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 A B E

LAC Z POLYLINKER
LAC Z
PHAGE F1

pGEM-3ZfP
3199 bp

Ampicillin Resistence
 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
• DNA concentration (Genomics Center)
Ampicillin Resistence

• 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

EcoRI (5735) HindIII (20)

T7 promoter 6xHis
lac operator HindIII (394)

ApaLI (4936) ClaI (401)

ApaLI (755)

lacI Amp(R)
Pst I (1185)

pET101/D-TOPO
5753 bp

(NOTE: We are using pET102, not the pET101 vector)

pBR322 origin
ApaLI (2001)

Ava I (3362) ApaLI (2498)

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  Clinical
 Gene cloning  DNA fingerprinting
 Crime scene analysis
Paternity testing
Real-time PCR


 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
 Mg++, buffer components, and water
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

Notice the production of double stranded, shortened PCR products (target sequence) that spans
the 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.

Hot springs at Yellowstone

 Taq polymerase is National Park, Wyoming.
stable at the high http://waynesword.palomar.edu/lmexer3b.htm

temperatures (~95oC)
used for denaturing
DNA.
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-main-page.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 nucleotides)

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”).

 If possible, avoid primer-dimer formation.

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’ GGGGATTCTACCCCACGATATAGCA-5’
(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?????