Polymerase Chain Reaction (pcr)
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Polymerase Chain Reaction (PCR) BCH/BIOL 406 Lecture 7
Midterm
Pick up test in class
Everyone has 4 points extra credit on webCT that is not part of their posted test score.
Questions or problems? Talk to me in lab this week.
Lab Report Update
Grades are posted-Returned this week
Next Monday-include tips for improving report #2 in lecture.
The second lab report due date has been moved back from Tues 4/10 to Thurs 4/12.
Recap of Previous lab Lux operon from Vibrio fischeri R
I
C
D
LAC Z
A
POLYLINKER LAC Z
PHAGE F1
pGEM-3ZfP 3199 bp
Ampicillin Resistence
B
E
Recap of Previous lab R
I C D A B E LAC Z
POLYLINKER LAC Z
• plasmid miniprep (Qiagen Kit)
PHAGE F1
pGEM-3ZfP 3199 bp
Ampicillin Resistence
• DNA concentration (Genomics Center) • Restriction digests using SacI, SalI and KpnI to check recombinant plasmid • Problems w/ KpnI digest
Next Goal: Subclone Lux AB fragment using PCR and clone it into an expression vector to produce protein. A
B
TOPO binding site
TOPO binding site
Eco RI (5735)
Hin dIII (20)
T7 promoter
6xHis
lac operator Apa
Hin dIII (394)
LI (4936)
Cla I (401) Apa
lacI
LI (755)
Amp(R) Pst I (1185)
pET101/D-TOPO 5753 bp
(NOTE: We are using pET102, not the pET101 vector) pBR322 origin Apa
Ava
I (3362)
Apa
LI (2498)
LI (2001)
Schedule for This Week
Tuesday
Read over revisions for quiz + notebook check Set up and run PCR reactions
Thursday
Quiz: Study material from today’s lecture Run agarose gel Purify PCR fragment using Qiagen PCR kit Aliquot 10ul of purified fragment for quantitation and sequencing at the Genomics Center.
Impact of PCR “ PCR has transformed molecular biology through vastly extending the capacity to identify, manipulate and reproduce DNA.” -Paul Rabinow, UC Berkeley Making PCR, A Story of Biotechnology, University of Chicago Press, 1996
History of PCR
PCR technique was invented by Kary Mullis and co-workers in 1985.
Goal: Make millions of copies of DNA from trace amounts of DNA starting material.
Only specific pieces of DNA are amplified.
Uses for PCR
Research
Gene cloning
Clinical
DNA fingerprinting
Real-time PCR
Crime scene analysis Paternity testing Archeological finds
DNA sequencing
Genetically inherited diseases
What is Polymerase Chain Reaction (PCR)?
In vitro DNA synthesis
Components include:
Heat-stable DNA polymerase (Taq polymerase) Two Primers (DNA oligonucleotides) Deoxynucleotides –dATP, dTTP, dCTP, dGTP DNA template ++
How does PCR work? One PCR Cycle:
How does PCR work?
One PCR cycle: What the products really looks like… Template Strand
4 DNA strands
Template Strand
Biology Animation Library: http://www.dnalc.org/ddnalc/resources/pcr.html
How does PCR work?
Two cycles: What the products really looks like…
8 DNA strands
How does PCR work?
Three cycles…
16 DNA strands
otice the production of double stranded, shortened PCR products (target sequence) that sp he two primers. Our target sequences will contain the LUX AB genes.
How does PCR work?
Four cycles…
32 DNA strands The number of DNA strands doubles after each cycle. Target sequence predominates.
How does PCR work? After 30 cycles…
Target sequence increases exponentially.
Review: 3 steps for each PCR cycle
Each PCR cycle includes:
A denaturation step (92-96oC) separates the two DNA strands.
A primer annealing step (40-75oC) which is a few degrees below the Tm of the primers.
A primer extension step (72oC) which is the optimal temperature for Taq DNA polymerase activity.
Our Reaction Conditions
94oC for 2 minutes
Followed by 30 cycles of:
94oC for 40 seconds 48oC for 2 minutes 72oC for 3 minutes
Followed by 1 cycle of: 72oC for 3 minutes.
PCR Thermocycler
http://biology.clc.uc.edu/fankhauser/Labs/Genetics/PCR/PCR_Protocol.htm
Components for PCR
Heat-stable DNA polymerase (Taq or Pfu polymerase) Two Primers (DNA oligonucleotides) Deoxynucleotides –dATP, dTTP, dCTP, dGTP DNA template Mg++, buffer components, and water
Heat-stable DNA polymerase
Taq DNA polymerase was isolated from the bacterium Thermus aquaticus. Taq polymerase is stable at the high temperatures (~95oC) used for denaturing DNA.
Hot springs at Yellowstone National Park, Wyoming. http://waynesword.palomar.edu/lmexer3 b.htm
Limitations of Taq Polymerase
Error rate for Taq= 1/5000 nucleotides
Does not have 3’ 5’ exonuclease activity for proofreading.
Pfu DNA polymerase can be substituted for Taq polymerase for better proofreading due to 3’ 5’ exonuclease activity. Pfu is slower than Taq and more expensive.
Limitations of Taq Polymerase
Pfu gives blunt end PCR products. (Use blunt end cloning strategy).
Taq adds an extra “A” to the 3’ end of PCR products. (Use “T-A” cloning vectors)
Pfu can remove “A overhangs” on Taq PCR products.
What will we be using today?
Platinum Taq
Taq has an antibody (Ab) bound to it. Ab prevents Taq activity at RT, but not after heating. Platinum Taq has Pfu added. Why would this be helpful?
Components
Heat-stable DNA polymerase (Taq polymerase)
Two Primers (DNA oligonucleotides) Deoxynucleotides –dATP, dTTP, dCTP, dGTP DNA template Mg++, buffer components, and water
Primers
Two oligonucleotides of different sequences. Each are typically 18-25 nucleotides long. (Ours are 24 [Forward] and 20 [Reverse]) Primers complementary base pair (“hybridize” or “anneal”) to template DNA.
General Example of Primers http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2002/Robinson/Isocitrate-mainpage.html
Lux AB Primers 3’ TACTTCAAACCTTTATAAAC 5’ 5’ CACCATGAAGTTTGGAAATATTTG 3’ (Forward Primer)
(Reverse Primer)
3’ TTTTAGCTTTACTTAAATGG 5’ 5’ AAAATCGAAATGAATTTACC 3’
Forward Primer = nucleotides 4230-4249 in template (+ 4 additional nucleotide Reverse Primer = nucleotides 6290-6310 in template Total length PCR product = 2080 base pairs long
Review: Annealing Temperature
The primer annealing temperatures typically range from 55-65oC based on length and G-C content. (Ours are 56oC [Forward] and 47oC [R])
Annealing temp should be a few degrees below the lowest melting temperature (Tm) for the two primers. (Ours is 48oC)
Tm of two primers should be within 5oC of each other. (Ours are 56oC and 47oC)
Tips: Successful Primer Design
3’ end should have exact homology to the template DNA.
Try to have 50-60% G-C composition.
Avoid 4 or more single nucleotides in a row.
Avoid complementary base pairing within the primer (“stem-loop” or “hairpins”).
Hairpin Structure TC C AGAAGGTGACCAAGTTCAT-3’ I I I I I I I C TCTTCCA-5’ CA
Primer-Dimers
Check Your Knowledge
3’ GCATTGCTACAT 5’
(Only 12 nucleotides long. Should be at least 18 nucleotides in length)
3’ GCCGGAGTCTGGCGCGCGCGC ‘5
(Too G-C rich. Will have a high Tm value.)
3’ GGGGATTCTACCCCACGATATAGCA5’
(Hairpin formation between GGGG and CCCC. Also, you want to avoid 4 or more G’s or C’s in a row.)
Components
Heat-stable DNA polymerase (Taq polymerase) Two Primers (DNA oligonucleotides)
Deoxynucleotides –dATP, dTTP, dCTP, dGTP DNA template Mg++, buffer components, and water
Deoxynucleic Acids
dATP, dTTP, dGTP and dCTP should be present in equal amounts.
10X dNTP mix is the least stable component.
Store frozen in small aliquots Keep dNTP’s on ice!
Components
Heat-stable DNA polymerase (Taq polymerase) Two Primers (DNA oligonucleotides) Deoxynucleotides –dATP, dTTP, dCTP, dGTP
DNA template
Mg++, buffer components, and water
Template DNA
Minimum…50,000 copies/PCR reaction (2 Kb fragment = 0.1 pg)
1ng-1ug template DNA
Higher concentrations for total genomic Lower concentrations for plasmid DNA
Use 20ng of lux operon plasmid
Template DNA
Always add template DNA last to your reaction vial to avoid contamination.
Always run controls
(+) cloned template (if available) (-) water only control (-) vector only control (pGEM) (-) forward primer control (-) reverse primer control
Components
Heat-stable DNA polymerase (Taq polymerase) Two Primers (DNA oligonucleotides) Deoxynucleotides –dATP, dTTP, dCTP, dGTP DNA template
Mg++, buffer components, and water
Mg++, Buffer, and Water
Mg+2 is an essential cofactor for Taq & Pfu DNA polymerase activity. Final [Mg+2] = 1.5mM
10X PCR buffer=100mM Tris, pH 8.3 + 500mM KCl.
Mg++, Buffer, and Water
Water should be ultrapure (MilliQ water) with no salts or DNA contamination.
Template DNA and primers should be resuspended in MilliQ water to avoid high concentrations of EDTA.
Any questions?????
Midterm
Pick up test in class
Everyone has 4 points extra credit on webCT that is not part of their posted test score.
Questions or problems? Talk to me in lab this week.
Lab Report Update
Grades are posted-Returned this week
Next Monday-include tips for improving report #2 in lecture.
The second lab report due date has been moved back from Tues 4/10 to Thurs 4/12.
Recap of Previous lab Lux operon from Vibrio fischeri R
I
C
D
LAC Z
A
POLYLINKER LAC Z
PHAGE F1
pGEM-3ZfP 3199 bp
Ampicillin Resistence
B
E
Recap of Previous lab R
I C D A B E LAC Z
POLYLINKER LAC Z
• plasmid miniprep (Qiagen Kit)
PHAGE F1
pGEM-3ZfP 3199 bp
Ampicillin Resistence
• DNA concentration (Genomics Center) • Restriction digests using SacI, SalI and KpnI to check recombinant plasmid • Problems w/ KpnI digest
Next Goal: Subclone Lux AB fragment using PCR and clone it into an expression vector to produce protein. A
B
TOPO binding site
TOPO binding site
Eco RI (5735)
Hin dIII (20)
T7 promoter
6xHis
lac operator Apa
Hin dIII (394)
LI (4936)
Cla I (401) Apa
lacI
LI (755)
Amp(R) Pst I (1185)
pET101/D-TOPO 5753 bp
(NOTE: We are using pET102, not the pET101 vector) pBR322 origin Apa
Ava
I (3362)
Apa
LI (2498)
LI (2001)
Schedule for This Week
Tuesday
Read over revisions for quiz + notebook check Set up and run PCR reactions
Thursday
Quiz: Study material from today’s lecture Run agarose gel Purify PCR fragment using Qiagen PCR kit Aliquot 10ul of purified fragment for quantitation and sequencing at the Genomics Center.
Impact of PCR “ PCR has transformed molecular biology through vastly extending the capacity to identify, manipulate and reproduce DNA.” -Paul Rabinow, UC Berkeley Making PCR, A Story of Biotechnology, University of Chicago Press, 1996
History of PCR
PCR technique was invented by Kary Mullis and co-workers in 1985.
Goal: Make millions of copies of DNA from trace amounts of DNA starting material.
Only specific pieces of DNA are amplified.
Uses for PCR
Research
Gene cloning
Clinical
DNA fingerprinting
Real-time PCR
Crime scene analysis Paternity testing Archeological finds
DNA sequencing
Genetically inherited diseases
What is Polymerase Chain Reaction (PCR)?
In vitro DNA synthesis
Components include:
Heat-stable DNA polymerase (Taq polymerase) Two Primers (DNA oligonucleotides) Deoxynucleotides –dATP, dTTP, dCTP, dGTP DNA template ++
How does PCR work? One PCR Cycle:
How does PCR work?
One PCR cycle: What the products really looks like… Template Strand
4 DNA strands
Template Strand
Biology Animation Library: http://www.dnalc.org/ddnalc/resources/pcr.html
How does PCR work?
Two cycles: What the products really looks like…
8 DNA strands
How does PCR work?
Three cycles…
16 DNA strands
otice the production of double stranded, shortened PCR products (target sequence) that sp he two primers. Our target sequences will contain the LUX AB genes.
How does PCR work?
Four cycles…
32 DNA strands The number of DNA strands doubles after each cycle. Target sequence predominates.
How does PCR work? After 30 cycles…
Target sequence increases exponentially.
Review: 3 steps for each PCR cycle
Each PCR cycle includes:
A denaturation step (92-96oC) separates the two DNA strands.
A primer annealing step (40-75oC) which is a few degrees below the Tm of the primers.
A primer extension step (72oC) which is the optimal temperature for Taq DNA polymerase activity.
Our Reaction Conditions
94oC for 2 minutes
Followed by 30 cycles of:
94oC for 40 seconds 48oC for 2 minutes 72oC for 3 minutes
Followed by 1 cycle of: 72oC for 3 minutes.
PCR Thermocycler
http://biology.clc.uc.edu/fankhauser/Labs/Genetics/PCR/PCR_Protocol.htm
Components for PCR
Heat-stable DNA polymerase (Taq or Pfu polymerase) Two Primers (DNA oligonucleotides) Deoxynucleotides –dATP, dTTP, dCTP, dGTP DNA template Mg++, buffer components, and water
Heat-stable DNA polymerase
Taq DNA polymerase was isolated from the bacterium Thermus aquaticus. Taq polymerase is stable at the high temperatures (~95oC) used for denaturing DNA.
Hot springs at Yellowstone National Park, Wyoming. http://waynesword.palomar.edu/lmexer3 b.htm
Limitations of Taq Polymerase
Error rate for Taq= 1/5000 nucleotides
Does not have 3’ 5’ exonuclease activity for proofreading.
Pfu DNA polymerase can be substituted for Taq polymerase for better proofreading due to 3’ 5’ exonuclease activity. Pfu is slower than Taq and more expensive.
Limitations of Taq Polymerase
Pfu gives blunt end PCR products. (Use blunt end cloning strategy).
Taq adds an extra “A” to the 3’ end of PCR products. (Use “T-A” cloning vectors)
Pfu can remove “A overhangs” on Taq PCR products.
What will we be using today?
Platinum Taq
Taq has an antibody (Ab) bound to it. Ab prevents Taq activity at RT, but not after heating. Platinum Taq has Pfu added. Why would this be helpful?
Components
Heat-stable DNA polymerase (Taq polymerase)
Two Primers (DNA oligonucleotides) Deoxynucleotides –dATP, dTTP, dCTP, dGTP DNA template Mg++, buffer components, and water
Primers
Two oligonucleotides of different sequences. Each are typically 18-25 nucleotides long. (Ours are 24 [Forward] and 20 [Reverse]) Primers complementary base pair (“hybridize” or “anneal”) to template DNA.
General Example of Primers http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2002/Robinson/Isocitrate-mainpage.html
Lux AB Primers 3’ TACTTCAAACCTTTATAAAC 5’ 5’ CACCATGAAGTTTGGAAATATTTG 3’ (Forward Primer)
(Reverse Primer)
3’ TTTTAGCTTTACTTAAATGG 5’ 5’ AAAATCGAAATGAATTTACC 3’
Forward Primer = nucleotides 4230-4249 in template (+ 4 additional nucleotide Reverse Primer = nucleotides 6290-6310 in template Total length PCR product = 2080 base pairs long
Review: Annealing Temperature
The primer annealing temperatures typically range from 55-65oC based on length and G-C content. (Ours are 56oC [Forward] and 47oC [R])
Annealing temp should be a few degrees below the lowest melting temperature (Tm) for the two primers. (Ours is 48oC)
Tm of two primers should be within 5oC of each other. (Ours are 56oC and 47oC)
Tips: Successful Primer Design
3’ end should have exact homology to the template DNA.
Try to have 50-60% G-C composition.
Avoid 4 or more single nucleotides in a row.
Avoid complementary base pairing within the primer (“stem-loop” or “hairpins”).
Hairpin Structure TC C AGAAGGTGACCAAGTTCAT-3’ I I I I I I I C TCTTCCA-5’ CA
Primer-Dimers
Check Your Knowledge
3’ GCATTGCTACAT 5’
(Only 12 nucleotides long. Should be at least 18 nucleotides in length)
3’ GCCGGAGTCTGGCGCGCGCGC ‘5
(Too G-C rich. Will have a high Tm value.)
3’ GGGGATTCTACCCCACGATATAGCA5’
(Hairpin formation between GGGG and CCCC. Also, you want to avoid 4 or more G’s or C’s in a row.)
Components
Heat-stable DNA polymerase (Taq polymerase) Two Primers (DNA oligonucleotides)
Deoxynucleotides –dATP, dTTP, dCTP, dGTP DNA template Mg++, buffer components, and water
Deoxynucleic Acids
dATP, dTTP, dGTP and dCTP should be present in equal amounts.
10X dNTP mix is the least stable component.
Store frozen in small aliquots Keep dNTP’s on ice!
Components
Heat-stable DNA polymerase (Taq polymerase) Two Primers (DNA oligonucleotides) Deoxynucleotides –dATP, dTTP, dCTP, dGTP
DNA template
Mg++, buffer components, and water
Template DNA
Minimum…50,000 copies/PCR reaction (2 Kb fragment = 0.1 pg)
1ng-1ug template DNA
Higher concentrations for total genomic Lower concentrations for plasmid DNA
Use 20ng of lux operon plasmid
Template DNA
Always add template DNA last to your reaction vial to avoid contamination.
Always run controls
(+) cloned template (if available) (-) water only control (-) vector only control (pGEM) (-) forward primer control (-) reverse primer control
Components
Heat-stable DNA polymerase (Taq polymerase) Two Primers (DNA oligonucleotides) Deoxynucleotides –dATP, dTTP, dCTP, dGTP DNA template
Mg++, buffer components, and water
Mg++, Buffer, and Water
Mg+2 is an essential cofactor for Taq & Pfu DNA polymerase activity. Final [Mg+2] = 1.5mM
10X PCR buffer=100mM Tris, pH 8.3 + 500mM KCl.
Mg++, Buffer, and Water
Water should be ultrapure (MilliQ water) with no salts or DNA contamination.
Template DNA and primers should be resuspended in MilliQ water to avoid high concentrations of EDTA.
Any questions?????
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