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![Rules of thumb for PCR conditions
Add an extra 3-5 minute (longer for Hot-start Taq) to your cycle
profile to ensure everything is denatured prior to starting the PCR
reaction
Approximate melting temperature (Tm) = [(2 x (A+T)) +(4 x
(G+C))]ºC
If GC content is < 50% start 5ºC beneath Tm for annealing temperature
If GC content ≥ 50% start at Tm for annealing temperature
Extension @ 72ºC: rule of thumb is ~500 nucleotide per minute.
Use 3 minutes as an upper limit without special enzymes
“Special” PCR cycling protocols
Touchdown PCR](https://image.slidesharecdn.com/random-230312082532-d05d764a/75/PCR-pdf-31-2048.jpg)
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مي....PCR.pdf
- 1. Polymerase chain Reaction (PCR) MaiMasri Faculty of Science Department of Zoology
- 2. Polymerase Chain Reaction (PCR) One of the most powerful tools in molecular biology Invented by Kary Mullis in 1983. This process acts as a “copying machine” for DNA 1993 Nobel Prize in Chemistry
- 3. What is PCR Anin vitro (cloning) method for the enzymatic synthesis of specific DNA sequences. Selective amplification of target DNA from a heterogeneous, complex DNC/cDNA population
- 4. Why PCR (amplify) DNA? 1. Simple 2. Powerful A. Sensitive B. Specific C. Reliable , fidelity 3. Fast
- 5. The reaction mixture Free nucleotides (A, G, T, C) DNA polymerase DNA (purified or a crude extract) Primers specific for the target DNA Buffer (containing magnesium)
- 6. The basic protocol—what’sin the tube Target DNA 5’ 3’ 3’ 5’ primers A B Free nucleotides Taq DNA polymerase Mg2+ Mg2+ Mg2+ Mg2+ Mg2+ Mg2+ Buffer containing magnesium
- 7. The basic protocol-- denaturation TargetDNA 95oC 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’
- 8. The basic protocol--annealing ~55oC 5’3’ 3’ 5’ 5’ 3’ 3’ 5’ 5’ 5’ Target DNA A B primers A B
- 9. The basic protocol--extension 72oC 5’3’ 3’ 5’ 5’ 3’ 3’ 5’ 5’ 5’ Target DNA Taq polymerase
- 10. The basic protocol--extension 72oC 5’3’ 3’ 5’ 5’ 3’ 3’ 5’ 5’ 5’ Target DNA
- 11. The basic protocol 1.Denaturation of DNA to single strands 2. Annealing of primers to DNA 3. Extension by polymerase 4. Repeat 30-35 times
- 12. One One billionin about 2 hours! At the end of each cycle, the amount of DNA has doubled In theory, each target DNA molecule is duplicated during every cycle. This means that 30 cycles would produce 230 (=109) molecules from a single target molecule. In reality, the yield is lower, especially in later cycles. 230=1,073,741,824
- 13. PCR - beforethe thermocycler 8 BORING hours per PCR! 95º C 5 min 35 times 55º C 3 min 72º C 5 min
- 14. standard tube, volume,cost evaporation & heat transfer concerns thin walled tube, volume, cost evaporation & heat transfer concerns
- 15. Most enzymeswould be killed at 95oC. Taq was isolated from Thermus aquaticus, a bacteria that grows in hot springs (~75oC) in Yellowstone National Park This organism’s enzymes have adapted to the high temperature, so they can survive cycling through the high temperatures.
- 16. Primers Target DNA 5’ 3’ 3’5’ forward reverse
- 17. Primers Primers providespecificity Must have some information about sequence flanking your target Complementary to opposite strands with 3’ ends pointing towards each other Should have similar melting temperatures. Be in vast excess.
- 18. Primer Design *Typically 20to 30 bases in length *Annealing temperature dependent upon primer sequence (~ 50% GC content) *Avoid secondary structure, particularly 3’ *Avoid primer complementarity (primer dimer) *The last 3 nucleotides at the 3` end is the substrate for DNA polymerase - G or C *Good free software programs available
- 19. Templates for PCR Small amount of template, in theory a single molecule Do not need to isolate sequence of interest DNA template need not be highly purified DNA is stable in absence of nucleases
- 20. Templates Dried blood Semen stains Vaginal swabs Single hair Fingernail scrapings Insects in Amber Egyptian mummies Buccal Swab Toothbrushes
- 21. A simple thermocyclingprotocol annealing 94ºC 94ºC 55ºC 72ºC 4ºC 3 min 1 min 45 sec 1 min ∞ hold Initial denaturation of DNA 1X 35X 1X extension denaturation
- 22. Typical PCR Temps/Times hold 4oC or 10 mM EDTA Stop reaction 5 – 10 min 70o – 75o C Final extension 0.5 – 2 min 70o – 75o C Primer extension 0.5 – 1 min 45o – 65o C Primer annealing 0.5 – 1 min 90o – 95o C Denature 1 – 3 min 90o – 95o C Initial denaturation 25 – 40 cycles
- 23.
- 24. The PCR machine Very rapidly changes the temperature between the various stages of the PCR process Programmable for use with many different cycling parameters
- 25. heated lid adjustable rampingtimes single/multiple blocks gradient thermocycler blocks Thermocyclers
- 26.
- 28. Advances due toPCR Generate very large quantities of specific DNA sequences, aiding in cloning Genomic sequencing DNA “fingerprinting”--forensic science Disease diagnosis Paternity determination Mutation detection Taxonomy Many more to come….
- 29. Basic Components ofPCR *Template DNA (0.5 - 50 ng) < 0.1 ng plasmid DNA, 50 ng to 1 μg gDNA for single copy genes *Oligonucleotide primers (0.1 – 2.0 μM) *dNTP’s (20 –250 μM) *Thermostable DNA pol (0.5 – 2.5 U/rxn) *MgCl2 (1 – 5 mM) affects primer annealing and Taq activity *Buffer (usually supplied as 10X) Working concentrations KCL (10 – 50 mM) Tris-HCl (10 mM, pH 8.3) NaCl2 (sometimes dNTPs Taq polymerase Primers DNA template Buffer + + A C T G MgCl2
- 30. Common PCR additives BSA(usually at 0.1 to 0.8 µg/µL final concentration) Stabilize Taq polymerase & overcome PCR inhibitors DMSO (usually at 2-5% v/v, inhibitory at ≤ 10% v/v) Denaturant - good at keeping GC rich template/primer strands from forming secondary structures. Glycerol (usually at 5-10% v/v) Increases apparent concentration of primer/template mix, and often increases PCR efficiency at high temperatures. Stringency enhancers (Formamide, Betaine, TMAC) Concentrations used vary by type Enhances yield and reduces non-specific priming Non-ionic detergents (Triton X, Tween 20 or Nonidet P-40) (0.1–1%) % of normal) 10 activity to ~ Taq % SDS cuts 01 . 0 SDS ( NOT Stabilize Taq polymerase & suppress formation of 2º structure
- 31. Rules of thumbfor PCR conditions Add an extra 3-5 minute (longer for Hot-start Taq) to your cycle profile to ensure everything is denatured prior to starting the PCR reaction Approximate melting temperature (Tm) = [(2 x (A+T)) +(4 x (G+C))]ºC If GC content is < 50% start 5ºC beneath Tm for annealing temperature If GC content ≥ 50% start at Tm for annealing temperature Extension @ 72ºC: rule of thumb is ~500 nucleotide per minute. Use 3 minutes as an upper limit without special enzymes “Special” PCR cycling protocols Touchdown PCR
- 32. PCR Problems Taq isactive at low temperatures At low temperatures mis-priming is likely Extension Rate Temp 0.25 nt/sec 22o C 1.5 nt/sec 37o C 24 nt/sec 55o C 150 nucleotides in 10 min
- 33. “Cheap” fixes Physical separation–”DNA-in-the-cap” Set up reactions on ice Hot-start PCR –holding one or more of the PCR components until the first heat denaturation Manually - delay adding polymerase Wax beads Touch-down PCR – set stringency of initial annealing temperature high, incrementally lower with continued cycling PCR additives 0.5% Tween 20 5% polyethylene glycol 400 betaine DMSO “Cures” for mis-priming