BC2004
Exercise 9:Polymerase Chain Reaction (PCR)
Spring 2005
weeks of 3/28-4/1 and 4/18-4/22/05
Once DNA is extracted from an organism, many different types of analyses can be performed.
Biologists are often interested in studying a specific gene or a specific chromosome region to
determine how that gene functions at the molecular level. To do this, the region containing the
gene of interest must be isolated from the rest of the DNA. A typical gene is composed of
approximately 1,000 to 3,000 nucleotide pairs of DNA, and most organisms have on the order of
106 to 1010 nucleotide pairs of DNA in each cell. In order to study one gene within the entire
DNA complement of an organism, it is necessary to go through several steps following DNA
extraction (as in Exercise 4) to isolate the small fragment of DNA for study.
The isolation of a specific DNA fragment is frequently achieved by cloning. This is a complex
and time-consuming process. To enable a fragment of DNA to be cloned, the DNA must be cut
into many small pieces (with restriction endonucleases; see Exercise 10), and then the pieces
must be sorted to find the one containing the gene of interest. In recent years, the need to clone a
fragment of DNA has been eliminated for many situations by the development of the Polymerase
Chain Reaction (PCR) by Kerry Mullis.
The advantage of PCR over cloning is that a specific gene can be targeted and millions of copies
of the gene made in vitro (in a test tube). The gene is thereby selectively amplified relative to the
rest of the genome; amplification eliminates the need to sort through all of the pieces of DNA
generated by restriction digestion. Enough must be known about the gene from previous work in
order for it to be targeted specifically, but with recent advances in genome sequencing, the
necessary information has become available for many genes. PCR is much quicker, easier, and
cheaper than cloning.
The polymerization reaction of DNA replication is catalyzed by an enzyme called DNA
polymerase, assisted in vivo (in the living cell) by many other proteins. During replication in
vivo, the two strands of the DNA molecule are separated for a short distance at a time, and each
exposed strand region is used as a template to make a complementary strand region. Ultimately,
all the new regions are united as one full strand, already hydrogen-bonded to an original,
template strand. The result is that two identical new strands of DNA are generated from one
original two-stranded molecule (this is called semi-conservative replication, see Figures 16.8,
16.9, and 16.10 on pp. 295-5 in Campbell 6th Edition). DNA polymerase exhibits two properties
of relevance to the use of PCR. First, the polymerase cannot initiate a new strand; it can only
extend a strand called the “primer.” In vivo, the priming strand is generated by a RNA
polymerase enzyme called primase, which synthesizes a short piece of RNA primer that is later
replaced by DNA. Second, it extends the primer strand by adding additional nucleotides to the
free hydroxyl group at its 3' end so that the new strand grows in the 5'  3' direction.
The PCR technique exploits the natural system of DNA replication. In the in vitro PCR
technique, the assisting proteins are not used; strand separation is achieved by heating the DNA
to break the hydrogen bonds that hold the strands together. The primer strands are short pieces of
DNA that are designed by and custom-synthesized for the experimenter, who designs the primer
BC2004, Spring Semester 2005, Lab Exercise 9-1
for each strand to be specific for the two ends of the region of template DNA that contains the
gene to be amplified (one is called the Forward (F) primer; the other is the Reverse (R) primer).
The two primers employed are different in sequence: one primer is complementary to one strand
of the template at one end of the gene, and the second is complementary to the other strand at the
other end of the gene. By designing the appropriate pair of primers, the experimenter is able to
target a specific gene for amplification by PCR.
To begin the PCR, many copies of the two primers are mixed with the template DNA, a mixture
of the four nucleotides dATP, dCTP, dGTP and dTTP, and a special heat-resistant DNA
polymerase (“Taq polymerase,” so called because it is prepared from the heat-loving bacterium
Thermus aquaticus).
The mixture is heated to 92 - 96o C to denature the DNA (i.e., separate the two strands by
breaking the hydrogen bonds that hold them together). The mixture is then allowed to cool so
that two-stranded DNA molecules re-form (“anneal”, through the re-formation of hydrogen
bonds). Some of two-stranded DNA that forms unites the primer molecules and the template
DNA in regions where the sequences of the molecules are complementary. The temperature is
then adjusted again to allow the Taq DNA polymerase to extends each primer using the
nucleotides in the mixture, producing a new copy of the specific area of the chromosome
downstream from where a primer and template DNA annealed.
This PCR reaction consists of many repetitions of three steps that together are referred to as a
cycle:
1. Heating (denaturation or strand separation).
This is usually achieved by 30 seconds at 95oC.
2. Cooling (annealing of the primers to the template DNA).
The temperature of annealing varies between 40 and 60oC and is specific for each
specific pair of primers. If the temperature is too high, the primers will not be
able to anneal to the template DNA even where they match exactly. If the
temperature is too low, the primers will anneal where they do not match exactly
and non-specific products will be amplified. If the temperature is just right, the
primers will stick when they do match the sequence to be amplified exactly.
3. Replication (primer extension).
This is typically from 1 to 3 minutes at 72oC, the temperature optimum for the
Taq DNA polymerase. The length of the extension time depends on the length of
the product being amplified; more time is necessary to amplify a longer product.
The cycle is repeated 30 to 40 times in a single PCR run. During each cycle, copies of the region
between the two primers become templates for subsequent cycles so that each cycle doubles the
number of copies of DNA in the region between (and including) the two primers. After 32
cycles, there should be approximately 230 copies of the gene. Although the original genomic
DNA is still present in the reaction mixture, it is an insignificant proportion of the total DNA
present; 99.99999 % of the DNA should be the gene of interest.
BC2004, Spring Semester 2005, Lab Exercise 9-2
Procedure (work in pairs)
1.
Label two sterile 500-μl microfuge tubes: “N" and “R"; add initials to each label. Be sure
to label the side of the tubes rather than their tops (if you label the top, the ink will melt
off onto the top of the PCR machine and your samples will no longer be labeled!).
The N tube will contain all the components of the PCR reaction mixture except the
template DNA; it will serve as a negative control. The template DNA is from the
bacteriophage (virus that infects bacteria) lambda.
PCR is so sensitive that a single template molecule is sufficient to result in a product.
Therefore, it is critical to ensure that the template you add is the only template available
for reaction. If there are any contaminating DNA molecules in any of the other reagents
that can cause a reaction, their amplified products will appear in the negative control. If a
product is observed in the N1 mixture, the results in the R mixture cannot be used for
further study; the desired amplification product will be contaminated with the undesirable
amplification product. If, on the other hand, no product is observable in the Negative
control, but a product has formed in the Reaction tube, this can be regarded as evidence
that the reaction amplified the intended template and nothing else.
2.
3.
To each of your two labeled tubes, add:
i.
8 μl dNTP mix (this contains dATP, dTTP, dCTP, and dGTP).
ii.
26 μl buffer mix (this contains the appropriate mixture of chemicals and ions that
are necessary for the enzymatic activity of Taq polymerase).
To the tube labeled N, add 11 μl of Primer-N Mix (containing Forward Primer, Reverse
Primer, and sterile distilled water).
The sequence of your Forward Primer is 5’ - ATG GCA TTC AGA ATG AGT G - 3’.
This anneals to the sequence of the Lambda genome between 21,973 bp and 21, 991 bp
(out of a total of 48,502 bp). The sequence of your Reverse Primer is 5’ – GCT TAT
GCA GCT GAC AGA GCC – 3’. If we were to tell you precisely where in the lambda
sequence this primer anneals, you could calculate the exact length (in bp) of your
expected amplified PCR product.
If you were a molecular biologist, you would often choose your primers to be upstream
and downstream of a gene you were interested in studying. You could then amplify the
gene in order to transform it into bacteria or to do other exciting things with in. In this
laboratory exercise, we are not amplifying a specific gene, but just a sequence found in
the lambda genome. The product of this PCR amplification is the same genetic
information that has been inserted into the pBLU plasmid you are using in your Exercise
8 Bacterial Transformation lab.
BC2004, Spring Semester 2005, Lab Exercise 9-3
If you want to see the entire 48,502 bp of the lambda genome, you can visit the following
website: http://www.ncbi.nlm.nih.gov/ of the National Center for Biotechnology
Information. Click on “Entrez”, then “Genome”, then “Viruses”, then “complete
alphabetical list”, and finally “bacteriophage lambda.”
You may want to revisit the NCBI website once you have completed this laboratory. You
can do a BLAST search for the reverse primer to find its exact location within the lambda
genome and use this to calculate the size that your PCR amplified product should be in
exact numbers of base pairs. Does this number match what you calculated from your gel?
To do a BLAST search, return to the website address listed above. Click on “BLAST”,
then “Nucleotide-nucleotide BLAST (blastn) [this will compare the nucleotide sequence
you type in to all the publically available nucleotide sequences, even those from humans.]
Type the sequence of the reverse primer into the box that says “search” to its left. Then,
click on the “BLAST!” button. A new window will open. It will tell you to wait a certain
amount of time before clicking on the “format” button, which will give you your results
page. Scroll down the results page until you see your sequence aligned with a sequence
from bacteriophage lambda.
4.
To the tube labeled R, add 11 μl of Primer-R Mix (containing Forward Primer, Reverse
Primer, and template DNA (in this case DNA from the bacteriophage Lambda).
5.
Add 5 μl of diluted Taq polymerase to each tube.
7.
Place your both of your tubes in the thermocycler. When tubes from all the groups have
been placed in the thermocycler, the cycler will be turned on. The PCR machine will first
heat the tubes to 95oC for 30 seconds and then to 80oC for 10 minutes. The first step
denatures any contaminating proteins that could interfere with the amplification reactions.
8.
The thermocycler will then proceed through 35 cycles of the following three steps:
denaturation, step 1: 30 seconds at 95o C.
annealing, step 2: 1.5 minutes 42o C.
primer extension, step 3: 1.5 minutes at 72o C.
The tubes will be left to go through the 35 cycles, and then the PCR machine will hold them at 4o
C overnight. The tubes will be removed and stored for you to analyze using agarose gel
electrophoresis later in the semester (Exercise 11).
BC2004, Spring Semester 2005, Lab Exercise 9-4
Materials
per PAIR of students:
2
1
1
1
sterile, nuclease-free 500-μl microfuge tubes (extras should also be available)
micropipettor, 5-50 uL
micropipettor, 0.5-10 uL
box sterile micropipette tips for EACH size of micropipettor
per group of FOUR students
1
ice bucket containing the following LABELED tubes
(a key may be provided on the lid of the ice bucket if necessary):
dNTP mix
buffer mix
Primer-N mix (primers only)
Primer-R mix (primers + DNA template)
diluted Taq polymerase
Thermalcycler, program:
35 cycles:
step 1:
step 2:
step 3:
step 4 :
step 5 :
50 μl
150 μl
30 μl
30 μl
25 μl
95oC
80oC
95oC
42oC
72oC
30 sec.
10 min.
30 sec.
1.5 min.
1.5 min.
step 6: Hold at 4oC until machine is stopped.
BC2004, Spring Semester 2005, Lab Exercise 9-5
This set-up will be done for you in advance of your lab section:
dNTP
Buffer Mix
Primer Mix N
Primer Mix R
Dilute Taq
Each
group of
FOUR
50 μl
150 μl
30 μl
30 μl
25 μl
Per section
(rounded up)
Per week (13 sections)
(rounded up)
220 μl
620 μl
150 μl
150 μl
100 μl
3 mL
9.0 mL
2.5 mL
2.5 mL
1.5 mL
dNTP mix, this recipe makes 1.0 mL (make three of these for the week)
Per sterile 1.5ml-microcentrifuge tube make 1.25mM dNTPstock:
add: 125l of each dATP (10mM solution)
dGTP (10mM solution)
dCTP (10mM solution)
dTTP (10mM solution)
add: 500l QH2O (sterile, nuclease-free)
mix, aliquot, label, and store in freezer
Buffer mix (4.5 : 3 : 18.5), this recipe makes 4.5 mL (make two of these for the week)
Per 15-mL sterile centrifuge tube, add:
779 l
10X Taq buffer
520 l
MgCl2, 25 mg/mL
3201 l
QH2O (sterile. nuclease-free)
Mix, label, aliquot, and store in freezer
DNA(1:50), makes 250 uL total (make once for the week), mix in sterile tube:
31 l Lambda DNA (506g/ml)
219 l QH2O (sterile)
Primers BIO2004F and BIO2004R, makes 2.5 mL total (make once for the week), mix in sterile
tube:
Want 5pmol/l so dilute the 200pmol/l stock of each
63 l
Primer stock
2437 l
QH2O (sterile, nuclease-free)
Primer Mix N (5:5:1), makes 2.5 mL (make once for the week)
1136 l
Primer BIO2004F
1136 l
Primer BIO2004R
228 l
QH2O (sterile, nuclease-free)
Mix in sterile tube, label, aliquot, and store in freezer
BC2004, Spring Semester 2005, Lab Exercise 9-6
Primer Mix R (5:5:1), makes 2.5 mL (make once for the week)
1136 l
Primer BIO2004F
1136 l
Primer BIO2004R
228 l
diluted Lambda DNA
Mix in sterile tube, label, aliquot, and store in freezer
Taq dilution (7 : 2 : 1, H2O : Taq polymerase : Taq Buffer), makes 100 l, make one for each
section, then aliquot into 25-uL aliquots for each group of 4 students.
700l QH2O (sterile, nuclease-free)
100l 10X Taq Buffer
Distribute 80l to LABELED 1.5ml centrifuge tubes and freeze.
On day of lab, thaw and add 20l Taq. Mix thoroughly, and divide into four 25-uL
aliquots, one for each group of FOUR students. Keep on ice after adding Taq.
BC2004, Spring Semester 2005, Lab Exercise 9-7