Upload
mahdi-zarei
View
2.243
Download
0
Tags:
Embed Size (px)
Citation preview
به نام خداPolymerase chain reaction
PCR by : Mahdi zarei
M.Sc. Student ,clinical
biochemistry
Ferdowsi university of mashhad
History:• By 1971 researchers in Khorana's project, concerned over their
yields of DNA, began looking at "repair synthesis" - an artificial system of primers and templates that allows DNA polymerase to copy segments of the gene they are synthesizing. Although similar to PCR in using repeated applications of DNA polymerase, the process they usually describe employs just a single primer-template complex, and therefore would not lead to the exponential amplification seen in PCR.
• Kary Mullis is generally credited with inventing PCR in 1983 while working for Cetus Corporation in Emeryville, California. While driving on Highway 128 from San Francisco to Mendocino, Mullis made an intellectual leap. He reasoned that by using two opposed primers, one complementary to the upper strand and the other to the lower, then performing multiple cycles of denaturation, annealing and polymerization he could exponentially amplify the piece of DNA between the primers.
PCR was invented in 1983 by ( Kary mullis ) & he received the Nobel Prize in chemistry in 1993, for his invention.
It revolutionized biological methods specially in molecular cloning in a way that it has became an inseparable & irreplaceable part of molecular investigations.
3
DEVELOPMENT OF PCR
PCR work was first published (1985)using Klenow polymerase
unstable with heat New enzyme had to be added manually at each
step Maximum length 400bp
– not very practical
First reports using DNA polymerase from Thermus aquaticus (1988)
• Taq-polymerase (Saiki et al, 1988) from Yellowstone National Park hot springs
Development….
Thermostable Polymerases Polymerase T ½,
95oC Extension
Rate (nt/sec) Type of
ends Source
Taq pol 40 min 75 3’A T. aquaticus
Amplitaq (Stoffel
fragment)
80 min >50 3’A T. aquaticus
Vent* 400 min >80 95% blunt
Thermococcus litoralis
Pfu >120 min 60 Blunt Pyrococcus furiosus
Tth* (RT activity)
20 min >33 3’A T. thermophilus
*Have proof-reading functions and can generate products over 30 kbp
Automation of PCR
Developed automatic “thermocycler” programmable heat block…
• The early PCR experiments , researchers had to rely on a series of water baths to maintain the different temperatures required by the procedure . “cycling” involved manual transfer of samples from one water bath to another at specified times.
• In 1988, perkin-elmer introduced the thermal cycler , a revolutionary device that automatically and repetitively raised and lowered the temperature of the samples during PCR cycles . This allowed the PCR technique to be automated. Subsequent refinements of this device extended the flexibility and accuracy of pcr.
• While in some old machines the block is submerged in an oil bath to control temperature, in modern PCR machines a Peltier element is commonly used.
PCR Thermocycler
Stage of pcr Exponential amplification:
At every cycle, the amount of product is doubled (assuming 100% reaction efficiency). The reaction is very sensitive.
Leveling off (linear)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.
COMPONENT OF PCR REACTION&PCR PROCEDURE
Components of PCR Reaction
• Template DNA• Flanking Primers• Thermo-stable polymerase
• Taq Polymerase
• dNTP • (dATP, dTTP, dCTP, dGTP)
• PCR Buffer (mg++)• Thermocyler
Thermus aquaticus
primers
1. PCR primers should be 10-24 nucleotides in length.
2. The GC content should be 40%-60%.
3. The primer should not be self-complementary or complementaryto any other primer in the reaction mixture, to prevent primer-dimer and hairpin formation.
4. Melting temperatures of primer pairs should not differ by more than 5°C, so that the GC content and length must be chosen accordingly.
5. The melting and annealing temperatures of a primer are estimated as follows: if the primer is shorter than 20 nucleotides, the approximate melting temperature is calculated with the formula:
Tm = 4(G + C) + 2 (A + T)
6. The annealing temperature should be about 5°C lower than the melting temperature.
PCR Buffer Basic Components
20mM Tris-HCL pH 8.4 50mM KCl 1.5 mM MgCl2
Magnesium – Since Mg ions form complexes with dNTPs, primers and DNA templates, the optimal concentration of MgCl2 has to be selected for each experiment. Too few Mg2+ ions result in a low yield of PCR product, and too many increase the yield of non-specific products and promote mis incorporation.
Potential Additives Helix Destabilisers - useful when target DNA is high G/CWith NAs
of high (G+C) content. dimethyl sulphoxide (DMSO), dimethyl formamide (DMF), urea formamide
Long Targets >1kb. Formamide and glycerol Low concentration of template: Polyethylene glycol (PEG)
Temperature
Denaturation Trade off between denaturing DNA and not
denaturing Taq Polymerase Taq half-life 40min at 95 °, 10min at 97.5°
95° Annealing
Trade off between efficient annealling and specificity
2-5 ° below Tm Extension
Temperature optimum for Taq Polymerase 72 °
PCRMelting
94 oC
Tem
pera
ture
100
0
50
T i m e
5’3’
3’5’
PCRMelting
94 oC
Tem
pera
ture
100
0
50
T i m e
3’5’
5’3’
Heat
PCRMelting
94 oCAnnealing
Primers50 oC
Extension72 oC
Tem
pera
ture
100
0
50
T i m e
3’5’
5’3’5’
5’
Melting94 oC
PCRMelting
94 oCMelting
94 oCAnnealing
Primers50 oC
Extension72 oC
Tem
pera
ture
100
0
50
T i m e
30x
3’5’
5’3’
Heat
Heat
5’
5’
5’
PCRMelting
94 oCMelting
94 oCAnnealing
Primers50 oC
Extension72 oC
Tem
pera
ture
100
0
50
T i m e
30x
3’5’
5’3’5’
5’
5’
5’
5’
5’
PCRMelting
94 oCMelting
94 oCAnnealing
Primers50 oC
Extension72 oC
Tem
pera
ture
100
0
50
T i m e
30x
3’5’
5’3’ 5’
5’5’
5’
5’
5’
Heat
Heat
PCRMelting
94 oCMelting
94 oCAnnealing
Primers50 oC
Extension72 oC
Tem
pera
ture
100
0
50
T i m e
30x
3’5’
5’3’ 5’
5’5’
5’
5’
5’
5’
5’
5’
5’
Fragments of defined length
PCRMelting
94 oCMelting
94 oCAnnealing
Primers50 oC
Extension72 oC
Tem
pera
ture
100
0
50
T i m e
30x
3’5’
5’3’ 5’
5’5’
5’
5’
5’
5’
5’
5’
5’
More Cycles = More DNA
Number of cycles 0 10 15 20 25 30
SizeMarker
Detection of pcr productDetection Visualization
Agarose gel and/or polyacrylamide gel electrophoresis
-EtBr staining (UV transilluminator, image analyzer) -Southern blotting (hybridization with labeled probe) -Silver staining
Restriction endonuclease digestion
-Agarose or polyacrylamide gel -HPLC
Dot blots Hybridization with labeled probe
High-pressure liquid chromatography UV detection
ElectrochemiluminescenceVoltage-initiated chemical reaction/photon detection
Direct sequencingRadioactive or fluoescent-based DNA sequencing
Controls for PCR
Blank reaction
Controls for contamination
Contains all reagents except DNA template
Negative control reaction
Controls for specificity of the amplification reaction
Contains all reagents and a DNA template lacking the target sequence
Positive control reaction
Controls for sensitivity
Contains all reagents and a known target-containing DNA template
Interpretation of the PCR Results
The PCR product should be of the expected size.
No product should be present in the reagent blank.
Misprimes may occur due to non-specific hybridization of
primers.(pcr product present in the negative control)
Primer dimers may occur due to hybridization of primers
to each other.
Variations of the PCR
Hot Start PCR
It is a method for increasing specificity of PCR
reactions.
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
Ampliwax or antibody are used in hot start pcr
Nested PCR
It is a method for increasing specificity of PCR
reactions.
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 - 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
Touchdown PCR
It is a method for increasing specificity of PCR reactions.
Touchdown PCR uses a cycling program where the annealing
temperature is gradually reduced (e.g. 1-2°C /every second cycle).
The initial annealing temperature should be several degrees
above the estimated Tm of the primers. The annealing
temperature is then gradually decreased until it reaches the
calculated annealing temperature of the primers or some degrees
below. Amplification is then continued using this annealing
temperature.
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.
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.
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.
Real-time PCR
real-time polymerase chain reaction, also called
quantitative real time polymerase chain reaction
(qPCR) or kinetic polymerase chain reaction.
Real-time PCR detects and measures the
amplification target DNA as they are produced.
Unlike, conv. PCR, real-time PCR uses an
oligonucleotide probe labeled with fluorescent dyes
or an alternative chemistry, and a thermocycler
equipped with the ability to measure fluorescence.
Real time PCR in comparison with other technical methods
amplification can be monitored real-time no post-PCR processing of products
(high throughput, low contamination risk)
Less time to getting results No gel-based analysis at the end of the pcr reaction Computer based analysis of the cycle-fluorescence
time course most specific, sensitive and reproducible not much more expensive than conventional PCR
(except equipment cost)
The Basic of Real time PCR
Two method for quantify real time pcr results:
ABSOLUTE QUANTIFICATION&
RELATIVE QUANTIFICATION
Absolute quantification
Relative quantification This involves comparing the Ct values of the samples of interest with
a control or calibrator such as a non-treated sample or RNA from
normal tissue. The Ct values of both the calibrator and the samples
of interest are normalized to an appropriate endogenous
housekeeping gene(GAPDH ,rRNA ,…).
The comparative Ct method is also known as the 2-ΔΔCt method :
ΔΔct=Δct sample – Δct reference
Here, Δct,sample is the Ct value for any sample normalized to the
endogenous housekeeping gene and ΔCt, reference is the Ct value for
the calibrator also normalized to the endogenous housekeeping gene.
Detection in real time PCR Uses fluorescence as a reporter by Three
general methods :
1. DNA-binding agents
(SYBR Green)less accuracy.
2. Hydrolysis probes(TaqMan)
3. Hybridization probes
(Light Cycler) most accurate & specific.
Beacons, Scorpions
DNA binding dye Binds to minor groove
(dsDNA) Emits light when bound More double stranded DNA =
more binding = more fluorescence
Forensically, can be used to calculate how much DNA was present before reaction.
Unspecific Melting curve analysis
1.SYBR® green
The advantage of this technique is that it is
relatively cheap as it can be used with any pair
of primers for any target. However, as the
presence of any dsDNA generates
fluorescence, specificity of this assay is
greatily decrease due to amplification of
nonspescific PCR products and primer-dimers.
Generating and comparing melting curves
using the light cycler is one method of
increasing the specificity of the reaction.
Melting curve analysis
Hydrolysis probe(taqman)
Dual hybridization probe
Molecular beacon
scorpions
references
MT Rahman , MS Uddin , R Sultana , A Moue , M Setu .
Polymerase chean reaction (PCR) : A Short Rewiew . AKMMC J
2013: 4(1): 30-36
Manit A,Iqubal S ,Magali W ,Lyndon G et all .basic principles of
real-time quantification pcr .mol.diagn.5(2),209-219.(2005)
http://www.nature.com/nprot/journal/v1
http://www.cryst.bbk.ac.uk/pps97/assignments/projects
http://www.invitrogen.com