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DNA amplification In vivo In vitro(Cloning) (PCR)
Method first proposed by H. G. Khorana & colleagues in 1970’s. 15 years later the idea was independently conceived by Karry Mullis in 1983. Used the Klenow fragment of E. coli DNA polymerase to describe the in-vitro amplification of genes. Saiki et al in 1988 used the thermostable DNA polymerase from Thermus aquaticus and greatly increased the efficiency of PCR. In 1989, Science magazine selected PCR as the major scientific development and Taq DNA polymerase as the molecule of the year. Karry Mullis was awarded the Noble prize for chemistry in 1993.
An in vitro method for enzymatically synthesizing defined sequences of DNA The technique has been a revolution in molecular biology and now is so pervasive that it is difficult to imagine life without it. The problem of insufficient DNA is no longer a problem in molecular biology research or DNA- based diagnostics.
It’sWhy Polymerase? a means of selectively amplifying a particular segment of DNA. It is called “polymerase” because the only enzyme used in this reaction is DNA The segment may represent a small part of a large polymerase. and complex mixture of DNAs: Why Chain? e.g. a specific exon of a human gene. It is called “chain” because the products of the first reaction become substrates of the It can be thought ofandaso on. following one, as molecular photocopier. The “Reaction” components
Essential components required: Template DNA A thermostable DNA polymerase A pair of synthetic oligonucleotide primers. Divalent cations (Mg 2+ ) dNTPs Buffer to maintain pH
Various types • Single or double stranded DNA • Genomic, cloned, bacterial, viral • RNA/cDNA Closed circular DNA templates are amplified slightly less efficiently than linear ones. Amplification depends on the number of copies of the target DNA seeded into the reaction.
Needs a pre-existing DNA to duplicate ◦ Cannot assemble a new strand from components ◦ Called template DNA Can only extend an existing piece of DNA ◦ Called primers 5’ 3’3’ 5’
PCR uses the enzyme DNA polymerase that directs the synthesis of DNA from deoxynucleotide substrates on a single-stranded DNA template A wide range of thermostable polymerases are available, which vary in their fidelity, efficiency and ability to synthesize large DNA products. Taq polymerase isolated from Thermus aquaticus is the first isolated and best known enzyme.
Taq polymerase Source Thermus aquaticus Activity 5’ – 3’ polymerase activity, but lacks 3’ – 5’ exonuclease activity (no proofreading) Stability Half life of <5 min at 100 C, but retains activity up to 40 min at 95°C Error rate 2 x 10-4 errors / base Fidelity lowWhen greater fidelity is required, other thermostableenzymes may have significant advantages.
Enzyme Source Optimum Fidelity Proofreading temp. CrTth T. thermophilus 75-80 Low nonePfu Pyrococcus 72-78 High Yes furiosusPwo P. woesei 60-65 High YesDeep Pyrococcus 70-80 High YesVent strain GB-D Cocktails of different enzymes are also available that allow desired features like high efficiency, high fidelity, proofreading and generation of high yields of long targets. For e.g. a mixture of Pfu and Taq allows generation of products as long as 35 kb.
All polymerases require free divalent cations – usually Mg 2+ for activity. Some require Mn 2+ (Tth for RT action). Cofactor in the catalytic addition of deoxynucleoside monophosphates to the 3’ end of the growing DNA chain
• A pair of synthetic primers is required to prime DNA synthesis. A forward and a reverse primer.• Primers anneal to the flanking regions by complementary- base pairing (G=C and A=T) using hydrogen bonding.• The most crucial factor in PCR is the design of the oligonucleotide primers. Careful design of primers is required to, Obtain desired products in high yields. Suppress amplification of unwanted sequences. Facilitate subsequent manipulation of the amplified product.
Base composition: G+C content between 40% to 60%. Length: 18-25 nucleotides long. Members of a primer pair should not differ in length by >3 bp. Complementarity: The 3’ terminal sequence of one primer should not be complementary to any site on the other primer. Melting temperature (Tm): The calculated Tm values of a primer pair should not differ by >5 C. 3’ termini: If possible, the 3’ base of each primer should be G or C.
Wallace rule: This equation can be used to calculate the Tm of duplexes 15-20 nucleotides in length in solvents of high ionic strength (e.g. 1M NaCl). Tm (in C) = 2 (A+T) +4 (G+C) Bolton and McCarthy (1962): The equation predicts the melting temperature of oligonucleotides 14-70 nucleotides in length in cation concentrations of 0.4 M or less: Tm (in C) = 81.5 C + 16.6 (log10 [K+]) + 0.41 (% (G+C) – 675/n)
Many computer programs are available that generatepotentially specific primers whose melting temperatureshave been calculated. • GeneFisher Interactive Primer Design Tool • OligoAnalyzer • Oligocalc • PCR Optimization Program Helper • Webprimer
The PCR usually consists of a series of 30 to 35 cycles. Mostcommonly, PCR is carried out in three steps, often preceded by onetemperature hold at the start and followed by one hold at the end. Atypical PCR cycle has following stepsDenaturation (94-95 C, for ~ 30 s)The template is denatured by heatAnnealing (55-60 C, for ~ 30 s)Annealing of oligonucleotide primers to single stranded targetsequencesElongation (72 C)Extension of annealed primers by a thermostable polymerase
Following PCR, the amplification product can be detected using gelelectrophoresis where visualization of a band containing DNAfragments of a particular size indicates the presence of the targetsequence in the original starter DNA sample.
Prevent contamination in PCR with exogenous DNA sequences. Always run negative controls. Use sterilized filter tips and positive displacement pipettes. Can use UV cabinets.Although the PCR concept is simple, successfulperformance of a PCR reaction depends on a numberof factors
For a standard Taq PCR reaction of 30 cycles , the reaction volumeof 25- 50 μl contains 1 pg – 1 μg of DNA 0.5 – 2.5 U of Taq polymerase 0.1 –1μM of each primer 1.5 mM of MgCl2 200 – 250 μM of each dNTP 50 Mm KCl PCR buffer (Tris-Cl pH 8.3 – 8.8)When setting up a reaction for the first time with newtemplate, new primers, new enzyme etc. amplification will be lessthan optimal.Optimization of the reaction to suppress non specific amplificationor enhance the yield of product is required.
Template:The primer / template ratio strongly influences thespecificity of the PCR and should be optimized empirically.Mg 2+ concentration:0.5 mM – 5 mM. Influences enzyme activity. Theconcentration of free Mg 2+ depends on conc. of dNTPs, freepyrophosphates and EDTA.Has to be optimized for every assay.dNTP concentration:Imbalanced dNTP mixtures (unequal conc. of four) willreduce Taq fidelity.
Annealing temperature of primers Primers have a calculated annealing temperature (e.g. 54°C). Temperature must be confirmed practically. Temperature steps of 2°C above and below (touch down PCR) Use gradient cycler.
Problem Explanation Suggested optimizationBands are sharp but Insufficient Use different conc. offaint priming or the two primers/ Mg 2+ extensionBands in negative Contamination of Make fresh reagentscontrols DNA and prevent cross contamination of tubesUndesired products Non specific Decrease theon gel priming annealing time and increase temp.
Possible causes of Remedies to rectificationnon detectableamplificationDefective reagents Compare yields from fresh & old reagentsSuboptimal annealing Optimize primer conc., recalculate the TmconditionsSuboptimal extension Optimize conc. of dNTPs, DNA, MgCl2, test pH, use fresh DNA polymerase, add an enhancer etc.Ineffective Denaturation Increase time & temp. of denaturationPrimer distance too Use polymerases capable of longlong amplification
When multiple amplification products are formed: Optimize concentrations of MgCl2, dNTPs, template DNA and polymerase. Use touchdown PCR. Verify concentration of primers and optimize if necessary. Carry out nested PCR.
Minute amounts of DNA template may be used from as little as a single cell. DNA degraded to fragments only a few hundred base pairs in length can serve as effective templates for amplification. Large numbers of copies of specific DNA sequences can be amplified simultaneously with multiplex PCR reactions. Contaminant DNA, such as fungal and bacterial sources, will not amplify because human-specific primers are used. Commercial kits are now available for easy PCR reaction setup and amplification.
The target DNA template may not amplify due to the presence of PCR inhibitors in the extracted DNA Amplification may fail due to sequence changes in the primer binding region of the genomic DNA template Contamination from other human DNA sources besides the forensic evidence at hand or previously amplified DNA samples is possible without careful laboratory technique and validated protocols
Generation of probes Generation of cDNA libraries Production of DNA for sequencing Analysis of mutations Diagnosis of monogenic diseases (single gene disorders) PCR use in Pre-implantation Genetic Diagnosis (PGD). PCR in forensic science Comparison of gene expression Cloning novel members of protein families using homology PCR Detection of bacteria and viruses
The speed and ease of use, sensitivity, specificity and robustness of PCR has revolutionised molecular biology and made PCR the most widely used and powerful technique with great spectrum of research and diagnostic applications. It enables the scientist to quickly replicate DNA and RNA on the benchtop. PCR and its related applications are rapid and convenient alternatives to traditional methods such as southern / northern blotting and molecular cloning.
Heating separates the double stranded DNA ◦ Denaturation Heat Cool Slow cooling anneals the two strands ◦ Renaturation
Two primers are supplied in molar excess They bind to the complementary region As the DNA cools, they wedge between two template strands Optimal temperature varies based on primer length etc.Typical temperature from 40 to 60 C
DNA polymerase duplicats DNA Optimal temperature 72C