Lab 8:
Amplification of the tPA Locus
using the Polymerase Chain Reaction (PCR)
Pre Lab Readiness
•
Genetics is the study of heredity: How biological information is
transferred from one generation to the next as well as how that
information is expressed within an organism.
•
DNA Replication is the process of making an identical copy of a
section of (double-stranded) DNA, using existing DNA as a template
for the synthesis of new DNA strands. In humans and other eukaryotes,
replication occurs in the cell nucleus.
•
Genes are units of information about specific traits. They are passed
from parents to offspring. Each gene has a specific location on a
chromosome.
•
Genotype is the genetic constitution (the genome) of an individual or
an organism.
Pre Lab Readiness (continued)
•
Phenotype is the observable physical or biochemical characteristics of an
individual or organism.
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Alleles are alternative forms of a gene. If two alleles of a pair are the same, it is a
homozygous condition. If the two alleles are different, this is called a
heterozygous condition.
•
Polymerase Chain Reaction (PCR) is an in vitro process that yields millions of
copies of desired DNA through repeated cycling of a reaction involving the DNA
polymerase enzyme.
•
Thermalcycler is a laboratory apparatus used to amplify segments of DNA via
the polymerase chain reaction (PCR) process. The device has a thermal block
with holes where tubes holding the PCR reaction mixtures can be inserted. The
cycler then raises and lowers the temperature of the block in discrete, preprogrammed steps.
Why are we doing this?
To use a very powerful
technique to amplify buccal
cell DNA and determine
student genotypes!
Lab 8 terms
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Buccal Cells are cells from the inner cheek lining.
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Chelex beads bind divalent magnesium ions (Mg++) which
serve as cofactors for nucleases that will degrade DNA.
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Nuclease a family of enzymes that will degrade nucleic
acids (DNA).
•
Amplification an increase in the number of copies of a
specific DNA fragment.
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Intron segment of a gene that does not code for protein.
Introns are transcribed into mRNA but are removed before
being translated into protein.
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Exon segment of a gene that encodes regions of protein
Obtaining your DNA Sample
1.
2.
3.
4.
5.
6.
7.
Obtain numbered chelex tube (record the number in your notebook)
Use a sterile pipette tip to scrap the inside of both cheeks
Add cheek cells to Chelex tube
Boil for 10 minutes (lyse cells and destroy nuclease)
Centrifuge for 5 minutes at ~10,000 rpm
Obtain a PCR tube. Label top and side with the number on chelex tube
Transfer 5uL of DNA to PCR tube. AVOID chelex beads!
What is PCR?
•
PCR is a an extraordinarily powerful technique used to
amplify a small sample of DNA by repeated cycles of
denaturing and replication to an amount large enough to
visualize. Visualization of the sample is generally achieved
by ethidium bromide staining using agarose gel
electrophoresis.
The PCR technique was invented by Dr. Kary Mullis in 1983.
He was awarded the Nobel Prize in Chemistry in 1993.
How and Where is PCR used?
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PCR is commonly used to produce many copies of a
selected gene segment or locus of DNA.
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In criminal forensics, PCR is used to amplify DNA
evidence from small samples that may have been left
at a crime scene.
•
PCR can be used to amplify DNA for genetic disease
screening.
?
How Does PCR Work
The PCR process usually consists of a series
of twenty to thirty-five cycles. Each cycle
consists of three steps.
Step 1: Denaturing temperature is raised to 9496°C to break hydrogen bonds
Step 2: Annealing temperature is lowered to
56°C to allow primers to attach to the target
sequence
Step 3: Elongation or Extension temperature is
Raised 72°C Taq polymerase binds and adds
nucleotides to build new DNA strands
Building new DNA Fragments
PCR : What do we need?
Template DNA – Which contains the DNA fragment to be amplified
Primers – are 2 short single stranded polynucleotides that flank the sequence to be amplified
 Forward
 Reverse
Nucleotides – the building blocks for new DNA strands dATP, dCTP, dGTP, dTTP
Magnesium chloride - enzyme cofactor helps Taq Polymerase work efficiently
Buffer – a solution which maintains the pH and provides a suitable chemical environment for PCR
Taq DNA polymerase is a temperature resistant enzyme which builds DNA strands. Taq was isolated from the
bacterium Thermus aquaticus, which normally lives in hot springs in temperatures around 100° C. Taq is stable
under the extreme temperature conditions of PCR.
What do we use?
Genomic DNA sample (5 µL)
Master mix (20 µL/reaction):
*2.5 µL 10x PCR buffer w/o MgCl2 OR 3.25 µL 10x PCR
buffer w/ MgCl2
0.5 µL dNTP’s (10 mM)
2.5 µL Forward primer (4pM/ µL)
2.5 µL Reverse primer (4pM/ µL)
0.15 µL Taq polymerase
11.1 µL ddH2O
*Note: if you are using 10x PCR buffer w/o MgCl2, you will need to add 0.75 µL
MgCl2 (50 mM)
Chromosome 8
The region we will be amplifying is located in an intron (non
translated region), of the tPA gene located on Chromosome 8.
Chromosome 8 and the tPA Gene
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The diagram indicates the intron
we will be targeting for PCR.
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The intron that we will be
targeting for amplification is
dimorphic, which means the
locus has two forms.
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one form carries a 300 bp DNA
fragmentknown as an Alu
element
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the second form of the locus
does not carry this fragment.
The tPA Gene
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The locus (ALU) we will amplify is located in the tissue
Plasminogen Activator (tPA) gene.
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This gene is on chromosome 8.
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The gene codes for a protein that is involved with dissolving
blood clots.
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tPA is a protein given to heart attack victims to reduce the
incidence of strokes.
What are ALU elements?
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Alu elements are short, around 300
bp, DNA fragments that are distributed
throughout our genome.
•
Estimated that we may carry over
1,000,000 copies of this fragment.
Possible Genotypes and Expected
Results:
1. Marker
2. Homozygous Alu +
(400bp sequence)
3. Homozygous Alu –
(100bp sequence)
4. Heterozygous
(400bp sequence and
100bp sequence)
Loading and Running Gels
• Carefully remove comb from gel, put dams down on both ends
of the gel tray.
• Place gel tray into gel box with buffer ensuring that the wells
are closest to the black electrode!
• Add 4ul of orange G (loading dye) to your PCR sample and
load 20ul of your sample into one of the wells.
• Once everyone has loaded their sample plug red electrode to
red and black electrode to black on power supply. Be sure the
power on the power supply is turned OFF before connecting
electrodes!
• Adjust voltage to 135-160 volts and allow gel to run for about
15-30 minutes.
Loading and Running Gels
continued
• After gel run is complete, turn off power
supply and unplug electrodes
• Your gel is now ready to be stained and
photographed
• Answer questions at the end of Lab 8
Gel Loading Techniques
Gel Loading Techniques