Biology 4900
Dr. Marshall’s Materials – Day
3
August 17th 2005
POLYMERASE CHAIN REACTION
à In vitro amplification of DNA
* Invented/discovered (?) by Kary Mullis, 1993 Nobel
Laureate
* An iterative process - PCR Animation
I. Principle and Optimization of
PCR
A. Steps, Reactants, and Number of Cycles
Number of cycles, temperature & duration of each step, and reactants
can be varied to achieve optimal DNA amplification
1. Each cycle of the polymerase chain reaction has three steps
i. Denaturation of the template by heat
·
94-95oC for 5-45 sec, often an initial
denaturation step for a few min (15 min at 95oC for Hot Start)
·
Temperature is the highest the Taq DNA polymerase enzyme can take for >30 cycles
·
May need to be higher for >55% GC templates
·
Longer times required for longer templates
ii. Annealing of the primers to the template DNA
·
Annealing temperature is critical to successful PCR
à If too high, primers won’t
anneal and yield will be low
à If too low, nonspecific
annealing occurs and unwanted segments of DNA are amplified
·
The optimal annealing temperature (TA) is
usually about 3-5oC lower than the melting temperature (TM)
-
à The calculated temperature at
which the primers dissociate from the template
àMore on primers and TA
below
·
Annealing usually takes about 15-45 sec
iii. Extension from the oligonucleotide primers
·
Carried out near the optimal temperature of DNA
synthesis catalyzed by the thermostable polymerase
à 72-78oC for Taq
·
The polymerization rate of Taq is ~ 2000 nucleotides/min, but extension is usually a minute
per 1000 bp
2. Number of cycles
25 - 35 cycles tend to be sufficient (see Table 8.2 in handout HDM#5)
·
Depends on number of copies of template DNA,
efficiency of the polymerase (Taq
efficiency is 0.7), how much product is required
·
After about 30 cycles, one of the reactants usually
becomes limiting
·
Keep in mind that PCR proceeds geometrically
3. A PCR has seven essential ingredients
i. Template DNA
·
5-100 ng, or at least several thousand copies. As
little as one copy works in theory and as much as 1 μg can be used.
·
In theory piece of DNA cannot be too long
ii. A pair of synthetic oligonucleotide primers – A “forward” and a
“reverse” flanking the gene of interest.
·
Typically 0.1-0.5 μM of each primer – higher
concentrations favour mispriming.
·
This amount is nonlimiting for at least 30 cycles.
iii. A mixture of the four deoxynucleoside triphosphates (dNTPs)
·
Typical concentrations of 200-250 μM but can be
as low as 20 μM.
·
Higher concentrations can be inhibitory (may sequester
Mg2+).
iv. Divalent cations (usually Mg2+)
·
Are required for polymerase activity
·
Are bound by phosphate groups on excess dNTPs &
primers so concentration must be in excess
·
Often 1.5 mM is included in the PCR buffer, but the
optimal concentration may be as high as 6 mM
·
Needs to be empirically determined for a new
combination of primers and template
v. A buffer to maintain pH – usually 10 mM Tris-Cl, pH 8.3-8.8 at RT
vi. Monovalent cations – 50 mM KCL
vii. A thermostable polymerase
·
A wide variety are available that vary in fidelity
(proofreading) and efficiency
·
Taq polymerase at 0.5 – 2.5U per
25 μL is often the enzyme of choice
·
Other enzymes or combinations of enzymes are used in
certain cases, for example to amplify larger fragments
|
A unit of Qiagen HotStar Taq
is defined as the amount of enzyme that will incorporate 10 nmol of dNTPs
into acid-insoluble material within 30 min at 72oC. |
4. Optional PCR ingredients
·
BSA, TMAC, ammonium sulfate, may increase the
efficiency of amplification and reduce mispriming
·
Optimize the PCR if possible without these additional
ingredients – they are inhibitory at the wrong concentrations
5. PCR inhibitors
·
Anything in excess
·
Proteinase K, phenol, EDTA
·
Other contaminants in the DNA
B. Designing Primers for PCR and Determining the Annealing
Temperature
Primers are the most critical element to the success of PCR
·
They need to match the template DNA, especially at the
3’ end
·
They also need to be specific - unique in the genome (a function of length)
Good PCR primers
Are about 20-30 nucleotides long
Are 40-60% G+C
Do not form stable secondary structure, are not self-complementary or
complementary to their partner primer
Do not reside in regions of the genome that contain homopolymeric tracts
Have a G or a C as the 3’ base, and the five most 3’ bases should
comprise 2-3 Gs or Cs
The two primers of a pair should not differ by more than 3-5oC
in their melting temperatures
Determining an appropriate annealing temperature
Rule of thumb:
Tm = 2(A + T) + 4(G + C), calculate for each primer
TA is a few degrees lower than the lowest of the two Tms
Software is available to do all this for us
C. Wolffish PCR
For the wolffish we will be amplifying three gene regions
1. The mitochondrial control region (Template)
Primers
2. A portion of a 1.9 kb
antifreeze protein gene (Template)
Primers
WAF1.9F 5’-AGCACATGAACCTGTCCTGTCAGAAGTCTC-3’
WAF1.9R 5’-GTTTGTGACAAAAACAAGGTTAATTGTGAG-3’
3. A portion of a 1.5 kb
antifreeze protein gene (Template)
Primers
WAF1.5F 5’-GAAAGTGACAAAAACAAGGTTAATTGTGAG-3’
WAF1.5R 5’-TAG
GAAACGGGATATGCCGGTTAAGTCCTC-3’
à Wolffish
Antifreeze Proteins - Genbank Accession #
M22125
à Evaluate these
primer combinations using OLIGO
D. Keeping Your PCRs Free of Contamination
When setting up a PCR, use aerosol-barrier tips
Use sterile DNase-free ingredients & plastics
Aliquot reagents so they can regularly be discarded
Wear gloves and keep your work area clean
Have separate pre- and post-PCR micropipettors, and equipment &
reagents
Perhaps even separate work area or room for PCR
|
Always run a negative
control with a PCR reaction. When you start to get a PCR product in the
negative control reaction, get a new set of reagent aliquots and sterilize
your equipment. If it is persistent you can try sterilizing the PCR cocktail
by exposure to UV (BEFORE adding Taq). |
à Other methods of
preventing false positives - Rys
and Persing 1993
II. PCR Protocol
|
~A note on Hot Start PCR – A 15 min 95oC step before the
cycling begins is necessary to deactivate a protein bound to the Taq enzyme. The protein keeps the Taq inactive while the reaction is
being set up, helping to keep nonspecific PCR products (amplicons) to a
minimum~ |
1. Obtain your DNA samples from yesterday, as well as the following
supplies:
10 μM aliquots of three
pairs of primers (F & R for each pair)
CR primers
AF1.5 primers
AF1.9 primers
10 mM dNTPs (2.5 mM each of
dA, dC, dG, and dTTP)
10X PCR buffer
A supply of fresh, distilled
& autoclaved water
2. When the frozen ingredients have thawed, vortex and spin them down.
3. Label three 1.5 mL tubes CR, AF1.5, AF1.9. Make up the following PCR
cocktail for each primer pair, in
the appropriate tube.
5 X 2.5 = 12.5μL 10X
buffer
5 X 0.5 = 2.5 μL 10mM
dNTP mix
5 X 1 = 5 μL F primer
5 X 1 = 5 μL R primer
5 X 19 = 95 μL water
5 X 0.2 = 1 μL 5U/μL
HotStar Taq polymerase (get this from
us when you are ready)
à Vortex and spin
4. Have 12 0.2 mL tubes ready, labeled 1-12 with your initials.
Dispense 24 μL of the CR cocktail into the first four 0.2 mL tubes
- one tube for each of your samples and the 4th as a negative
control.
Repeat with the AF1.5 cocktail for tubes 5-8, and the AF1.9 cocktail for
tubes 9-12.
5. Add 1μL your first DNA sample to tubes 1, 5, & 9; 1 μL
of your second to tubes 2,6, &10; and 1 μL of your third to tubes 3,
7, & 11.
No DNA gets added to tubes 4, 8,
&12, your negative controls.
Vortex and spin.
6. Place samples in the Mastercycler and run the following profile:
Hot start: 95oC for
15 min
Denaturation: 93oC
for 30 sec
Annealing: 54oC for
30 sec
Elongation: 72oC
for 1 min
à 35 cycles, heated lid at 104oC
IV. Gel Electrophoresis Protocol
This procedure is virtually identical to yesterday, except load 3 µL of
PCR product and 1µL (roughly) of gel-loading buffer.
The gels can be poured immediately after the PCR cycle has started, and
left to sit at room temperature until needed.
IV. PCR Product Purification
Before using amplified DNA for other purposes such as sequencing, it
needs to be purified –
that is, extra reactants and salts need to be removed, especially extra
primer.
The method and principle for this are very similar to those for DNA
extraction –
the filter is modified to bind DNA over a different molecular weight
range.
à We will be using the
Qiagen QIAquick PCR Purification kit & protocol.
1. Make sure the remainder PCR product is at the bottom of the tube by
quickly spinning the tubes.
2. Add 110 µL of buffer PB to the PCR product.
3. Mix and pipette onto the Qiagen QIAquick column.
4. Centrifuge for 1 min. Discard collection tube contents and place
column back in the same collection
tube.
5. Add 750 μL of buffer PE.
6. Centrifuge for 1 min. Discard collection tube contents, place column
back in collection tube, and spin for another 30 sec.
7. Transfer column to a labeled 1.5 mL tube. Label tube with sample #,
gene region, date, initials.
8. Add 30 μL water directly onto filter and incubate at RT for 5
min.
9. Centrifuge for 1 min to elute DNA.
10. Store at -20oC for sequencing next Thursday.
V. Some PCR Innovations
Reverse Transcriptase PCR
Quantitative PCR
5’ and 3’ RACE
Inverse PCR
Differential Display PCR
Long PCR
Whole-Genome PCR
Amplification of Ancient DNA
Multiplex PCR
VI. Chemicals and Equipment for DNA Extraction
A. Chemicals and Reagents
MSDS Sheets are available for:
Ethidium bromide
Tris, Boric Acid, and EDTA
Bromophenol blue
Agarose
Qiagen QIAquick PCR
Purification kit components
HotStar Taq
10X PCR Buffer
B. Equipment
i. Set of micropipettors.
ii. Vortex.
iii. Programmable thermal cycler. The one we use is an Eppendorf
Mastercycler Thermocycler from Fisher
Scientific.
iv. Microcentrifuges … VWR is a good
supplier.
v. Electrophoresis chambers. The ones we
are using came from Owl Scientific.
vi. Electrophoresis power supply (“power pack”) – VWR sells these, as does Fisher
Scientific
viii. PhosphoImager. A transilluminator
and camera connected to a computer to adjust the image & print on a thermal
printer.
VII. Referencs
1. HotStar Taq PCR Handbook. Qiagen, Inc. 1999-2002.
2. QiaQuick Spin Handbook. Qiagen, Inc. 2001.
3. Weissensteimer T, Griffen HG, and Griffen A. 2004. PCR Technology: Current Innovations. CRC
Press.
4. Sambrook J and Russell DW (2001) Molecular Cloning: A Laboratory
Manual, Third Edition.