Lab 8: Tpa Locus: PCR

Lab 8: Tpa Locus: PCR
Laboratory 1
8
Genomic DNA
Extraction from Buccal Epithelial Cells
The purpose of this lab is to collect a DNA sample
from the cells that line the inside of your mouth and
to use this sample to explore one of the most powerful
techniques in molecular biology—the Polymerase Chain
Reaction (PCR). Although PCR has many applications,
it is commonly used to produce many copies of a
selected gene segment or locus of DNA. In criminal
forensics, for example, PCR is used to amplify DNA
evidence from small samples that may have been left at
a crime scene. A skilled technician can even obtain a
DNA sample left by the tongue on the back of a postage
stamp used to send a letter. DNA samples obtained in
this manner have been used for PCR in several highprofile criminal cases.
To obtain your DNA sample, you’ll use a toothpick
to obtain some buccal epithelial cells. The cells will be
transferred to a solution containing Chelex beads. The
Chelex beads will bind divalent magnesium ions (Mg++).
These ions often serve as cofactors for nucleases that
will degrade your DNA sample and may interfere with
the enzyme (Taq polymerase) used in the reaction. By
removing magnesium ions, the degradation of genomic
DNA by nucleases is reduced. This mixture will be
placed into boiling water to lyse the cells and liberate
the DNA.
8.1
The mixture of your genomic DNA, cell debris and
Chelex beads is then centrifuged to pellet the cell debris
and Chelex, while keeping your genomic DNA in the
supernatant. This is a quick and easy way to separate
genomic DNA from the cell debris. The DNA sample,
however, is far from pure as it contains proteins and
nucleic acids from organisms that were in your mouth
at the time of sampling (mostly bacteria and food).
Generally, these contaminants do not inhibit PCR
because the process uses specific primers, short segments
of DNA about 25 nucleotides in length that can be
made to target only human genomic DNA. Therefore,
if the supernatant carries some foreign DNA, it should
not interfere with the targeting of the human-specific
primers. A more detailed description of PCR and the
role that primers play will be discussed later in this lab.
Although we are using buccal epithelia as a DNA
source, other tissues could have been used. Here are some
DNA yields from other human tissues: Blood yields 40
μg/ mL; hair root yields around 250 ng/mL; muscle yields
around 3 μg/ mL; and sperm yields 3.3 pg/cell.
The second part of this lab involves the actual PCR.
You will use the sample of genomic DNA you just
collected as a target for the PCR reaction.
Version 07/01/2012
Laboratory 1
8
Materials
Reagents
0.5 mL of 10% Chelex solution
Master mixes I and II
Equipment & supplies
Boiling water bath
Microcentrifuge
Thermal cycler
Microfuge tube
Permanent marker
Sterile toothpicks
Methods
Getting your sample ready...

1 O
btain a Chelex tube. Note that this tube is identified with a number and
letter. Record this number and letter in your notebook. Only you will know this
anonymous code.
2 T o collect buccal epithelial cells, use a sterile toothpick or yellow pipette

tip to gently scrape the inside of both cheeks. This procedure should be
noninvasive, so don’t draw blood.
3 T ransfer the cells that you have removed from the toothpick to the Chelex

tube. Vigorously twirl the toothpick with the Chelex resin to knock off the cells
from the toothpick.
This is important; you want to get as many cells off the toothpick and into the
Chelex tube as possible.
4 C
lose the Chelex tube tightly. Take the tube to the boiling water bath or 100°C hot

block. Boil or heat the cells for 10 minutes. This heating will lyse the cells and
help to destroy some of the nucleases, which degrade the DNA
5 Use the high-speed centrifuge to spin down the Chelex and cell debris.

6 Using
the P-20 pipette and a clean pipette tip, carefully remove 20 μL of

supernatant and place it into a clean 1.5 mL microfuge tube. Avoid aspirating
Chelex beads as this will inhibit the downstream PCR procedure. Label this
tube with your personal, anonymous code (number).
7 I f your sample is not used immediately, leave this sample at the front of

the room in the rack labeled “Genomic DNA Samples.” These samples will be
placed into the refrigerator overnight and returned to you for the next lab.
Your genomic DNA sample can be kept in the refrigerator at 4°C
or freezer at _–20°C until you are ready to run the PCR reaction.
8.2
8
Laboratory 1
Amplification of the tPA Locus using the
Polymerase Chain Reaction
The polymerase chain reaction, PCR, is a molecular
biology technique that was discovered by Kary Mullis
during the early 1980s. The technique uses some elegant
chemistry and precise thermal cycling of reactants to
target and amplify a specific location (locus) along the
DNA molecule. From a single DNA target, PCR can
produce more than one billion copies of the target in
about 1.5 hours. This powerful chemistry proved to be
so significant that Mullis was awarded the Nobel Prize
for Chemistry in 1993. Today, PCR is considered to be a
standard protocol in molecular biology and hundreds of
scientific papers using this technique are published each
year.
The locus we will amplify is located in the t issue
P lasminogen A ctivator (tPA) gene. This gene is carried
on chromosome 8, the gene codes for a protein that is
involved with dissolving blood clots. tPA is a protein
administered to heart attack victims to reduce the
incidence of strokes. The region we will be amplifying,
however, is located in an intron (nontranslated region)
of the tPA gene.
Chromosome 8
,
3,
e
5
Exon
Intron
i
tPA gene
e
i
e
i
e
,
5,
3
300bp
primer
Alu element
primer
Alu+ allele
400bp
primer
primer
Alu- allele
100bp
The intron that we will be targeting for amplification
is dimorphic, which means the locus has two forms. One
form carries a 300 bp DNA fragment known as an Alu
element and the second form of the locus does not carry
this fragment. Therefore, when we examine this locus,
8.3
we find that it may or may not carry an Alu element. The
figure below indicates the intron we will be targeting for
PCR.
Alu elements are short, around 300 bp, DNA fragments
that are distributed throughout our genome. It has been
estimated that we may carry more than 1,000,000 copies
of this fragment. The Alu element appears to be a part
of the DNA coding for an RNA molecule that aids in
the secretion of newly formed polypeptides from the
cell. Unless it happens to become inserted into an exon
or coding region, it has little if any effect on protein
function.
Amplification of DNA by PCR is dependent upon
primers that target specific loci. The two primers that we
will be using have unique nucleotide sequences that are
complementary to only one locus in the human genome.
The primer sequences are:
Forward primer: 5’ G T A A G A G T T C C G T A A C G G A C A G C T 3’
Reverse primer: 5’ C C C C A C C C T A G G A G A A C T T C T C T T 3’
Laboratory 1
8
Methods
1
Obtain the genomic DNA sample with your number and the
PCR tube labeled with your anonymous code number.
2
The PCR tube already contains Master
mix I. Master mix I contains the two
primers that target the tPA locus, dNTP’s
(deoxynucleotide triphosphates: ATP, TTP, CTP
and GTP), PCR buffer, molecular grade water
(very pure) and Taq polymerase.
Code
Number
3
Using a clean pipette tip, add 5 µL of your genomic DNA to
this PCR tube. Carefully add your DNA sample directly into
the 10µL of Master mix I. Do this without creating bubbles.
4
Carefully cap the PCR tube. This is a very thin walled tube
so avoid crushing it, but make sure that the cap is firmly
seated over the opening of the tube.
5
Place your PCR tube into the ice bucket by the thermal
cycler.
6
The instructor will add 10µL of Master mix II, containing
MgCl2, just before placing your samples into the thermal
cycler. Taq polymerase, an enzyme from the bacterium
Thermus aquaticus, requires Mg++ ions as cofactors to
activate it.
7
Discard your genomic DNA sample.
8
Your instructor will run the PCR reaction at another time.
9
After the PCR run, 15 µL of your PCR product will be loaded
into a 2% agarose gel. The gel will be stained and photodocumented. These steps will be done by your instructor.
8.4
8
Laboratory 1
Conclusions
These questions should be answered after you have seen the PCR results.
1
What is your genotype with respect to the tPA gene?
2
With respect to the tPA gene, how many genotypes are possible?
3
Complete the following table using the class information.
Begin by determining the number of each genotype present in your class.
Genotypes
Alu+ alleles*
Alu– alleles
Total Alu+ alleles
Total Alu– alleles
Alu+ Alu+
Alu+ Alu–
Alu– Alu–
* The term “alleles” refer to forms of a gene or DNA sequence.
4
Calculate the frequency (percentage) of each genotype present.
5
Calculate the frequency of each allele present in your class.
6
Compare your results with other classes.
Is there a difference in relative frequencies of genotypes and alleles?
8.5
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