POLYMERASE CHAIN REACTION29-07-2011 Presented by: Dr.Praveenkumar Doddamani Department of Microbiology M.R.Medical College,Gulbarga.
INTRODUCTION HISTORY WHT IS PCR? PRINCIPLE OF PCR EQUIPMENTS & ELEMENTS OF PCR STEPS OF PCR CYCLE VARIATIONS OF PCR ADVANTAGES OF PCR LIMITATIONS OF PCR APPLICATIONS OF PCR
INTRODUCTION PCR is powerful Method of In-vitro DNA synthesis. PCR has revolutionized molecular biology and is used in virtually every area of natural science and medicine . This technique has cut across the boundaries separating basic and applied research, commercial technology and
History 1983—Kary Mullis, a scientist working for the Cetus Corporation was driving along US Route 101 in northern California when he came up with the idea for the polymerase chain reaction 1985—the polymerase chain reaction was introduced to the scientific community at a conference in October.
Polymerase Chain ReactionMethodology: A Mile stone in MedicalHistory He had the idea to use a pair of primers to bracket the desired DNA sequence and to copy it using DNA polymerase, a technique which would allow a small strand of DNA to be copied almost an infinite number of times. Cetus took Mullis off his usual projects to concentrate on PCR full-time
Cetus rewarded Kary Mullis with a $10,000 bonus for his invention Later, during a corporate reorganization, Cetus sold the patent for the PCR process to a pharmaceutical company Hoffmann-LaRoche for $300 million. 1993 Kary Mullis got NOBEL PRIZE for chemistry.
Dr. Kary Mullis, wins Nobel Prize in1993 Kary received a Nobel Prize in chemistry in 1993, for his invention of the polymerase chain reaction (PCR). The process, which Kary Mullis conceptualized in 1983, is hailed as one of the monumental scientific techniques of the twentieth century.
What is PCR? PCR is an exponentially progressing synthesis of the defined target DNA sequences in vitro.
Why “Polymerase”? It is called ―polymerase‖ because the only enzyme used in this reaction is DNA polymerase.Why “Chain”? It is called ―chain‖ because the products of the first reaction become substrates of the following one, and so on.
The ―Reaction‖ Components1) Target DNA - contains the sequence to be amplified.2) Pair of Primers - oligonucleotides that define the sequto be amplified.3) dNTPs - deoxynucleotidetriphosphates: DNA building4) Thermostable DNA Polymerase - enzymethat catalyzes the reaction5) Mg++ ions - cofactor of the enzyme6) Buffer solution – maintains pH and ionicstrength of the reaction solution suitable forthe activity of the enzyme
Principle ofPCR The principle of PCR is rather simple and involves enzymatic amplification of a DNA fragment flanked by two oligonucleotides (primers) hybridized to opposite strands of the template with the 3’ends facing each other. DNA polymerase synthesizes new DNA starting from the 3’ end of each primer. Repeated cycles of heat denaturation of the template, annealing of the primers and extension of the annealed primers by DNA polymerase results in amplification of the DNA fragment. The extension product of each primer can serve as
The Reaction PCR tube THERMOCYCLER
Elements of standard PCRreaction Components of a standard PCR reaction are: Thermostable DNA polymerase, DNA template, primers, dNTP substrate, MgCl2 buffer and salt. In addition, PCR reactions frequently include compounds that stabilize the enzyme and reagents that help DNA dissociation or primer annealing.
DNA polymeraseMost common thermostable DNA polymerase is Taq polymerase. ( Thermus aquaticus)Properties that make it less than ideal: First, the enzyme has very high error rates due to the lack of 3’to 5’exonuclease activity. Second, the enzyme adds nucleotides to 3’ ends in a template-independent manner, making the amplification product difficult to clone. Third, the enzyme is quite expensive.
Variety of thermostable DNA polymerases from thermophilic or hyperthermophilic bacteria. Standard PCR reactions (e.g., Tli, Pvo) , Sequencing (e.g., Pvo, AmpliTherm), Long PCR reaction mixtures(e.g.Tth). A used concentration is 2.0 units/100 µl reaction.
DNA template One of the most important features of PCR is that it can be performed with very small quantities of relatively impure DNA. Even degraded DNA can be successfully amplified. Therefore a number of simple and rapid protocols to purify DNA for PCR have been developed. Numbers of contaminants efficiency of amplification. Ex: urea, SDS, sodium acetate and some components eluted from Agarose can interfere with PCR. Most of these impurities can be removed by washing with 70% ethanol or by reprecipitation of DNA in the presence of ammonium acetate.
Primers: guidelines Optimal primer set should hybridize to the template efficiently with negligible hybridization to other sequences of the sample. primers should be at least 20 to 25 bases in length. However, RAPD analysis primers are usually 8 to 10 bases in length. Primers, if possible, should have GC content similar to that of the target & both primers similar GC. Primers should not have sequences with significant secondary structure. No simple repeats or palindromic sequences. Primer pairs should not contain complementary sequences to each other. primers with less 3’ overlaps
Primers That Form Hairpins Primers can have self-annealing regions within each primer (i.e. hairpin and fold back loops) A primer may be self-complementary and be able to fold into a hairpin: 5´-GTTGACTTGATA ||||| T 3´-GAACTCT The 3´ end of the primer is base-paired, preventing it annealing to the target DNA.
Substrate Conc.of each of dNTP in PCR should not exceed 200 µM. 200 µM dNTP’s→12.5 µg of DNA when half of the nucleotides are incorporated. All 4 dNTPs should be used at equivalent concentrations , This will minimize the error rate of the enzyme. An excess of nucleotides inhibits enzyme activity and can contribute to the appearance of false products.
MgCl2 Concentration Mg ion is a required co-factor for all DNA polymerases. magnesium ion concentration may affect the following: Primer annealing. Temperature of strand dissociation for template and product. Product specificity. Formation of primer-dimer artifacts and enzyme fidelity. Many templates require optimization of magnesium ion concentration for efficient,
Buffer A standard buffer for PCR is 10 to 50 mM Tris- HCL. The optimum pH is between 8-9 for most thermophilic polymerases. Since the Δ pKa for Tris is high(-0.031/ C), the true pH of the reaction mixture during a typical thermal cycle varies considerably (approximately 1 to 1.5 pH units).
SALTS : in Buffer The salt used in most reactions is K /Na, added to facilitate correct primer annealing. For Taq polymerase, the conc.is 50 mM. Other components stabilize the enzyme are: gelatin, bovine serum albumin or nonionic detergent(Tween 20 or Triton X 100). Most protocols work well without. When using DNA template(high GC content), the reaction mixture also includes reagents to lower the Tm (melting temp) of the template. Among these are DMSO, acetamide or glycerol.
Thermal cycling profileStandard PCR consists of three steps; Denaturation, Annealing and Extension. These steps are repeated or cycled 25-30 times. Most protocols also include a single Denaturation step (Initial Denaturation) before cycling begins and single long extension step (final extension) at the end.
Steps in PCR Denaturation 93 to 95 C 1min Annealing 50 to 55 C 45sec Elongation 70 to 75 C
Denaturation of DNADenaturation is the first step in PCR, in whichthe DNA strands are separated by heating to95°C.The Hydrogen bonds between the two strandsbreaks down and the two strands separates.
Annealing Annealing is the process of allowing two sequences of DNA to form hydrogen bonds. The annealing of the target sequences and primers is done by cooling the DNA to 55°C. Time taken to anneal is 45 seconds
Taq polymerase binds ….Taq polymerase binds to the template DNAand starts adding nucleotides that arecomplementary to the first strand.This happens at 72°C as it is the optimumtemperature for Taq Polymerase.
Elongation step In this step, DNA polymerase synthesizes a new DNA strand by extending the 3’ end of the primers. Time of the elongation depends on the length of the sequence to be amplified. Since Taq polymerase can add 60-100 bases per second under optimal conditions, synthesis of a 1Kbp fragment should require a little less than 20 seconds. most protocols recommend 60 seconds per 1 Kbp DNA to account for time needed to reach the correct temperature and to compensate for other unknown factors that can affect reaction rate. The shortest possible time should be used to preserve
The target product is made only in the third cycle 5’ 3’ 3’ 5’Cycle 1 5’ 3’ 3’ 5’ 3’ 5’ 3’ 5’Cycle 2 5’ 3’ 3’ 5’ 3’ 5’Cycle 3 5’ 3’ 3’ 5’ 5’ 3’
After 30cycles this becomesone billion! 230
DNA copies vs Cycle number 2500000 2000000DNA copies 1500000 1000000 500000 0 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
The PCR process can be divided intothree stages 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 need to 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
PCR Cycles Review Denaturation: 94 - 95 C Primer Annealing: 55 - 65 C Elongation of DNA: 72 Number of Cycles: 25-40 No target products are made until the third cycle. At 30 cycles there are 1,073,741,764 target copies (~1 109).
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
Advantages of PCR Speed Ease of use Sensitivity Robustness
Limitations of PCR Need for target DNA sequence information Primer Designing for unexplored ones. Boundary regions of DNA to be amplified must be known. Infidelity of DNA replication. Taq Pol – no Proof reading mech – Error 40% after 20 cycles 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.
Applications of PCR Medical applications Infectious disease applications Forensic applications Research applications
Applications of PCR Basic Research Applied Research • Mutation screening • Genetic matching • Drug discovery • Detection of pathogens • Classification of organisms • Pre-natal diagnosis • Genotyping • DNA fingerprinting • Molecular Archaeology • Gene therapy • Molecular Epidemiology • Molecular Ecology • Bioinformatics • Genomic cloning • Site-directed mutagenesis • Gene expression studies
Applications of PCRMolecular Identification Sequencing Genetic Engineering• Molecular Archaeology • Bioinformatics • Site-directed mutagenesis• Molecular Epidemiology • Genomic cloning • Gene expression studies• Molecular Ecology • Human Genome Project• DNA fingerprinting• Classification of organisms• Genotyping• Pre-natal diagnosis• Mutation screening• Drug discovery• Genetic matching• Detection of pathogens
MICROBIOLOGICAL APPLICATION OF PCR:•A•B•C•D•E
ConclusionThe speed and ease ofuse, sensitivity, specificity androbustness of PCR has revolutionisedmolecular biology and made PCR the mostwidely used and powerful technique with greatspectrum of research and diagnosticapplications.
Colony PCRColony 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.
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 Specialized enzyme systems have been developed that inhibit the polymerases activity at ambient temperature, either by the binding of an antibody or by the presence of covalently bound inhibitors that only dissociate after a high-temperature activation step DNA Polymerase- Eubacterial type I DNA polymerase, Pfu These thermophilic DNA polymerases show a very small polymerase activity at room temperature.
Asymmetric PCR Asymmetric PCR is used to preferentially amplify one strand of the original DNA more than the other. It finds use in some types of sequencing and hybridization probing where having only one of the two complementary stands is ideal. PCR is carried out as usual, but with a great excess of one primers for the chosen strand.
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
AFLP PCRAFLP is a highly sensitive PCR-based methodfor detecting polymorphisms in DNA. AFLPcan be also used for genotyping individualsfor a large number of loci•Genomic DNA is digested with one or morerestriction enzymes. tetracutter (MseI) and ahexacutter (EcoRI).•Ligation of linkers to all restriction fragments.• Pre-selective PCR is performed using primerswhich match the linkers and restriction sitespecific sequences.•Electrophoretic separation and amplicons on agel matrix, followed by visualisation of the bandpattern.
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. Applications Amplification and identification of sequences flanking transposable elements, and the identification of genomic inserts.
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.
In Situ PCR In Situ PCR (ISH) is a polymerase chain reaction that actually takes place inside the cell on a slide. In situ PCR amplification can be performed on fixed tissue or cells. Applies the methodology of hybridization of the nucleic acids. Allows identification of cellular markers Limited to detection of non-genomic material such as RNA, genes or genomes
In Situ PCR
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 not possible with conventional PCR.
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 complementary DNA.
Allele-specific PCR Used for identify of SNPs. It requires prior knowledge of a DNA sequence, including differences between alleles. Uses primers whose 3 ends encompass the SNP PCR amplification under stringent conditions is much less efficient in the presence of a mismatch between template and primer Successful amplification with an SNP-specific primer signals presence of the specific SNP in a sequence
What is Real Time PCR?Real Time PCR is a technique in whichfluoroprobes bind to specific target regions ofamplicons to produce fluorescence during PCR.The fluorescence, measured in Real Time, isdetected in a PCR cycler with an inbuilt filterflurometer.