Polymerase Chain Reaction PCR

Transcription

1 1

2 Description of Module Subject Name Paper Name Module Name/Title Dr. Vijaya Khader Dr. MC Varadaraj 2

3 1. Objectives 1. To understand principle of 2. Types 3. Applications 2. Lay Out 3

4 Types of Qualitative Quantitative Qualitative RT- Nested Multiplex Touchdown Hotstart Quantitative Real Time 3. History was invented in 1984 by American Biochemist Kary mullis & he received the Nobel Prize in chemistry in 1993, for his invention. 4

5 It revolutionized biological methods specially in molecular cloning in a way that it has became an inseparable & irreplaceable part of molecular investigations. Introduction Nowadays, advances and applications of research in biochemistry and genetic play an important role in the field of health sciences. This has become necessary a molecular approach of the disease for a better interpretation of processes and as horizon in the development of new diagnostic and therapeutic strategies. Therefore, techniques in molecular biology have modified diagnosis, prevention and control of diseases in living beings. Molecular technology has become a crucial tool for identifying new genes with importance in medicine, agriculture, animal production and health, environment and the industry related to these areas. Among the applications of molecular techniques is important to highlight the use of the () in the identification and characterization of viral, bacterial, parasitic and fungal agents It is Used to amplify specific sequences simply and quickly by a direct enzymatic process. amplification requires a small amount of nucleotide sequence at each end of the region to be amplified Oligonucleotides complementary to that sequence are synthesized, typically nucleotides long. These oligonucleotides are used as primers for enzymatic amplification. A reaction mixture is set up containing a sample of DNA that includes the region to be amplified, the primers in large molar excess, deoxynucleoside triphosphates (dntps) and a heat-stable DNA polymerase. The most common enzyme for this purpose is the Taq polymerase, which is a DNA polymerase isolated from the thermophilic bacterium Thermus aquaticus, which can be grown routinely in the laboratory at 75 o C or more. 4. Principle of Denaturation Annealing Extension Final Extension The mixture is heated to a temperature sufficient to denature the sample DNA. After this it is cooled to a temperature low enough for the primers to anneal to the DNA. It is possible for the sample DNA simply to self-anneal, but this is rendered less likely by the large molar excess of the primers. 5

6 The mix is then incubated at a temperature sufficient for the polymerase to synthesize a complementary strand to each piece of sample DNA, starting with the primers and using the dntps provided. Components of a DNA template that contains the DNA region (target) to amplify Two primers that are complementary to the 3' (three prime) ends of each of the sense and anti-sense strand of the DNA target Taq polymerase or another DNA polymerase with a temperature optimum at around 70 C Deoxynucleoside triphosphates (dntps, sometimes called "deoxynucleotide triphosphates"; nucleotides containing triphosphate groups), the building-blocks from which the DNA polymerase synthesizes a new DNA strand Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase Bivalent cations, magnesium or manganese ions; generally Mg 2+ is used, but Mn 2+ can be used for mediated DNA mutagenesis, as higher Mn 2+ concentration increases the error rate during DNA synthesis Monovalent cation potassium ions Stages of Exponential amplification: At every cycle, the amount of product is doubled (assuming 100% reaction efficiency). The reaction is very sensitive: only minute quantities of DNA must be present. Leveling off stage: The reaction slows as the DNA polymerase loses activity and as consumption of reagents such as dntps and primers causes them to become limiting. Plateau: No more product accumulates due to exhaustion of reagents and enzyme 6

7 Figure 1. Diagrammatic representation of 7

8 A typical set of reactions might have an initial melting carried out for 5 min at 94 0 C followed by approx. 30 cycles each comprising melting for 1 min at 94 0 C renaturation for 1 min at approx C and DNA synthesis for 1.5 min at 72 0 Cnand then after the 30 cycles a final extended round of DNA synthesis for 10 min. This would result in a theoretical amplification of over fold. Figure 2. Thermal Cycler 8

9 Enzyme Used: Taq Polymerase The thermostability of the Taq enzyme helps in its purification after expression in E. coli, since contaminating E. coli proteins can be inactivated by heating. The enzyme has 5-3 DNA polymerase and 5-3 exonuclease activities. It polymerizes about nucleotides per second. Therefore extension time is given 1 min for 1kb Disadvantages of Taq Taq polymerase has no proof-reading (3-5 exonuclease) activity. Incorporates a wrong nucleotide in 10kb Taq polymerase has relatively low processivity, Amplifies only upto 5kb DNA Taq polymerase is not fully heat stable, It has a half-life of about 40 min at 95 o C, which means there will be significant loss of activity over the 30 or so cycles used in a typical experiment. Taq polymerase incorporates an extra A residue, This may help in cloning Other Polymerases Tfl and Tth enzymes from Thermus flavus and Thermus thermophilus respectively. These generally do not have 3-5 proof-reading activity. Proof-reading enzymes include Tli and Vent R from Thermococcus litoralis, Pfu from Pyrococcus furiosus, and DeepVent R from Pyrococcus sp. These marine bacteria generally grow at even higher temperatures than Thermus aquaticus, and the polymerases are more thermostable than the Taq enzyme Primer A primer is a strand of short nucleic acid sequences (generally about 10 base pairs) that serves as a starting point for DNA synthesis. 9

10 Primer Synthesis Length. A short primer may offer sufficient specificity when amplifying using a simple template such as a small plasmid but a long primer may be required when using eukaryotic genomic DNA as template. In practice, nucleotides is generally satisfactory. Mismatches. Primers do not need to match the template completely The 3 end of the primer should be correctly base-paired to the template, otherwise the polymerase will not be able to extend it. It is often beneficial to have C or G as the 3 terminal nucleotide. This makes the binding of the 3 end of the primer to the template more stable than it would be with A or T at the 3 end. Primer Designing Melting temperature. The temperatures at which the two primers can associate with the template should be relatively similar to ensure that they both bind at about the same time as temperatures are being lowered during annealing. The similarity of melting temperatures is likely to mean that the primers have a similar nucleotide composition. Tm = 2 (A+T) + 4 (C+G) Internal secondary structure. This should be avoided, or a primer may fold back on itself and not be available to bind to the template. As an intra-molecular reaction, self-annealing is likely to take place in preference to intermolecular annealing of the primer to the template. Primer-primer annealing. It is also important to avoid the two primers being able to anneal to each other. Extension by DNA polymerase of two self-annealed primers leads to formation of a primer 10

11 dimer. These will be very efficient templates for amplification in subsequent rounds of, as they are small. Degenerate primers Sometimes degenerate primers are used. These are actually mixtures of similar, but not identical primers. They may be convenient if the same gene is to be amplified from different organisms, as the genes themselves are probably similar but not identical. Applications DNA sequencing. Diagnostic. Amplified fragment length polymorphism (AFLP) Archeology & evolution RT- 5. Types of This is usually done by having a round of reverse transcription, using a reverse transcriptase enzyme and a single primer, to make a single strand of cdna prior to the itself. The primer for reverse transcription could be oligo-dt for general cdna synthesis from polyadenylated messages, or it could be specific to a particular message. 5.1 Nested Nested polymerase chain reaction (Nested ) is a modification of polymerase chain reaction intended to reduce non-specific binding in products due to the amplification of unexpected primer binding sites. Nested polymerase chain reaction involves two sets of primers, used in two successive runs of polymerase chain reaction, the second set intended to amplify a secondary target within the first run product. 11

12 Figure 3. Diagrammatic Representation of Nested 12

13 5.2 Multiplex Multiplex polymerase chain reaction (Multiplex ) refers to the use of polymerase chain reaction to amplify several different DNA sequences simultaneously (as if performing many separate reactions all together in one reaction). Figure 4. Diagrammatic Representation of Multiplex 13

14 5.3 Touchdown In this, a high annealing temperature is used initially (at which even correct binding may not be possible). The annealing temperature is reduced in subsequent rounds. There will, therefore, come a point at which correctly matched primer-template annealing is just possible, but incorrect matching is not. DNA synthesis can therefore start. Although later cycles may be under less stringent conditions, the early cycles will have been carried out under the most stringent conditions and the desired products will be the most abundant. Figure 5. Diagrammatic Representation of Touch Down 14

15 5.4 Hot Start As soon as the reagents have all been mixed together, it is possible for the DNA polymerase to start synthesis. This may happen while the reaction mixture is being heated for the first time, and is at a temperature low enough to allow non-specific annealing of primer to template, generating a range of non-specific products. This problem would be prevented if DNA synthesis could not take place until the first cycle had reached its maximum temperature. This is the basis of hot-start. In the simplest form, the DNA polymerase is not added to the reaction tubes until they have reached the DNA melting temperature of the first cycle Figure 6. Diagrammatic Representation of Hot-Start 15

16 6. Quantitative (Real Time ) There are two main approaches to estimating the abundance of a molecule. One is to use a standard and visualize the amount of the product of interest by gel electrophoresis, comparing it with suitable controls or standards. This is an end-point measurement of the amount of target sequence. The second approach is to quantify the reaction in real time (i.e. while the is in progress). 6.1 SYBR Green This can be done in two ways. In the first, a fluorescent, double-stranded DNA (dsdna)-binding dye (such as SYBR green) is present in the. As dsdna product accumulates, the amount of fluorescence from the dye increases, and this can be detected. The experiment requires a machine that is also equipped with a fluorescence measurement facility. This approach is adequate if the generates the product of interest very specifically. The method simply detects dsdna, it measures the amount of product at a given time regardless of whether it is from the correct region. 6.2 Quencher and reporter approach The second approach to real-time allows detection of a specific product, rather than dsdna in general, It uses a specially synthesized probe oligonucleotide. This probe is designed to anneal within the region to be amplified and carries a fluorescent reporter dye at one end and a quencher at the other end of the molecule. 16

17 If the quencher and the reporter are in close proximity (i.e. attached to the same oligonucleotide), then the quencher stops the reporter from fluorescing. During, the probe will anneal to single-stranded DNA within the target region. When the polymerase meets the annealed probe, the 5-3 exonuclease activity of the enzyme degrades the probe, liberating the reporter from the quencher. Thus, the fluorescent reporter accumulates during the course of the. 6.3 TaqMan TaqMan probes consist of a fluorophore covalently attached to the 5 -end of the oligonucleotide probe and a quencher at the 3 -end. Several different fluorophores (e.g. 6-carboxyfluorescein, acronym: FAM, or tetrachlorofluorescin, acronym: TET) and quenchers (e.g. tetramethylrhodamine, acronym: TAMRA, or dihydrocyclopyrroloindole tripeptide minor groove binder, acronym: MGB) are available. The quencher molecule quenches the fluorescence emitted by the fluorophore when excited by the cycler s light source via FRET (Fluorescence Resonance Energy Transfer) As long as the fluorophore and the quencher are in proximity, quenching inhibits any fluorescence signals TaqMan Denaturation and hybridization of probe. Extension of primer and strand displacement of probe. Cleavage of probe and fluorescence from the reporter dye. Fluorescence from reporter dye is directly proportional to the number of amplicons generated 17

18 Figure 7. TaqMan Approach 18

19 6.4 Molecular beacons Loop: This is the base pair region of the molecular beacon which is complementary to the target sequence. Stem: The beacon stem is formed by the attachment, to both termini of the loop, of two short (5 to 7 nucleotide residues) oligonucleotides that are complementary to each other. 5' fluorophore: At the 5' end of the molecular beacon, a fluorescent dye is covalently attached. 3' quencher (non fluorescent): The quencher dye is covalently attached to the 3' end of the molecular beacon. When the beacon is in closed loop shape, the quencher resides in proximity to the fluorophore, which results in quenching the fluorescent emission of the latter. Figure 8. Molecular Beacons If the nucleic acid to be detected is complementary to the strand in the loop, the event of hybridization occurs. The duplex formed between the nucleic acid and the loop is more stable than that of the stem because the former duplex involves more base pairs. This causes the separation of the stem and hence of the fluorophore and the quencher. Once the fluorophore is distantiated from the quencher, illumination of the hybrid with light results in the fluorescent emission. 19

20 The presence of the emission reports that the event of hybridization has occurred and hence the target nucleic acid sequence is present in the test sample. Baseline The baseline phase contains all the amplification that is below the level of detection of the real time instrument. Threshold where the threshold and the amplification plot intersect defines C T. Can be set manually/automatically C T (cycle threshold) the cycle number where the fluorescence passes the threshold R n (R n-baseline) NTC no template control DRn is plotted against cycle numbers to produce the amplification curves and gives the C T value. 20

21 Real Time in comparison with other Technical Methods 21