Transcript Document
Polymerase Chain Reaction
(basic principal)
Polymerase Chain Reaction (PCR)
Developed by Kary Mullis and described in 1985 (Saiki et al., 1985)
Mimics repeated cycles of DNA replication to amplify specific sequences of DNA
Subsequently modified to use RNA as the template for replication
WHAT IS PCR ?
Within a dividing cell, DNA replication involves series of enzymes mediated reaction :
for unwind double helix DNA (helicases) For synthesis of primers (a RNA polymerase / primase) For DNA synthesis (DNA polymerase)
In PCR :
High temperature to denature dsDNA
ssDNA Primers: synthetically pre-made DNA polymerase : thermostable
Taq –
DNA polymerase
PCR PCR is a technique that takes a specific sequence of DNA of small small amounts and amplifies it to be used for further works The Polymerase Chain Reaction (PCR) provides an extremely sensitive means of amplifying relatively large quantities of DNA First described in 1985, Nobel Prize for Kary Mullis in 1993 The technique was made possible by the discovery of Taq polymerase, the DNA polymerase that is used by the bacterium Thermus aquaticus that was discovered in hot springs
What is PCR ?
Method for exponential amplification of DNA or RNA sequences
Basic requirements
template DNA or RNA
2 oligonucleotide primers complementary to different regions of the template
heat stable DNA polymerase
4 nucleotides and appropriate buffer
The primary materials, or reagents, used in PCR are:
DNA nucleotides, the building blocks for the new DNA Template DNA, the DNA sequence that you want to amplify Primers, oligonucleotides that are complementary to a short region on either side of the template DNA DNA polymerase, a heat stable enzyme that drives, or catalyzes, the synthesis of new DNA The targets in PCR are the sequences of DNA on each end of the region of interest, which can be a complete gene or small sequence.
The Cycling Reactions : There are three major steps in a PCR (denaturation, annealing & extension), which are repeated for 20 to 40 cycles. This is done on an automated Thermo Cycler, which can heat and cool the reaction tubes in a very short time. Denaturation at around 94 ° C : During the denaturation, the double strand melts open to single stranded DNA, all enzymatic reactions stop (for example the extension from a previous cycle). Annealing at around 54 ° C : Hydrogen bonds are constantly formed and broken between the single stranded primer and the single stranded template. If the primers exactly fit the template, the hydrogen bonds are so strong that the primer stays attached Extension at around 72 ° C : The bases (complementary to the template) are coupled to the primer on the 3' side (the polymerase adds dNTP's from 5' to 3', reading the template from 3' to 5' side, bases are added complementary to the template)
The PCR Cycle
The different steps of PCR
PCR
Exponential increase of the number of copies during PCR
PCR Every cycle results in a doubling of the number of strands DNA present After the first few cycles, most of the product DNA strands made are the same length as the distance between the primers The result is a dramatic amplification of a the DNA that exists between the primers. The amount of amplification is 2 raised to the n power; n represents the number of cycles that are performed. After 20 cycles, this would give approximately 1 million fold amplification. After 40 cycles the amplification would be 1 x 10 12
Verification of PCR product
Critical Factors for Successful PCR
Equipment
Thermal cycling profile & cycle number
Enzyme concentration
MgCl
2
Concentration
dNTPs Conc.
Primer sequences
Template
.Equipment :
a. Type of thermocycler : - accurately & reproducibly 3 PCR incubation temperature - Ramp - cycle between temperatures repeatedly & reproducibly b. Type of reaction tubes: affect heat transfer - fit precisely into wells - thin walls
PCR Optimization
Yield
Specificity
Fidelity
Size of product
Robustness of reaction
Yield
Optimum yield for a given number of cycles can be approximated using the formula: 2 n x initial number of copies. n = # of cycles There is an exponential phase of amplification in PCR until the number of product copies reaches approximately 10 off dramatically and the product stops accumulating 12 . After this point the accumulation of product amplification generally drops exponentially. The number of cycles needed to reach this point can be approximated with the formula: N f = N o (1 + Y) n Where N f N o is the final number of the double stranded target sequence is the original number of target copies Y is the efficiency per cycle of the polymerase n is the number of cycles
Factors That Influence Specificity
Primer design
Enzyme/reaction set-up
Reaction optimization
Magnesium concentration
Buffer formulation
Annealing temperature
Factors That Influence Fidelity
Number of cycles
Magnesium concentration
Nucleotide concentration and balance
Enzyme choice
Cycle Number
Standard Range:
20-30 cycles The plateau effect encourages nonspecific amplification and so increasing the number of cycles does not increase specificity or efficiency of your PCR.
Improve specificity:
Reduce number of cycles. Reduce cycle segment lengths.
Improve efficiency:
For amplifying large fragments (> 1 kb) increase the duration of each thermal step.
The “Plateau Effect”
The plateau effect
: attenuation in the exponential rate of product accumulation in late stages of a PCR, when product reaches 0.3-1.0 nM.
This may be caused by degradation of reactants (dNTPs, enzyme); reactant depletion (primers, dNTPs - former a problem with short products, latter for long products); end-product inhibition (pyrophosphate formation); competition for reactants by non-specific products; competition for primer binding by re-annealing of concentrated (10nM) product.
Several conditions can effect the plateau:
The utilization of substrates, either primers or dNTPs. The stability of the reactants. End product inhibition. Competition for reactants by nonspecific products or primer-dimers. Reannealing of product at higher concentrations which prevents the extension process. Incomplete denaturation at higher product concentration.
Primer design
General notes on primer design in PCR Perhaps the most critical parameter for successful PCR is the design of primers Primer selection Critical variables are: - primer length - melting temperature (T
m
) - specificity - complementary primer sequences - G/C content - 3’-end sequence
Primer design
Primer length
specificity and the temperature of annealing are at least partly dependent on primer length oligonucleotides between 20 and 30 (50) bases are highly sequence specific primer length is proportional to annealing efficiency: in general, the longer the primer, the more inefficient the annealing the primers should not be too short as specificity decreases
Primer design Specificity Primer specificity is at least partly dependent on primer length: there are many more unique 24 base oligos than there are 15 base pair oligos Probability that a sequence of length n will occur randomly in a sequence of length m is: P = (m – n +1) x (¼) n Example: the mtDNA genome has about 20,000 bases, the probability of randomly finding sequences of length n is: n 5 10 15 P n 19.52
1.91 x 10 -2 1.86 x 10 -5
Primer design Complementary primer sequences
- primers need to be designed with absolutely no intra-primer homology beyond 3 base pairs. If a primer has such a region of self-homology, “snap back” can occur - another related danger is inter-primer homology: partial homology in the middle regions of two primers can interfere with hybridization. If the homology should occur at the 3' end of either primer, primer dimer formation will occur
G/C content
- ideally a primer should have a near random mix of nucleotides, a 50% GC content - there should be no PolyG or PolyC stretches that can promote non-specific annealing
3’-end sequence
- the 3' terminal position in PCR primers is essential for the control of mis-priming - inclusion of a G or C residue at the 3' end of primers helps to ensure correct binding (stronger hydrogen bonding of G/C residues)
Primer design Melting temperature (T
m
)
the goal should be to design a primer with an annealing temperature of at least 50°C the relationship between annealing temperature and melting temperature is one of the “Black Boxes” of PCR a general rule-of-thumb is to use an annealing temperature that is 5°C lower than the melting temperature the melting temperatures of oligos are most accurately calculated using nearest neighbor thermodynamic calculations with the formula:
T m
= H [S+ R ln (c/4)] –273.15 °C + 16.6 log 10 [K + ] (H is the enthalpy, S is the entropy for helix formation, R is the molar gas constant and c is the concentration of primer) a good working approximation of this value can be calculated using the Wallace formula:
T m
= 4x (#C+#G) + 2x (#A+#T) °C both of the primers should be designed such that they have similar melting temperatures.
If primers are mismatched in terms of Tm, amplification will be less efficient or may not work: the primer with the higher Tm will mis-prime at lower temperatures; the primer with the lower Tm may not work at higher temperatures.
Uniqueness
There shall be one and only one target site in the template DNA where the primer binds, which means the primer sequence shall be unique in the template DNA.
There shall be no annealing site in possible contaminant sources, such as human, rat, mouse, etc. (BLAST search against corresponding genome) Template DNA 5’...TCAACTTAGCATGATCGGGTA...GTAGCAGTTGACTGTACAACTCAGCAA...3’ AGTTG TGCTA CTAC AACTG CAGTC AGTTG TGCT A Primer candidate 1 Primer candidate 2 5’-TGCTAAGTTG-3’ 5’ CAGTCAACTGCTAC-3’ NOT UNIQUE!
UNIQUE!
Length
Primer length has effects on uniqueness and melting/annealing temperature. Roughly speaking, the longer the primer, the more chance that it’s unique; the longer the primer, the higher melting/annealing temperature.
Generally speaking, the length of primer has to be at least 15 bases to ensure uniqueness. Usually, we pick primers of 17-28 bases long. This range varies based on if you can find unique primers with appropriate annealing temperature within this range.
Base Composition
Base composition affects hybridization specificity and melting/annealing temperature.
• Random base composition is preferred. We shall avoid long (A+T) and (G+C) rich region if possible.
Template DNA 5’...TCAACTTAGCATGATCGGGCA...AAGATGCACGGGCCTGTACACAA...3’ TGCT ATCA
G
T
GCCC
• Usually, average (G+C) content around 50-60% will give us the right melting/annealing temperature for ordinary PCR reactions, and will give appropriate hybridization stability.
Internal Structure
If primers can anneal to themselves, or anneal to each other rather than anneal to the template, the PCR efficiency will be decreased dramatically. They shall be avoided. However, sometimes these 2 structures are harmless when the annealing temperature does not allow them to take form. For example, some dimers or hairpins form at 30 C while during PCR cycle, the lowest temperature only drops to 60 C.
Primer Pair Matching
Primers work in pairs – forward primer and reverse primer. Since they are used in the same PCR reaction, it shall be ensured that the PCR condition is suitable for both of them.
One critical feature is their annealing temperatures, which shall be compatible with each other. The maximum difference allowed is 3 C. The closer their T anneal are, the better.
Summary ~ when is a “primer” a primer?
5’ 3’ 5’ 3’ 5’ 3’ 3’ 5’
1.
2.
3.
4.
5.
6.
7.
Summary ~ Primer Design Criteria
Uniqueness: ensure correct priming site; Length: 17-28 bases.This range varies; Base composition: average (G+C) content around 50-60%; avoid long (A+T) and (G+C) rich region if possible; Optimize base pairing: it’s critical that the stability at 5’ end be high and the stability at 3’ end be relatively low to minimize false priming. Melting Tm between 55-80 C are preferred; Assure that primers at a set have annealing Tm within 2 – 3 C of each other.
Minimize internal secondary structure: hairpins and dimmers shall be avoided.
MgCl2 concentration :
- Opt. 0.5-5.5mM, increase increase PCR product but decrease specificity Mg‡ : - influences enzyme activity - increase Tm dsDNA & forms soluble complexes with dNTPs to produce actual substrate that the polymerase recoqnize Conc. Mg‡ depends on conc.compound that binds the ion such as dNTPs, Ppi, EDTA
Magnesium Concentration
Standard Range: 0.5 - 10 mM Free Mg++ ions should exceed that of total dNTP concentration by 0.5 mM - 3.0 mM.
The main source of phosphate groups in a reaction is the dNTPs so any change in their concentration affects the concentration of available Mg++ since Mg++ form a soluble complex with dNTPs.
Mg++ has been shown to be a superior divalent cation compared to Mn++ and Ca++ and will bind to the template DNA, dNTPs, primers, and polymerase.
Its concentration affects product specificity, primer annealing, the formation of primer-dimer artifacts, melting temperature, and enzyme activity and fidelity. Thus, an important optimization strategy is to titrate your magnesium concentration for each DNA template, dNTP, primer and/or polymerase concentration change - especially if large products (>1 kb) are being amplified.
Improve specificity: Decrease concentration. Note that inadequate Mg++ concentration can result in lower efficiency/yields.
Improve efficiency: Increase concentration. specificity. However, excess Mg++ tends to cause nonspecific reactions and smeared electrophoretic bands, lowering
.
DNTPs concentration :
inbalanced dNTPs mixtures
reduce Taq fidelity dNTPs reduce free Mg‡
interfering polymerase activity & decreasing primer annealing
Deoxynucleoside Triphosphates (dNTPs)
Standard Range: 20 -200 together with the primers. If one dNTP is at a higher concentration it will be preferentially incorporated for this reason, the four dNTP concentrations (dATP, dCTP, dTTP, dGTP) should be the same so that accurate incorporation takes place. In addition, it is important to keep the four dNTP concentrations above the estimated K 15 µ M). µ M dNTP concentration should be titrated m of each dNTP (10 µ M Improve specificity: Reduce dNTP concentration but note that the Mg++ concentration should be lowered in an equimolar proportion. This is because the main source of phosphate groups in a reaction is the dNTPs so any change in their concentration affects the concentration of available Mg++ since Mg++ form a soluble complex with dNTPs. Improve efficiency: For a large template sequence, increase the amount of dNTP. Improve fidelity: Lower dNTP concentrations but note that the Mg++ concentration should be lowered in an equimolar proportion.
Annealing Temperature
Standard Range: 37 in the K m ° C - 65 ° C, 10 - 120 secs. During annealing, the primers are rapidly hybridized. Differences possessed by different DNA polymerases can change the effective primer annealing temperature. Annealing times for LA PCR can be approximated with the formula: 60 secs + (2.5 secs/100 bases) = approximate annealing time
Improve specificity:
2 ° -5 ° Increase the annealing temperature (increments of C are recommended) since it reduces the possibilities of non-specific priming and therefore nonspecific product formation. Reduce annealing times since very long annealing times normally do not improve yield, but rather produce an increase in spurious priming and thus greater amounts of nonspecific PCR products.
Annealing Temperature
•
Thermal cycling profile
Initial denaturation: completely denature complex genomic DNA accessible for primers • • Primer annealing: the most critical factor in designing a high specificity PCR Primer extension:- opt.temp for Taq DNA pol. ~ 72 ° C - +60 bases/sec.
20” ~ <500 bp . Denaturation step during cycling: - Usually 95 °C ~ 20” 30”, too low incomplete snaps back no acces for primers
Final extension :
usually 72 ° C 5 15’ Promote completion of partial extension products and annealing single stranded complementary products
Template:
conc.should optimized
Too little
primers may not able to find target
Too much
lead to increase mispriming
Template purity
influences outcome of reaction.
General work strategies to avoid contamination
Laboratory bench & equipments
Cross contamination between samples
Product from previous PCR amplification
Various quantity of templates
DNA Pol
Pfu Pfu (exo-) Psp Psp (exo-) Pwo Taq Taq (N term del) Tbr Tfl Tli Tli (exo-) Tma Tth
Enzyme Choice
Trade Name
Deep Vent ProofStart DeepVent exo Klen-Taq DyNAzyme Vent Vent exo UIITma
Product End
Blunt Blunt Blunt Blunt Blunt 3’ A 3’ A 3’ A Blunt Blunt Blunt Blunt 3’ A
Exo Activity
3’-5’ proofread No 3’-5’ proofread No 3’-5’ proofread 5’-3’ No 5’-3’ 3’-5’ proofread No 3’-5’ proofread 5’-3’
Example of a PCR Protocol
COMPONENT VOLUME FINAL CONCENTRATION
1.autoclaved ultra-filtered water (pH 7.0) 20.7µL 2.10x PCR Buffer* 3.dNTPs mix (25 mM each nucleotide) 2.5µL 0.2µL 1x 200 µM (each nucleotide) 4.primer mix (25 pmoles/µL each primer) 5.Taq DNA polymerase (native enzyme) 0.4µL 0.2µL 0.4 µM (each primer) 1 Unit/25 µL 6.genomic DNA template (100 ng/µL) 1.0µL 100 ng/25 µL * The 10x PCR buffer contains: 500 mM KCl; 100 mM Tris-HCl (pH 8.3); 15 mM MgCl2 (the final concentrations of these ingredients in the PCR mix are: 50 mM KCl; 10 mM Tris HCl; 1.5 mM MgCl2).
Troubleshooting
Non-Specific Product Yields
Components Primer concentration is too high. Primer degeneracy is too high. Nested primers are required. New primers are required. Template denaturation efficiency is too low. Mg++ concentration is too high. dNTP concentration is too high. Polymerase concentration too high. pH is suboptimal.
Conditions Annealing temperature is too low.
Too many cycles. Ramp speed is too slow. Inhibitors are present and/or concentrations are too high. Enhancers are required. Contaminants are present. Hot Start required.
or Touchdown PCR is
Little or No Product Yield
Components Primer concentration is too low. Primer concentrations not balanced.
New primers are required.
Nested primers are required. Contaminated primer.
Template concentration is too low. Template concentration is too high. Template is degraded.
Template: Target sequence is not present in target DNA. Mg++ concentration is too low. Mg++ is unevenly mixed in source solution. dNTP concentration is too low. dNTPs degraded. pH of the reaction buffer is too high. Reaction mixture incomplete degraded.
Buffer isn't diluted enough.
Conditions Denaturing temperature is suboptimal. Annealing temperature is too high. Inhibitors are present. Enhancers needed. Mineral oil problem. Reaction tubes are contaminated. Too few cycles. Thermalcycler didn't cycle. Thermalcycler was programmed incorrectly. Thermalcycler temperatures are too low in some positions. Thermalcycler top was left open.
Multiple Product Yields or High Molecular-Weight Smear Observed
Components Primer concentration is too high. New primers are required. Nested primers are required. Template: Check the target DNA sequence to see if there are known mispriming areas. Template: Band purification followed by reamplification is required. Template is degraded. Template concentration is too high. Mg++ concentration is too high. Polymerase concentration is too high.
Conditions Denaturation temperature is too low. Annealing temperature is too low. Annealing incubation times are too long.
Extension incubation times are too long.
Two temperature PCR protocol is required. Hot Start required . or Touchdown PCR is Too many cycles.
Product is the Wrong Size
Components The primers have homology with a repetitive DNA sequence in the template.
Primer-Dimers
Components The 3' ends of the primers are complimentary. Primers need to be longer. Primer concentration is too high. Target template concentration is too low. Conditions The published size for expected products is only a rough estimate. Result Plausibility: Repeat PCRs with primers from an independent but related sequence.
Conditions Annealing temperature is suboptimal. Too many cycles.
Inhibitors
You can test for inhibition from endogenous substances known in the sample by spiking a control reaction with a known amplifiable target and its respective primers. Re-extraction, ethanol precipitation, and/or centrifugal ultrafiltration may correct the problem chloroform SDS concentrations of as low as 0.01% are inhibitory phenol heparin xylene cyanol bromphenol blue inosine deoxyuridine
PCR and Contamination The most important consideration in PCR is contamination Even the smallest contamination with DNA could affect amplification For example, if a technician in a crime lab set up a test reaction (with blood from the crime scene) after setting up a positive control reaction (with blood from the suspect) cross contamination between the samples could result in an erroneous incrimination, even if the technician changed pipette tips between samples. A few blood cells could volitilize in the pipette, stick to the plastic of the pipette, and then get ejected into the test sample Modern labs take account of this fact and devote tremendous effort to avoiding cross-contamination
Sources of Contaminants
Components Reagents Purified restriction fragment of target sequence Plasmid DNA that contains target sequence Post PCR contamination from the handling of PCR products.
Conditions Biological samples (e.g. patients, animal, etc.) Sample collection methods Lab Staff Lab environment Liquid nitrogen/ice Tissue homogenizer Gloves Pipetts/pipette tips Reaction tubes/glassware Recombinant or biological products (e.g. gelatin, bovine serum albumin, restriction enzymes) Hood or fume-cupboard filters Centrifuges/centrifuge tubes Microtome blades Thermal cycler, heating blocks, water baths UV transilluminator Electrophoresis apparatus Dot-blot apparatus Razor and/or microtome blades Microcentrifuges Concentrators and vacuum bottles Gel apparatus or UV light box Dry ice/ethanol or water baths
Contoh aplikasi PCR
1. Deteksi penyakit infeksi 2. Deteksi kontaminasi agen infeksius 3. Deteksi penyakit genetik 4. Deteksi onkogen 5. Deteksi polimorfisme dalam populasi 6. Analisis evolusi 7. Keperluan penyidikan 8. Melakukan mutagenesis 9. Keperluan riset lain
MISAL DALAM DIAGNOSA LEPTOSPIRA
Contoh aplikasi PCR dalam diagnosa leptospirosis
COMPETITIVE PCR FOR QUANTITATION OF CHLAMYDIA TRACHOMATIS
Teknik analisis polimorfisma berbasis PCR
•
PCR dengan primer spesifik
•
PCR dengan primer semispesifik (rep-PCR)
•
PCR dengan primer acak:
-Arbitrarily Primed PCR(AP-PCR) (Welsh & McCelland, 1990) -Random Asmplified Polymorphic DNA-PCR (RAPD-PCR)
(William et al, 1990 ) Keuntungan RAPD-PCR: -menganalisis variasi genom suatu spesies dengan cepat & efisien -seluruh proses (isolasi DNA
dokumentasi) dilakukan < 24 jam -tidak memerlukan bahan kimia berbahaya -memungkinkan memproses sampel secara serentak
Amplifikasi sekuen microsatelite
Applications of PCR on VNTR
Three VNTR loci from suspects, along with the DNA from the scene are run through PCR amplification, and then through electrophoresis. This gives six bands, which can have common bands for some individuals, but the overall pattern is distinctive for each person.
Applications of PCR
Single – Strand Conformation Polymorphism ( SSCP )
First introduced by Orita
et al
(1989) Based on the fact that electromobility of short DNA fragment on non denaturing gel: varied, depend on nucleotides sequence Can be used to screen mutation at certain DNA fragment denatured dsDNA ssDNA non-denaturing ssDNA secondary structured (Stabilized by intramolecule interaction) PAGE mobility shift (mutant)
SSCP can be used to analized PCR product PCR-SSCP Factors influenzed the sensitivity : -size of DNA fragments ( 150 – 250bp ), > 550 bp can not be detected -Position of mutation : should be not at/near the tip of DNA fragment -Temperature -Ionic strength -Pore size, conc. of poliacrylamide gel -Addition of neutral comp. (glycerol)
SSCP results
Lane 1 : sensitive isolate Lane 2-6 : resistant isolates
PCR product of pfcrt gene fragments
Plasmodium falciparum chloroquine resistant transporter