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

Eco RI (5735) Hin dIII (20)

T7 promoter 6xHis
lac operator Hin dIII (394)

Apa LI (4936) Cla I (401)

Apa LI (755)

lacI Amp(R)
Pst I (1185)

pET101/D-TOPO
5753 bp

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

pBR322 origin
Apa LI (2001)

Ava I (3362) Apa LI (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
 Real-time PCR 
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.
Hot springs at Yellowstone
National Park, Wyoming.
 Taq polymerase is http://waynesword.palomar.edu/lmexer3
b.htm

stable at the high

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 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’
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?????