PCR lecture.ppt
- 1. In Vitro Amplification of DNA by PCR Arif Ullah Ph.D. Zoology Presented to: Dr. Dil Ara Abbas Bukhari Course title: Molecular Biology Applied
- 2. Contents Introduction PCR components PCR steps ◦ Denaturation ◦ Annealing ◦ Extension PCR types ◦ Nested PCR ◦ RT-PCR
- 3. Polymerase Chain Reaction (PCR) Technique widely used in molecular biology to make multiple copies of a specific DNA segment. In vitro technique for amplification of a region of DNA whose sequence is known or which lies between two regions of known sequence.
- 4. Short history of PCR 1983: Dr. Kary Mullis developed PCR 1985: First publication of PCR by Cetus Corporation appears in Science. 1986: Purified Taq polymerase first used in PCR 1988: PerkinElmer introduces the automated thermal cycler. 1993: Dr. Kary Mullis shares Nobel Prize in Chemistry for conceiving PCR technology.
- 5. Application PCR is molecular technique to amplify segment of DNA Used in clinical and research laboratories for a broad range of applications ◦ Diagnosis of genetic diseases ◦ Genetic fingerprints ◦ Detection and diagnosis of infectious diseases ◦ Detection of infection in the environment
- 6. Principles of PCR Based on DNA replication in vivo DNA is unwound to single strand, duplicated, rewound Amplify DNA in short period of time Amplify DNA fragment between 0.1 to 10kbp Some allow to amplify up to 40 kbp
- 7. Basic requirements for PCR reaction A DNA template: DNA target region to amplify. Size of template can be <0.1 to few kilobase Total amount of DNA for PCR is 0.05-0.1ug A DNA polymerase: An enzyme that polymerizes new DNA strands; heat-resistant Taq polymerase is especially common, as it is more likely to remain intact during the high-temperature DNA denaturation process. Primers: 16-30 nucleotides long primers are used Complementary to the 3' ends of each of the sense and anti-sense strands of the DNA target; without primers there is no double-stranded initiation site at which the polymerase can bind
- 8. Cont… Deoxynucleotide triphosphates (dNTPs): Building blocks to synthesizes a new DNA strand. Reaction Buffer: Providing a suitable chemical environment for optimum activity and stability of the DNA polymerase. Bivalent cations: Mg++ Mg ion stimulate polymerase activity Increase melting temperature of primer Monovalent cations: Potassium (K) ions
- 9. PCR COMPONENTS Water 10x reaction buffer MgCl2 dNTPs Target DNA Forward primer Reverse primer Polymerase enzyme
- 10. Dream Taq Green PCR Master Mix Optimum PCR components are directly used in form of mastermix Contain 1. Dream Taq DNA polymerase 2. Optimized Dream Taq Green buffer 3. MgCl2 4. dNTPs Higher yields compare to conventional Taq DNA polymerase
- 11. Significance of master mix Direct loading of PCR prodcuts on gel High yield and high sensitivity of PCR Amplification of long targets upto 6kb from genomic DNA and up to 20 kb from viral DNA Incorporate modified nucleotides, but doesnot incorporate dUTP
- 12. Procedure of PCR Carried in reaction volume of 10-200ul in small tubes called PCR tubes PCR tubes 0.2-0.5mL Placed in thermocycler
- 13. Main steps Consists of series of 20-40 repeated temperature changes called cycles If 100% efficiency is assumed in each cycle there will be 220 fold amplification after 20 cycle of pcr
- 14. Steps in PCR Steps Temp. Duration Denaturation 92°C to 95°C 1min Annealing 50°C to 55°C 45sec Elongation 70°C to 75°C 1-2min
- 15. Steps in PCR reaction
- 20. Visualization of PCR products Gel electrophoresis is employed to check pcr products Visualized under x-rays Size is determined by comparing with ladder.
- 21. Types of PCR Allele-specific PCR Assembly PCR Asymmetric PCR Convective PCR Dial-out PCR Digital PCR (d PCR) Hot start PCR In silico PCR (digital PCR, virtual PCR, electronic PCR, e-PCR)
- 22. Cont… Intersequence-specific PCR (ISSR) Methylation-specific PCR (MSP) Multiplex-PCR Nanoparticle-Assisted PCR (nanoPCR) Nested PCR quantitative PCR (qPCR) Reverse Transcription PCR (RT-PCR) Touchdown PCR (Step-down PCR)
- 23. Allele-specific PCR A PCR application in which alleles that differ by one or more nucleotides can be distinguished on the basis of PCR amplification. It permits the detection of any mutation in human DNA by analysing the PCR products directly in an ethidium bromide-stained agarose or polyacrylamide gel. Used in DNA-based diagnostic techniques involving the diagnosis of genetic and infectious diseases.
- 24. Hot start PCR Modified form of PCR that reduces non- specific amplification during the initial set up stages of the PCR. It may be performed manually by heating the reaction components to the denaturation temperature (e.g., 95 °C) before adding the polymerase.
- 25. Multiplex-PCR Consists of multiple primer sets within a single PCR mixture to produce amplicons of varying sizes that are specific to different DNA sequences. Annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction.
- 26. Nested PCR Increases the specificity of DNA amplification, by reducing background due to non-specific amplification of DNA. Two sets of primers are used in two successive PCRs: In the first reaction, one pair of primers is used to generate DNA products, which besides the intended target, may still consist of non-specifically amplified DNA fragments.
- 27. Cont… The product(s) are then used in a second PCR with a set of primers whose binding sites are completely or partially different from and located 3' of each of the primers used in the first reaction. Nested PCR is often more successful in specifically amplifying long DNA fragments than conventional PCR, but it requires more detailed knowledge of the target sequences.
- 28. Quantitative PCR (qPCR)/Real Time PCR Used to measure the quantity of a target sequence (commonly in real-time). It quantitatively measures starting amounts of DNA, cDNA, or RNA. quantitative PCR is commonly used to determine whether a DNA sequence is present in a sample and the number of its copies in the sample. Quantitative PCR has a very high degree of precision.
- 29. Reverse transcription PCR (RT-PCR) It is used for amplifying DNA from RNA. Reverse transcriptase reverse transcribes RNA into cDNA, which is then amplified by PCR. RT-PCR is widely used in expression profiling, to determine the expression of a gene or to identify the sequence of RNA transcript.
- 30. Touchdown PCR (step-down PCR) A variant of PCR that aims to reduce nonspecific background by gradually lowering the annealing temperature as PCR cycling progresses. The annealing temperature at the initial cycles is usually a few degrees (3–5 °C) above the Tm of the primers used, while at the later cycles, it is a few degrees (3–5 °C) below the primer Tm. The higher temperatures give greater specificity for primer binding, and the lower temperatures permit more efficient
- 31. heated lids adjustable ramping times single/multiple blocks gradient thermocycler blocks Thermocyclers
- 32. Detection of amplification products Gel electrophoresis Sequencing of amplified fragment Southern blot
- 33. Advantages of PCR Fairly simple to understand and to use. Produce Automated, fast, reliable results. Highly sensitive. Potential to produce millions to billions of copies of a specific product for sequencing, cloning, and analysis. Broad uses. Defined, easy to follow protocol.
- 34. Limitations of PCR Smallest amount of contaminated DNA can be amplified, resulting in misleading or ambiguous results. Need for target DNA sequence information Boundary regions of DNA to be amplified must be known. Infidelity of DNA replication: Taq Pol – no Proof reading– Error 40% after 20 cycles
- 35. Cont… Short size and limiting amounts of PCR product Up to 5kb can be easily amplified . Up to 40kb can be amplified with some modifications. Cannot amplify gene >100kb Cannot be used in genome sequencing projects.
- 36. Things to try if PCR does not work A) If no product ( of correct size ) produced: Check DNA quality. Reduce annealing temperature. Increase magnesium concentration. Add dimethyl sulphoxide ( DMSO ) to assay ( at around 10% ). Use different thermostable enzyme. Throw out primers - make new stocks.
- 37. Cont… B) If extra spurious product bands present: Increase annealing temperature Reduce magnesium concentration Reduce number of cycles Try different enzyme
- 38. Applications of PCR Common applications of PCR in various fields can be explained in following categories: Medical Applications Infectious disease Applications Forensic Applications Research and Molecular Genetics
- 39. Forensic applications Can be used as a tool in genetic fingerprinting. This technology can identify any one person from millions of others in case of: •crime scene. •rule out suspects during police investigation. •paternity testing even in case of availability of very small amount of specimens (stains of blood, semen, hair etc.).
- 40. Research and molecular genetics In genomic studies: to compare the genomes of two organisms and identify the difference between them. In phylogenetic analysis: minute quantities of DNA from any source like a fossilized material, hair, bones, mummified tissues. In study of gene expression analysis, PCR based mutagenesis. In Human genome project for aim to complete mapping and understanding of all genes of human beings.
- 41. Conclusion PCR is not only vital in the clinical laboratory by amplifying small amounts of DNA for STD detection, but it is also important for genetic predisposing for defects. The PCR technology can also be employed in law enforcement, genetic testing of animal stocks and vegetable hybrids, and drug screening along with many more areas.
- 42. References Molecular Cell Biology ( Lodish, Darnell..) https://sciencebasedmedicine.org/wp- content/uploads/2011/09/nested-pcr.gif http://11e.devbio.com/ch/03/wt/031005/figure1. jpg https://www.thermofisher.com/pk/en/home/bran ds/thermo-scientific/molecular- biology/molecular-biology-learning- center/molecular-biology-resource- library/basic-principles-rt-qpcr.html Paul, N. (2010). Hot start PCR. Methods in Molecular Biology. 630. pp. 301–318 Fundamentals of Biochem ( Voet, Voet, Pratt)