3’ end of the primers. With the primers in excess, these will bybridize during the annealing step (vertical lines), and the primers will be extended by the polymerase (dotted line) using the opposite primer as the template. The resulting product, denatured in the next cycle, will complete for primers with the intended template. DNA TEMPLATE • may be single- or double-stranded DNA • In a clinical sample: patient’s genomic or mitochondrial DNA or from viruses, bacteria, fungi, or parasites • best templates: o free of contaminating proteins o without nicks or breaks that can stop DNA synthesis DEOXYRIBONUCLEOTIDE BASES • An equimolar mixture of the four deoxynucleotidetriphosphates • Deoxynucleotidetriphosphates(dNTPs) is added DNA POLYMERASE • Taq polymerase o Thermostable enzyme o Isolated from the thermophilic bacterium, Thermus aquaticus. • Tth polymerase o From Thermus thermophiles o Has reverse transcriptase activity (reverse transcriptase PCR)-where the starting material is an RNA template PCR BUFFER • Provide the optimal conditions for • Potassium chloride (20 –100 mM), ammonium sulfate (15– 30 mM), or other salts of monovalent cations enzyme activity • ↑Salt concentration makes longer DNA products denature more slowly • Magnesium chloride also affects primer annealing and for enzyme activity • Tris buffer and accessory buffer components-for optimal enzyme activity and accurate amplification • Accessory components- used to optimize reactions o Bovine serum albumin o Dithiothreitol o Formamide o Chaotropic agents PCR STEPS/PROCEDURE • Step 1: Denaturation • Step 2: Annealing • Step 3: Extension • Elements of a PCR Cycle Step Temperature (°C) Time (sec) Denaturation 90-96 20-60 Annealing 50-70 20-90 Extension 68-75 10-60 STEP 1: DENATURATION • The double-stranded DNA → two single strands (in order to be replicated) • Accomplished by heating the sample at 94 –96C o Denaturation of the DNA target. The region to be amplified is shown in green. The primers are present in vast excess.
STEP 2: ANNEALING • Critical step for the specificity • The two oligonucleotides that will prime the synthesis of DNA anneal (hybridize) to complementary sequences on the template • The two oligonucleotides that will prime the synthesis of DNA anneal (hybridize) to complementary sequences on the template • Annealing temperatures will range 50 –70C o In the second step of the PCR cycle, annealing the primers hybridize to their complementary sequences on each strand of the denatured template. The primers are designed to hybridize to the sequences flanking the region of interest. STEP 3: EXTENSION • The polymerase synthesizes a copy of the template DNA by adding nucleotides to the hybridized primers. • DNA polymerase replicates the template DNA by simultaneously extending the primers on both strands of the template. • Occurs at the optimal temperature of the enzyme, 68 –72C. o DNA polymerase catalyzes addition of deoxynucleotide triphosphates (dNTPs) to the primers, using the sample DNA as a template. This completes one PCR cycle. Note how in the original template there was one copy of the green region. Now, after one cycle, there are two copies. PRODUCT OF PCR (AMPLICON) o The components and result of a PCR. Oligodeoxynucleotides (primers) are designed to hybridize to sequences flanking the DNA region under investigation. The polymerase extends the primers making many copies of the region flanked by the primer sequences, the PCR product. DISCUSSION NOTES • Taq - fluorescent o Q end - Quencher of fluorescence o E end - Reporter of fluorescence • Initial denaturation - initial unzipping • Fluorescence - directly proportional to virulence load o Released, Measured during PCR • DNA is compounded every cycle, increasing the amount of DNA • o Use tungsten-halogen lamp