Introduction:
The
molecular technique called PCR is in
vitro amplification of a specific segment of DNA using a thermostable
enzyme. Although it is a fairly new
technique, invented in 1985 by Cary Mullis, it is widely used in hundreds of
labs all over the world. The PCR process
makes millions of copies of DNA in just a few hours. It is a replication reaction, which uses
reagents very similar to what is needed for DNA replication inside a cell. Each strand serves as a template for synthesis
of its complementary strand.
PCR
has many applications. 1) It is a form of direct cloning of DNA (without the
need for bacteria). This is convenient
when there is little DNA to work with.
2) PCR can produce a DNA fingerprinting pattern for forensics purposes,
such as identifying blood at a crime scene.
3) PCR can be used in prenatal diagnosis of genetic diseases. 4) It can
be used for evolutionary analyses, to look at genetic relationships among or
within taxa (genera, species, populations).
5) PCR can detect allelic sequence variation and chromosomal
rearrangements. 6) It is involved in the
DNA sequencing process. 7) It is the
newest technique in detecting viral or bacterial infection within a host.
The
requirements for PCR are as follows: dsDNA, dNTP nucleotides (dATP, dGTP, dCTP,
dTTP), magnesium ion (Mg++),
a forward primer and a reverse primer, Taq
polymerase, and buffer. Taq polymerase is an enzyme extracted
from the bacterium, Thermus aquaticus. This bacterium lives in hot springs, thus it
can withstand high temperatures. Taq polymerase is thermostable at close
to boiling temperatures. During PCR, the
sample is heated to 90°C
(for 30 sec) to denature the DNA (separate the 2 strands). Then the sample is cooled down to 50°C
(for 1 min) to allow the 2 primers to anneal to each DNA strand. Finally, the sample is heated to 72°C
(for 2 min), which is the optimal temperature for Taq polymerase to add on nucleotides after each primer. This cycle
of denaturing, annealing and extending is repeated 30-40 times. For each cycle, the targeted gene is doubled
in number.
1 --> 2 --> 4 --> 8 --> 16
--> 32 --> 64 --> 128 --> 256 --> 512 --> 1024 -->
2048 --> 4096 -->
8192 --> 16,384 --> 32,768
--> 65,536 --> 131,072 --> 262,144 --> 524,288 --> 1,048,576
As shown above, after 20 cycles,
over a million copies of a gene from one molecule of DNA is generated. This logarithmic process is called
amplification.
Polymerase
Chain Reaction (PCR)
Materials:
micropipettors & tips
microfuge
thermocycler
microtubes (1.5 ml & 0.5 ml)
vortexer
PCR kit of reagents
waste beaker
kimwipes
tube of mineral oil
dsH2O
gloves
microtube rack
& marker
Procedure:
1) Put on gloves. Place the lid of the 0.5
ml tube container up-side-down on the lab bench. Pour some of the tubes slowly on top of the
lid. With gloves on and without touching
the lid, place 4 tubes in the blue rack.
They are for 2 samples, a positive control and a negative control. Pour the extra tubes back in the container
and close the lid. (This technique is
used to avoid any contamination of the tubes).
Label the top of the tubes with the permanent marker. Write +C and -C for the controls, and number
each sample.
2) Spin
down the PCR kit reagents, DNA samples, and
+C. Place all tubes in the gray
microfuge. Make sure they are
balanced. Every tube should have another
tube opposite it in the microfuge. If there
is an odd number of tubes to spin, then make a balance tube by adding some
water to an empty tube using a micropipettor.
Put pressure on the lid with one hand and press the green button to
start spinning. Press the red stop
button after ~10 sec.
3) Below
is a recipe of what your cocktail mix will contain. To determine the volumes of each item,
multiply the volume in the cocktail by the number of tubes you have in your
experiment, plus one more for pipette error (i.e. if you have 4 samples,
multiply each number by 5). Fill in the table below. This is a recipe for a 25 ml
PCR reaction.
Note****This
recipe is for DNA concentrations ranging from 10-150 ng/ml. If your DNA concentration is out of this
range, then adjust the amount of dsH2O
and DNA accordingly to reach a 25 ml volume reaction (DNA + dsH2O = 13.8 ml).
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1X Cocktail
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?X Volume Master Mix
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2.5 ml
buffer
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1.5 ml MgCl2
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0.5 ml dGTP
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0.5 ml dATP
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0.5 ml dTTP
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0.5 ml dCTP
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2.5 ml Osp2'
forward primer
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2.5 ml OspB3
reverse primer
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10.8 ml
dsH2O
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0.2 ml Taq polymerase
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4) Set
your micropipettors to the proper settings.
You will have to reset them as you make your cocktail.
5) In a 1.5 ml tube: add all of the items
in order of the recipe (table), using a
new sterile pipette tip every time. Avoid any contamination!!! Then ask the instructor for the Taq polymerase and add to your
tube. This is called your master mix.
Note**** The rest of the procedure needs to be
accomplished as quickly as possible (the enzyme is more active at room temp than
at 4°C).
6) Vortex
your master mix very gently using a slight touch, then spin briefly using a balance tube in the
microfuge.
7) Open
all PCR tubes. Aliquot 22 ml of the master mix to each tube, and close each
cap as you fill them. (Use the same
pipette tip, since the tubes are sterile and you are using the same mix. This saves time).
8) Open
each tube (except the -C), one at a time, and add 3 ml of each
appropriate DNA sample, including
the positive control. For this, you have
to change your pipette tip every time!!!
9) Make
sure the rotor inside the microfuge holds 0.5 ml tubes. If not, then change the rotor. The rotor pops right out as you lift it up,
and the replacement rotor will click into place as you push it down. Spin all PCR tubes briefly in the microfuge.
10) Place
tubes in a row, in the microtube rack.
Open all tubes and, using a yellow
pipette tip, add 2 small drops of oil to each, by holding the micropipettor
vertically above the tubes without touching them. Since you are not touching the tubes, you
will use the same yellow tip like an assembly line.
11) Place
tubes together in the thermocycler in a row angled slightly to aid in
fitting. Avoid the holes in the corners.
A Typical Temperature Profile:
40 cycles of:
93°C
/ 30 sec (DNA denaturation)
50°C
/ 1 min (primer annealing)
72°C
/ 2 min (DNA extension)
then
4°C
final holding temp
The
amplification process takes 3-4 hours.