Pcr
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- Pages: 33
Introduction to Polymerase Chain Reaction (PCR)
Review: The structure of DNA
Unzipping Antiparallel Strands
Review: Genome Sizes Pine: 68 billion bp Corn: 5.0 billion bp Soybean: 1.1 billion bp Human: 3.4 billion bp Housefly: 900 million bp Rice: 400 million bp E. coli: 4.6 million bp HIV: 9.7 thousand bp http://www.cbs.dtu.dk/databases/DOGS/abbr_table.txt
The Problem... How do we identify and detect a specific sequence in a genome?
The Problem... (How do we identify and detect a specific sequence in a genome?)
TWO BIG ISSUES:
There are a LOT of other sequences ) Y
T I C in a genome that we’re not IFI
EC P (S
interested in detecting. The amount of DNA in samples ) N O Iwe’re interested in is VERY small. T A
P M (A
IC F I L
Just How Big Is 3.4 Billion? Human genome is 3.4 billion bp If the bases were written in standard 10-point type, on a tape measure... ...The tape would stretch for 5,366 MILES = 8,636 km! Identifying a 500bp sequence in a genome would be like finding a section of this tape measure only 4 feet long = 122 cm!
How many molecules do we need to be able to see them? To be visible on an agarose gel, need around 10 ng DNA For a 500-bp product band, weighing 660 g/mol.bp, therefore need 10 x 109 / (500*660) = 3.03x 10-14 moles Avogadro’s number = 6.02 x 1023 Therefore need 1.8 x 1010 copies!
In other words, to “see” a single “gene”, the DNA in a sample of 100 cells would have to be multiplied 180 million times!!!!!
The Problem... (How do we identify and detect a specific sequence in a genome?)
TWO BIG ISSUES: There are a LOT of other sequences in Y a genome that we’re not T I C I F interested in detecting. CI E SP The amount of DNA in samples N O I Twe’re interested in is VERY small. A C I F LI P AM
PCR solves BOTH of these issues!!!
What is PCR? PCR (Polymerase Chain Reaction) is a process used to make many copies of select regions of DNA. Inventor: 1983 Kary Mullis
Nobel prize in chemistry in 1993
Some Uses of PCR Forensic DNA detection Proving innocence Identifying transgenic plants Detection of diseases Cloning Detection of ancient DNA Specific sequence targeting Assembling artificial sequences Endangered spsecies GMO detection
The Process of PCR Consists of three steps: Denaturation Annealing Extension
Denaturation The double-stranded DNA has to be heated to 94-96°C in order to separate the strands The more there is G or C, the higher Tm The longer the primers, the higer Tm
Annealing The temperature is lowered so the primers can attach themselves to the single DNA strands. The temperature depends on:
Concentration of primers Composition of nucleotides
Building starts from the 3’ end
Extension The DNA-Polymerase has to copy the DNA strands. It starts at the annealed primer and works its way along the DNA strand. The extension temperature depends on the DNA-Polymerase.
What time does it take? Denaturation: 30 - 60 sec Annealing: 30 - 60 sec Extension: 30 - 60 sec 25 - 35 cycles only (otherwise enzyme decay causes artifacts) 72oC for 5 min at end to allow complete elongation of all product DNA Altogether: 7 min ( 8,5 min) * 25 (35) = 3h5h
How PCR works The PCR cycle Denaturation 93°-95°C Annealing usually at 50° to 70°C depending on the Tm of the oligos Extension: DNA synthesis which is about 70 - 75°C.
How PCR works
How PCR works
How PCR works
PCR Reaction Components Water Buffer solution DNA template Primers Nucleotides Mg++ ions DNA Polymerase
PCR Reaction: Water Water
The medium for all other components.
PCR Reaction: Buffer Buffer
Stabilizes the DNA polymerase, DNA, and nucleotides 500 mM KCl 100 mM Tris-HCl, pH 8.3 Triton X-100 or Tween
PCR Reaction: Template DNA DNA template
Contains region to be amplified Any DNA desired Purity not required Should be free of polymerase inhibitors
PCR Reaction: Primers Primers
Specific for ends of amplified region Forward and Reverse Annealing temps should be known Depends on primer length,
GC content, etc.
Length 15-30 nt Conc 0.1 – 1.0 µM (pMol/µL)
PCR Reaction: Nucleotides Nucleotides
Added to the growing chain Activated NTP’s dATP, dGTP, dCTP, dTTP Stored at 10 mM, pH 7.0 Add to 20-200 µM in assay
PCR Reaction: Magnesium Mg++ ions
Essential co-factor of DNA polymerase Too little: Enzyme won’t work. Stabilizes the DNA doublehelix Too much: DNA extra stable, non-specific priming, band smearing Used at 0.5 to 3.5 µM in the assay
PCR Reaction: Polymerase DNA Polymerase
The enzyme that does the extension TAQ or similar Heat-stable Approx 1 U / rxn
A Typical PCR Reaction Sterile Water 38.0 10X PCR Buffer 5.0 MgCl2 (50mM) 2.5 dNTP’s (10mM each) 1.0 PrimerFWD (25 pmol/µL) 1.0 PrimerREV 1.0 DNA Polymerase 0.5 DNA Template 1.0 Total Volume
µL µL µL µL µL µL µL µL
50.0 µL
A Simpler PCR Reaction Sterile Water 38.0 10X PCR Buffer 5.0 MgCl2 (50mM) 2.5 dNTP’s (10mM each) 1.0 PrimerFWD (25 pmol/µL) 1.0 PrimerREV 1.0 DNA Polymerase 0.5 DNA Template 1.0 Total Volume
PREMIXES CAN REDUCE THE NUMBER OF ITEMS ADDED TO THE MIX
µL µL µL µL µL µL µL µL
50.0 µL
PREMIX Buffer MgCl2 dNTP’s DNA Polymerase “Enhancers” Sterile Water Primers FWD+Rev DNA Template
24.0 µL
Total Volume
50.0 µL
1.0 µL 25.0 µL
Using a PCR Mastermix Component 1X Sterile Water 38.0 10X PCR Buffer 5.0 MgCl2 (50mM) 2.5 dNTP’s (10mM each) 1.0 PrimerFWD (25 pmol/ul) 1.0 PrimerREV 1.0 DNA Polymerase 0.5 DNA Template 1.0 Total Volume
µL µL µL µL µL µL µL µL --
20X 760 ul 100 µL 50 µL 20 µL 20 µL 20 µL 10 µL
50.0 µL 980 µL
Aliquot 49 µL
Add DNA as last step
Typical Thermal Cycler Conditions 1. Initial Denaturation 2. DNA Denaturation
95o C 95o C
4 min 1 min
3. Primer Annealing
65o C
1 min
4. Primer Extension 72o C 1 min 5. Go to step #2, repeat 29 more times 6. Hold at 4o C 7. End
Thermal Cycler/PCR Machine
End of PCR Introduction
Review: The structure of DNA
Unzipping Antiparallel Strands
Review: Genome Sizes Pine: 68 billion bp Corn: 5.0 billion bp Soybean: 1.1 billion bp Human: 3.4 billion bp Housefly: 900 million bp Rice: 400 million bp E. coli: 4.6 million bp HIV: 9.7 thousand bp http://www.cbs.dtu.dk/databases/DOGS/abbr_table.txt
The Problem... How do we identify and detect a specific sequence in a genome?
The Problem... (How do we identify and detect a specific sequence in a genome?)
TWO BIG ISSUES:
There are a LOT of other sequences ) Y
T I C in a genome that we’re not IFI
EC P (S
interested in detecting. The amount of DNA in samples ) N O Iwe’re interested in is VERY small. T A
P M (A
IC F I L
Just How Big Is 3.4 Billion? Human genome is 3.4 billion bp If the bases were written in standard 10-point type, on a tape measure... ...The tape would stretch for 5,366 MILES = 8,636 km! Identifying a 500bp sequence in a genome would be like finding a section of this tape measure only 4 feet long = 122 cm!
How many molecules do we need to be able to see them? To be visible on an agarose gel, need around 10 ng DNA For a 500-bp product band, weighing 660 g/mol.bp, therefore need 10 x 109 / (500*660) = 3.03x 10-14 moles Avogadro’s number = 6.02 x 1023 Therefore need 1.8 x 1010 copies!
In other words, to “see” a single “gene”, the DNA in a sample of 100 cells would have to be multiplied 180 million times!!!!!
The Problem... (How do we identify and detect a specific sequence in a genome?)
TWO BIG ISSUES: There are a LOT of other sequences in Y a genome that we’re not T I C I F interested in detecting. CI E SP The amount of DNA in samples N O I Twe’re interested in is VERY small. A C I F LI P AM
PCR solves BOTH of these issues!!!
What is PCR? PCR (Polymerase Chain Reaction) is a process used to make many copies of select regions of DNA. Inventor: 1983 Kary Mullis
Nobel prize in chemistry in 1993
Some Uses of PCR Forensic DNA detection Proving innocence Identifying transgenic plants Detection of diseases Cloning Detection of ancient DNA Specific sequence targeting Assembling artificial sequences Endangered spsecies GMO detection
The Process of PCR Consists of three steps: Denaturation Annealing Extension
Denaturation The double-stranded DNA has to be heated to 94-96°C in order to separate the strands The more there is G or C, the higher Tm The longer the primers, the higer Tm
Annealing The temperature is lowered so the primers can attach themselves to the single DNA strands. The temperature depends on:
Concentration of primers Composition of nucleotides
Building starts from the 3’ end
Extension The DNA-Polymerase has to copy the DNA strands. It starts at the annealed primer and works its way along the DNA strand. The extension temperature depends on the DNA-Polymerase.
What time does it take? Denaturation: 30 - 60 sec Annealing: 30 - 60 sec Extension: 30 - 60 sec 25 - 35 cycles only (otherwise enzyme decay causes artifacts) 72oC for 5 min at end to allow complete elongation of all product DNA Altogether: 7 min ( 8,5 min) * 25 (35) = 3h5h
How PCR works The PCR cycle Denaturation 93°-95°C Annealing usually at 50° to 70°C depending on the Tm of the oligos Extension: DNA synthesis which is about 70 - 75°C.
How PCR works
How PCR works
How PCR works
PCR Reaction Components Water Buffer solution DNA template Primers Nucleotides Mg++ ions DNA Polymerase
PCR Reaction: Water Water
The medium for all other components.
PCR Reaction: Buffer Buffer
Stabilizes the DNA polymerase, DNA, and nucleotides 500 mM KCl 100 mM Tris-HCl, pH 8.3 Triton X-100 or Tween
PCR Reaction: Template DNA DNA template
Contains region to be amplified Any DNA desired Purity not required Should be free of polymerase inhibitors
PCR Reaction: Primers Primers
Specific for ends of amplified region Forward and Reverse Annealing temps should be known Depends on primer length,
GC content, etc.
Length 15-30 nt Conc 0.1 – 1.0 µM (pMol/µL)
PCR Reaction: Nucleotides Nucleotides
Added to the growing chain Activated NTP’s dATP, dGTP, dCTP, dTTP Stored at 10 mM, pH 7.0 Add to 20-200 µM in assay
PCR Reaction: Magnesium Mg++ ions
Essential co-factor of DNA polymerase Too little: Enzyme won’t work. Stabilizes the DNA doublehelix Too much: DNA extra stable, non-specific priming, band smearing Used at 0.5 to 3.5 µM in the assay
PCR Reaction: Polymerase DNA Polymerase
The enzyme that does the extension TAQ or similar Heat-stable Approx 1 U / rxn
A Typical PCR Reaction Sterile Water 38.0 10X PCR Buffer 5.0 MgCl2 (50mM) 2.5 dNTP’s (10mM each) 1.0 PrimerFWD (25 pmol/µL) 1.0 PrimerREV 1.0 DNA Polymerase 0.5 DNA Template 1.0 Total Volume
µL µL µL µL µL µL µL µL
50.0 µL
A Simpler PCR Reaction Sterile Water 38.0 10X PCR Buffer 5.0 MgCl2 (50mM) 2.5 dNTP’s (10mM each) 1.0 PrimerFWD (25 pmol/µL) 1.0 PrimerREV 1.0 DNA Polymerase 0.5 DNA Template 1.0 Total Volume
PREMIXES CAN REDUCE THE NUMBER OF ITEMS ADDED TO THE MIX
µL µL µL µL µL µL µL µL
50.0 µL
PREMIX Buffer MgCl2 dNTP’s DNA Polymerase “Enhancers” Sterile Water Primers FWD+Rev DNA Template
24.0 µL
Total Volume
50.0 µL
1.0 µL 25.0 µL
Using a PCR Mastermix Component 1X Sterile Water 38.0 10X PCR Buffer 5.0 MgCl2 (50mM) 2.5 dNTP’s (10mM each) 1.0 PrimerFWD (25 pmol/ul) 1.0 PrimerREV 1.0 DNA Polymerase 0.5 DNA Template 1.0 Total Volume
µL µL µL µL µL µL µL µL --
20X 760 ul 100 µL 50 µL 20 µL 20 µL 20 µL 10 µL
50.0 µL 980 µL
Aliquot 49 µL
Add DNA as last step
Typical Thermal Cycler Conditions 1. Initial Denaturation 2. DNA Denaturation
95o C 95o C
4 min 1 min
3. Primer Annealing
65o C
1 min
4. Primer Extension 72o C 1 min 5. Go to step #2, repeat 29 more times 6. Hold at 4o C 7. End
Thermal Cycler/PCR Machine
End of PCR Introduction
