PCR (Lecture 7)

CHE 554
BIOCHEMISTRY LAB

 PCR:
the polymerase chain reaction.

 Agarose Gel Electrophoresis

 Restriction Analysis

Professor
Testa

Experiment #24 in section V on

nucleic acids.
 The PCR Reaction
 The Polymerase Chain Reaction (PCR) is used for the
amplification of specific fragments of DNA.
– applications include detecting a DNA sequence of
interest,
– determining the nucleotide sequence of genes of interest

– obtaining large quantities of DNA of interest

– making specified minor changes to the sequence of DNA
– etc

 Short DNAs are used to ‘prime’ DNA synthesis. These

primers are designed to bind to DNA regions flanking your
desired product, so that the amplified product is what is
between (and including) the primers. See Figure on next
page.

 It is a three step, cyclic process

– Denaturation of all components [94 OC]
– Primer annealing to the template [55 OC]
– DNA synthesis (Elongation) [72 OC]

 PCR uses a thermally stable DNA polymerase to prevent its

degradation during the denaturation step.

 Temperature control is automated by a thermal cycler.

 Primed DNA
Synthesis

5’ to 3’

Garrett and Grisham Fig 11.2

 Cloning strategy-

PCR out regions of the DNA

that lie between the two
probes.

Primers also include sequences

for cloning into a vector
(pT7blue).
 PCR see also Fig 24.1

+
melt 95°C
Cool to 55 °C
primers bind +
incubate at 65 °C +
+ +
+
+
+
+
+
+
+
+ +
+
+
+ +
+
+
primers at +
+
+
x1000 +
+
+
 PCR, the video

Also http://www.youtube.com/watch?v=x5yPkxCLads
 Thermal cycler

Reagents:
These are used in
very small
quantities. Since
(in principle) a
single copy of a
gene suffices to
transform a
bacterium, this is
quantum biology.
 Plasmid Maps

Plasmids are small extra-chromosomal DNA

molecules that include a DNA sequence that
recruits a DNA polymerase and thus ensures
replication of the plasmid and thus its
maintenance in dividing cells. A gene for
antibiotic resistance ensures that cells lacking
the plasmid will die out of a culture grown in the
corresponding antibiotic.
 Primer design
Sequence of pBluescript
ctaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaat
aggccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttgg
aacaagagtccactattaaagaacgtggactccaacgtcaaagggcgaaaaaccgtctatcagggcgatggccc
actacgtgaaccatcaccctaatcaagttttttggggtcgaggtgccgtaaagcactaaatcggaaccctaaag
ggagcccccgatttagagcttgacggggaaagccggcgaacgtggcgagaaaggaagggaagaaagcgaaagga
gcgggcgctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgcttaatgcgcc
gctacagggcgcgtcccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgc
tattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtca
cgacgttgtaaaacgacggccagtgagcgcgcgtaatacgactcactatagggcgaattgggtaccgggccccc
cctcgaggtcgacggtatcgataagcttgatatcgaattcctgcagcccgggggatccactagttctagagcgg
ccgccaccgcggtggagctccagcttttgttccctttagtgagggttaattgcgcgcttggcgtaatcatggtc
atagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgta
aagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcggga
aacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttc
cgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcgg
taatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccagg
aaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacg
ctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgc
gctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttct
catagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccc
cgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgc
cactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtgg
tggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaa
aagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcaga
ttacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaa
aactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatg
aagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcac
ctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgg
gagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagc
aataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctatta
attgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggc
atcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatg
atcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcag
tgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtg
actggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaat
acgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaac
tctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatct
tttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgac
acggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatga
gcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgcca
c

From Fig. 24.3

Primer 1 = aaagggcgaaaaaccgtctatcag

Primer 2 = tttgccggatcaagagctaccaac complement = gttggtagctcttgatccggcaaa

9
 Primer design
Sequence of pBluescript
ctaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaat
aggccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttgg
aacaagagtccactattaaagaacgtggactccaacgtcaaagggcgaaaaaccgtctatcagggcgatggccc
actacgtgaaccatcaccctaatcaagttttttggggtcgaggtgccgtaaagcactaaatcggaaccctaaag
ggagcccccgatttagagcttgacggggaaagccggcgaacgtggcgagaaaggaagggaagaaagcgaaagga
gcgggcgctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgcttaatgcgcc
gctacagggcgcgtcccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgc
tattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtca
cgacgttgtaaaacgacggccagtgagcgcgcgtaatacgactcactatagggcgaattgggtaccgggccccc
cctcgaggtcgacggtatcgataagcttgatatcgaattcctgcagcccgggggatccactagttctagagcgg
ccgccaccgcggtggagctccagcttttgttccctttagtgagggttaattgcgcgcttggcgtaatcatggtc
atagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgta
aagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcggga
aacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttc
cgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcgg
taatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccagg
aaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacg
ctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgc
gctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttct
catagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccc
cgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgc
cactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtgg
tggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaa
aagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcaga
ttacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaa
aactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatg
aagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcac
ctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgg
gagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagc
aataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctatta
attgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggc
atcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatg
atcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcag
tgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtg
actggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaat
acgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaac
tctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatct
tttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgac
acggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatga
gcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgcca
c

From Fig. 24.3

Primer 1 = aaagggcgaaaaaccgtctatcag

Primer 2 = tttgccggatcaagagctaccaac complement = gttggtagctcttgatccggcaaa

10
 Plasmid Maps

What sizes of PCR products do you predict

in each case ?

In order to distinguish these two plasmids, we

need to exploit a means by which they differ.
Cut with KpnI or SstI. These are restriction
endonucleases.
 Restriction Endonucleases
 Restriction enzymes are enzymes that cut DNA at
specific sequences within double stranded DNA.

 Different enzymes cut DNA at different sequences.

 Target sequences are usually palindromic.

 These enzymes can be used to confirm the

presence of sequences (as in this experiment).

 These enzymes can also be used to produce

complementary overhangs “sticky ends”. These
can recombine to produce recombinant DNA
molecules from two pieces that have been
produced by the same restriction enzymes.

 We will be using KpnI (GGTACC) and SstI (GAGCTC)

5’CGTACCGATGGTACCGGAT3’
3’GCATGGCTACCATGGAATA5’
5‘CGTACCGATGG3’ + 5’TACCGGAT3’
3‘GCATGGCTACCAT5’ 3’GGAATA5’
 Plasmid Maps

What sizes of restriction fragments do you

predict in each case ?

How will you determine what the sizes

of your fragments are ?
 Agarose Gel Electrophoresis
 Agarose forms a highly hydrated matrix that is porous with
pores large enough to allow DNA to pass through. Thus it
can be thought of a solid solution.
 A block of agarose can be used to bridge two reservoirs of
buffer, one of which contains a negative electrode, and the
other of which contains a positive electrode. In this
configuration, the agarose is an element of an electrical
circuit. As highly charged polyanions, DNA molecules serve
as the charge carriers, and are drawn towards the positive
electrode by the electric field.
 Migration rate depends inversely on the size of the DNA
fragment. Small pieces will migrate faster than large ones.
Pieces of the same size will migrate at the same speed and
end up together as a band.
Agarose Gel Electrophoresis
 Detection of DNA via a
fluorescent ligand
• A small molecule intercalator (Ethidium Bromide) is added to
the gel, it fluoresces when is binds to DNA, concentrating
the fluorescent signal where DNA is.
• DNA is visualized by exposing the gel to ultraviolet light.
• Sizes of fragments are determined by comparison with size
markers (called a ladder)
• A Running dye is also used to observe the progress of the
gel directly without resorting to fluorescence. The dye is
chose to migrate with or ahead of the smallest pieces of
DNA.

http://pathmicro.med.sc.edu/
http://en.wikipedia.org/wiki/ pcr/realtime-home.htm
16
Ethidium_bromide (A nice tutorial !)
Agarose Gel Casting and
Electrophoresis
Documenting your gel
 The Experiment

 On day 1 you will set up the PCR reaction, using

as a template an unknown template, which will
be either the plasmid pBluescript II (K/S) or
pBluescript II (S/K).

 The PCR reaction will run overnight. Chris Noe

will store them in freezer the next morning.

 On day two you will first make the agarose gel.

 Also on day two you will expose some of your

PCR product to restriction enzymes and then
run them on the agarose gel (with size controls).

 From the sizes of the fragments produced by

your restriction enzymes, you will determine
which plasmid you started with.
 The Experiment
 We will make up two reactions and three control

 For the PCR reaction, we will make up our own ‘guru’ mix.
Make this in a 1.5 μL sterile eppendorf tube.
– Sterile water: 104 μL
– 10X Buffer: 65 μL
– 50 mM MgCl2: 51 μL
– 1.25 mM dNTPs: 68 μL each
– Total 492 μL
 Make up four reactions, each in a 0.5 μL sterile eppendorf tube.
– put 76 μL guru mix in each of the four tubes,
– Tube A: Add 6.8 μL Primer 1 + 10 μL template + 3 μL Taq +
5.5 μL sterile water.
– Tube B: Add 6.8 μL Primer 1 + 6.8 μL Primer 2 + 10 μL
template
– Tube C: Add 6.8 μL Primer 1 + 6.8 μL Primer 2 + 3 μL Taq
+ 10 μL template.
– Tube D: Add 6.8 μL Primer 1 + 6.8 μL Primer 2 + 3 μL Taq
+ 10 μL 10xtemplate.

– Taq DNA Polymerase is 1 U/μL

– Primers are each 10 μM.
– Template is 1 ng/μL, 10x template is 10 ng/μL

 Jennifer, Zach and Mike please use template A. Elani, Tara,

Jeremy and Govind please use template B.
PCR and restriction reactions
• For the PCR reaction, use 42 cycles of the { } loop:
– ramp to 94 °C over 50 sec

– 94 °C for 2 minutes
– {ramp to 52 °C over 30 sec.
– ramp to 55 °C over 30 sec.
– 55 °C for 1 minute
– ramp to 72 °C over 10 sec.
– 72 OC for 2 minutes
– ramp to 94 °C in 15 sec.
– 94 OC for 30 sec.}
– Hold at 4 OC

• For the restriction analysis prepare digests of the PCR products:

– PCR1 = 5 μL reaction D + 16.5 μL sterile water 2.5 μL 10x
KpnI buffer + 1 μL KpnI.
– PCR2 = 5 μL reaction D + 16.5 μL sterile water 2.5 μL 10x
SstI buffer + 1 μL SstI.

• Centrifuge briefly, to ensure all reagents are together in the

bottom of the tube.
• Reaction time begins when eppis are placed in 37 °C water bath.

• For the gel, make and load 12 μL samples instead of 6 μL.

– Make and load samples of PRC reaction A, B, C, D (10 μL
DNA + 2 μL 6x DNA sample buffer).
– Also make and load samples of PCR1, PCR2 (10 μL DNA + 2
μL 6x DNA sample buffer).
21 – Also make and load a sample of the 250 bp DNA ladder.
 Changes From the Book
 Day 1
– We will not use mineral oil. Our thermal
cycler has a heated lid, so no condensate
forms and liquid does not leave the reaction
mixture.
– Use the PCR conditions and parameters
under optimization right now by Chris Noe.
(For the purposes of the prelab, list the
conditions provided above “or are modified
to optimize performance”.)

Day 2
– Make a 0.7% agarose gel
 Follow steps 5-7 on day 2
 Use TAE buffer, not TBE
 Add 3 uL Ethidium Bromide to the gel solution
 Do not worry about evaporation

– Skip steps 10-11 (we will incorporate EtBr in

the gel)
– Skip steps 18 and 19.

 TAE = Tris-acetate (40 mM, pH 8.5) EDTA (1 mM).

 Thermocycler addresses

Column Name
1 Elani
2 Michael
4 Tara
5 Jeremy
7 Jennifer
8 Zach
10 Govind

23
 Safety Considerations
 Observe all normal laboratory safety practices.

 Wear gloves all the time when handling

ethidium bromide. Use appropriate waste
container to dispose an agarose gel!

 Do not expose your eyes and skin to UV light.

Wear gloves and safety goggles. Also don the
face mask when working with the trans-
illuminator.

 Be careful working around the agarose gel

box, it is an electrocution hazard.
 ETHIDIUM BROMIDE
UCSC Laboratory Safety Services Chemical Information Sheet
Providing a little slug of information on…..

Chemical Information Overview

Ethidium bromide is a large, flat basic molecule that resembles a DNA base pair. Because of its chemical structure, it
can intercalate (or insert) into a DNA strand. Ethidium bromide is commonly used in molecular biology laboratories to
stain electrophoresis gels. The compound forms fluorescent complexes with nucleic acids and these can be viewed
under UV light.

Ethidium bromide is a known mutagen in certain animal and microorganism test systems. Although the compound
has not been thoroughly evaluated in humans, based on current toxicity data and its interaction with DNA it should be
handled with considerable caution.

Chemical Name: Ethidium Bromide Synonyms: Ethobromide;

Chemical Formula: C21H20N3Br Dromilac;
Molecular Weight: 394.4 Homidium Bromide;
CAS Number: 1239-45-8 EtBr; RD 1572

Physical Data
Description: Compound may be ordered as a solid powder, tablets, or a stock solution of known concentration.
Melting Point (solid): 500o F (260o C)

Exposure Limits
Occupational exposure limits have not been established by OSHA, NIOSH, or ACGIH.

Toxicity Data
LD50 wt/kg oral LC50 4-hr inhalation
Descriptive Term
dose in rats dose in rats
Acute Animal Toxicity Extremely Toxic < 1 mg < 10 ppm
Oral; rat: LD50 = 1503 mg/kg Highly Toxic 1-50 mg 10-100 ppm
Inhalation; rat LC50 = 0.0118-0.1340 ppm Moderately Toxic 50-500 mg 100-1,000 ppm
Subcutaneous; mouse: LD50 = 110 mg/kg Slightly Toxic 0.5-5 g 1,000-10,000 ppm
Intraperitoneal; mouse: LDLo = 20 mg/kg Practically Nontoxic 5-15 g 10,000-100,000 ppm
Relatively Harmless > 15 g > 100,000 ppm

Personal Protective Equipment (PPE)

Gloves: Wear Nitrile gloves to prevent hand contamination. Thin disposable gloves (such as 4, 6, or 8 mil blue
Nitrile gloves) used in laboratory operations provide a contact barrier only and should be disposed of
immediately when contamination is suspected. Disposable gloves should not be worn for protection from
hazardous chemicals without double gloving because of the potential for pinholes. Latex disposable gloves
are especially prone to defects and pinholes and are not recommended.

Glasses: Wear chemical safety glasses with side shields.

Lab Attire: Always wear long pants, closed toe shoes, and a lab coat when handling hazardous materials.

Health Hazard Data

Acute
• Material may be harmful by all routes of entry; inhalation, ingestion, or skin absorption.
• Material causes eye and skin irritation and is irritating to mucous membranes and upper respiratory tract.

25
Chronic
• This agent intercalates DNA strands and was mutagenic in a number of test systems (yeast cells).
• The chemical, physical, and toxicological properties have not been thoroughly investigated in humans.
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