Polymerase chain reaction principles and practice
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The document provides an overview of Polymerase Chain Reaction (PCR) including its history, components, and various applications across fields such as genetics and forensic science. It discusses PCR optimization techniques such as primer design, cycling conditions, and specific types of PCR like real-time and nested PCR. The document also highlights the advantages and challenges of PCR in applications such as disease detection and genetic testing.
Polymerase chain reaction principles and practice
- 2. What is PCR?What is PCR? It was invented in 1983 by Dr. Kary Mullis, for which he received the Nobel Prize in Chemistry in 1993. PCR is an exponentially progressing synthesis of the defined target DNA sequences in vitro.
- 3. What is PCR? :What is PCR? : Why “Polymerase”?Why “Polymerase”? It is called “polymerase” because the only enzyme used in this reaction is DNA polymerase.
- 4. What is PCR? :What is PCR? : Why “Chain”?Why “Chain”? It is called “chain” because the products of the first reaction become substrates of the following one, and so on.
- 5. What is PCR? :What is PCR? : The “Reaction” ComponentsThe “Reaction” Components 1) Target DNA - contains the sequence to be amplified. 2) Pair of Primers - oligonucleotides that define the sequ to be amplified. 3) dNTPs - deoxynucleotidetriphosphates: DNA building 4) Thermostable DNA Polymerase - enzyme that catalyzes the reaction 5) Mg++ ions - cofactor of the enzyme 6) Buffer solution – maintains pH and ionic strength of the reaction solution suitable for the activity of the enzyme
- 7. Denature (heat to 95o C) Lower temperature to 56o C Anneal with primers Increase temperature to 72o C DNA polymerase + dNTPs
- 11. DNA copies vs Cycle number 0 500000 1000000 1500000 2000000 2500000 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Cycle number DNAcopies
- 12. PCR Reagents 1X Buffer 10mM Tris-HCl, 50mM KCl MgCl2 1mM - 4mM (1.5mM) dNTPs 200μM Primers 100nM-1μM, 200nm (or less) for real time analysis DNA polymerase Taq DNA polymerase is thermostable 1-4 Units (1 unit) DNA 10pg-1μg (20ng)
- 13. Applications of PCRApplications of PCR Classification of organisms Genotyping Molecular archaeology Mutagenesis Mutation detection Sequencing Cancer research Detection of pathogens DNA fingerprinting Drug discovery Genetic matching Genetic engineering Pre-natal diagnosis
- 14. Different types of buffers
- 16. Fragments of defined length PCR Melting 94 o C Melting 94 o C Annealing Primers 50 o C Extension 72 o CTemperature 100 0 50 T i m e 30x 3’5’ 5’3’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’
- 17. Number of cycles 0 10 15 20 25 30 Size Marker
- 18. PCR Optimisation 1: Buffers Most buffers have only KCl (50mM) and Tris (10mM) Concentrations of these can be altered KCl facilitates primer binding but concentrations higher than 50mM inhibit Taq DMSO, BSA, gelatin, glycerol, Tween-20, Nonidet P-40, Triton X-100 can be added to aid in the PCR reaction Enhance specificity, but also can be inhibitory Pre-mixed buffers are available
- 19. PCR Optimisation 2: MgCl2 MgCl2: required for primer binding MgCl2 affects primer binding, Tm of template DNA, product- and primer-template associations, product specificity, enzyme activity and fidelity dNTPs, primers and template chelate and sequester the Mg ion, therefore concentration should be higher than dNTPs (as these are the most concentrated) Excess magnesium gives non-specific binding Too little magnesium gives reduced yield
- 20. PCR Optimisation 3: Primer Design Specific to sequence of interest Length 18-30 nucleotides Annealing temperature 50o C-70o C Ideally 58o C-63o C GC content 40-60% 3’ end critical (new strand extends from here) GC clamp (G or C at 3’ terminus) Inner self complementarity: Hairpins <5, dimers <9 3’ complementarity: <3-4 bases similar to other primer regions
- 21. PCR Optimisation 4: Cycling Conditions Denaturation: Some Taq polymerases require initial denaturation (hot start) Annealing temperature: ~ 5o C less than Tm of primers Tm = 4(G + C) + 2(A + T)o C (or use of primer software) Decrease in annealing temperature result in non-specific binding Increase in annealing temperature result in reduced yield
- 22. PCR Optimisation 5: Cycle Number 25-40 cycles Half-life of Taq is 30 minutes at 95o C Therefore if you use more than 30 cycles at denaturation times of 1 minute, the Taq will not be very efficient at this point Theoretical yield = 2n ie. cycle 1 = 2, cycle 2 = 4, cycle 3 = 8, etc eg. if you start with 100 copies after 30 cycles you will have 107, 374, 182, 400 copies
- 23. In summary Primer length should not exceed 30 mer. Tm, not more than 60 degree . GC Content should be in the range of 40-60 % for optimum PCR efficiency. Primers should end (3′) in a G or C, or CG or GC: this prevents “breathing” of ends and increases efficiency of priming.
- 25. Primer Problems primers should flank the sequence of interest primer sequences should be unique primers that match multiple sequences will give multiple products repeated sequences can be amplified - but only if unique flanking regions can be found where primers can bind
- 26. Sequence Specific Oligonucleotide (SSO) probe Amplified fragment-length polymorphism to generate finger prints Large VNTR regions (10-30 b.p. repeat) Short Tandem Repeats (STR) (2-7 b.p. repeat) RAPD using universal primers Rep- PCR (ERIC primers) PCR- Ribotyping (16S rDNA regions) PCR Based Methods
- 27. Variations of the PCR Colony PCR Nested PCR Multiplex PCR AFLP PCR Hot Start PCR In Situ PCR Inverse PCR Asymmetric PCR Long PCR Long Accurate PCR Reverse Transcriptase PCR Allele specific PCR Real time PCR
- 28. Types of PCR Long PCR: Used to amplify DNA over the entire length up to 25kb of genomic DNA segments cloned. Nested PCR: Involves two consecutive PCR reactions of 25 cycles. The first PCR uses primers external to the sequence of interest. The second PCR uses the product of the first PCR in conjunction with one or more nested primers to amplify the sequence within the region flanked by the initial set of primers. Inverse PCR: Used to amplify DNA of unknown sequence that is adjacent to known DNA sequence. Quantitative PCR: Product amplification w r t time, which is compared with a standard DNA. Hot start PCR: Used to optimize the yield of the desired amplified product in PCR and simultaneously to suppress nonspecific amplification.
- 29. Colony PCR Colony PCR- the screening of bacterial (E.Coli) or yeast clones for correct ligation or plasmid products. Pick a bacterial colony with an autoclaved toothpick, swirl it into 25 μl of TE autoclaved dH2O in an microfuge tube. Heat the mix in a boiling water bath (90-100C) for 2 minutes Spin sample for 2 minutes high speed in centrifuge. Transfer 20 μl of the supernatant into a new microfuge tube Take 1-2 μl of the supernatant as template in a 25 μl PCR standard PCR reaction.
- 30. Hot Start PCR This is a technique that reduces non-specific amplification during the initial set up stages of the PCR The technique may be performed manually by heating the reaction components to the melting temperature (e.g., 95°C) before adding the polymerase DNA Polymerase- Eubacterial type I DNA polymerase, Pfu These thermophilic DNA polymerases show a very small polymerase activity at room temperature.
- 31. Nested PCR Two pairs (instead of one pair) of PCR primers are used to amplify a fragment. First pair -amplify a fragment similar to a standard PCR. Second pair of primers-nested primers (as they lie / are nested within the first fragment) bind inside the first PCR product fragment to allow amplification of a second PCR product which is shorter than the first one. Advantage- Very low probability of nonspecific amplification
- 32. Multiplex PCR • Multiplex PCR is a variant of PCR which enabling simultaneous amplification of many targets of interest in one reaction by using more than one pair of primers.
- 33. Inverse PCR Inverse PCR (Ochman et al., 1988) uses standard PCR (polymerase chain reaction)- primers oriented in the reverse direction of the usual orientation. The template for the reverse primers is a restriction fragment that has been selfligated Inverse PCR functions to clone sequences flanking a known sequence. Flanking DNA sequences are digested and then ligated to generate circular DNA. Application Amplification and identification of flanking sequences such as transposable elements, and the identification of genomic inserts.
- 34. Long PCR Extended or longer than standard PCR, meaning over 5 kilobases (frequently over 10 kb). Long PCR is useful only if it is accurate. Thus, special mixtures of proficient polymerases along with accurate polymerases such as Pfu are often mixed together. Application- to clone large genes
- 35. Reverse Transcriptase PCR Based on the process of reverse transcription, which reverse transcribes RNA into DNA and was initially isolated from retroviruses. First step of RT-PCR - "first strand reaction“- Synthesis of cDNA using oligo dT primers (37°C) 1 hr. “Second strand reaction“-Digestion of cDNA:RNA hybrid (RNaseH)-Standard PCR with DNA oligo primers. Allows the detection of even rare or low copy mRNA sequences by amplifying its
- 36. Why real time PCR ? • QUANTITATION OF mRNA – northern blotting – ribonuclease protection assay – in situ hybridization – RT-PCR • most sensitive • can discriminate closely related mRNAs • technically simple • but difficult to get truly quantitative results using conventional PCR
- 37. Real-Time PCRReal-Time PCR Real-time PCR monitors the fluorescence emitted during the reaction as an indicator of amplicon production at each PCR cycle (in real time) as opposed to the endpoint detection
- 38. Traditional PCR has advanced from detection at the end-point of the reaction to detection while the reaction is occurring (Real-Time). Real-time PCR uses a fluorescent reporter signal to measure the amount of amplicon as it is generated. This kinetic PCR allows for data collection after each cycle of PCR instead of only at the end of the 20 to 40 cycles.
- 39. Real-time PCR advantagesReal-time PCR advantages * amplification can be monitored real-time * no post-PCR processing of products (high throughput, low contamination risk) * ultra-rapid cycling (30 minutes to 2 hours) * wider dynamic range of up to 1010 -fold * requirement of 1000-fold less RNA than conventional assays (6 picogram = one diploid genome equivalent) * detection is capable down to a two-fold change * confirmation of specific amplification by melting curve analysis * most specific, sensitive and reproducible * not much more expensive than conventional PCR (except equipment cost)
- 40. Real-time PCR disadvantagesReal-time PCR disadvantages * Not ideal for multiplexing * setting up requires high technical skill and support * high equipment cost * intra- and inter-assay variation * RNA liability * DNA contamination (in mRNA analysis)
- 41. Applications of PCRApplications of PCR Classification of organisms Genotyping Molecular archaeology Mutagenesis Mutation detection Sequencing Cancer research Detection of pathogens DNA fingerprinting Drug discovery Genetic matching Genetic engineering Pre-natal diagnosis
- 42. PCR Virtues High sensitivity Can detect and quantify specific events Higher stability of DNA permits analysis of food samples. Quantitative and qualitative
- 43. Some applications of PCR. Forensic medicine. Preimplantation Genetic Diagnosis (PGD). Archeology. Paternity testing.
- 44. Forensic uses of PCR PCR can be used to amplify DNA from a small amount of cells (about 1000 cells). The amplified DNA from cells can be used in DNA fingerprinting analysis to determine who was at the crime scene.
- 45. DNA fingerprinting using PCR in forensic investigations. DNA is isolated from blood at a crime scene and amplified by PCR. The amplified DNA is digested with restriction enzymes and resolved on an agarose gel. Southern blot analysis is performed to give a DNA fingerprint.
- 46. How reliable is DNA fingerprinting? DNA regions chosen are ones known to be highly variable from one person to another. In most forensic cases, the probability of two people having identical DNA fingerprints is between one chance in 100,000 and one in a billion. The exact number depends on the number of probes used to different regions of human chromosomal DNA.
- 47. Satellite DNA can be used as markers for DNA fingerprinting. Satellite DNA consists of tandemly repeated base sequences within the human genome. The most useful satellite DNA for forensic purposes are microsatellites having repeating units of only a few base pairs, and the number of repeats are highly variable from one person to another. Microsatellite DNA is also called a simple tandem repeats (STRs).
- 48. STRs in DNA fingerprinting. The greater the number of STRs analyzed in a DNA sample, the more likely the DNA fingerprint is unique to an individual. PCR is used to selectively amplify particular STRs before electrophoresis. PCR is especially valuable when DNA is in poor condition or available in minute quantities.
- 49. PCR use in Pre-implantation Genetic Diagnosis (PGD). PGD is a way to determine if human embryos from in vitro fertilization have genetic defects (for example, cystic fibrosis). A cell is removed from an eight cell embryo and the DNA is analyzed by PCR for genetic defects. Only healthy embryos are implanted into a mother’s uterus. Should this technology be used for things like gender selection?
- 50. Thank you
Editor's Notes
- #37: Northern blotting and RPAS are the gold standards, since no amplification is involved. In situ hybridization is qualitative rather than quantitative. Techniques such as Northern blotting and ribonuclease protection assays (RPAs) work very well, but require more RNA than is sometimes available. PCR methods are particularly valuable when amounts of RNA are low, since the fact that PCR involves an amplification step means that it is more sensitive. However, traditional PCR is only semi-quantitative at best, in part because of the insensitivity of ethidium bromide (however, there are more sensitive ways to detect the product) and, in part, as we shall discuss later, because of the difficulties of observing the reaction during the truly linear part of the amplification process. Various competitive PCR protocols have been designed to overcome this problem but they tend to be cumbersome. Real-time PCR has been developed so that more accurate results can be obtained. An additional advantage of real-time PCR is the relative ease and convenience of use compared to some of these older methods (as long as one has access to a suitable real-time PCR machine).