1. University of Missouri Columbia
DNA Van Program
University of Missouri Columbia
Thanks to Dr. Miriam Golomb
AND NATIONAL STARCH COMPANY
9/21/2018

2. DNA Fingerprinting and PCRTM VAN PROGRAM
TPA-25 ALU
DNA Fingerprinting and PCRTM VAN PROGRAM
INSTRUCTOR:
SUSIE HELWIG
NORTH KANSAS CITY HIGH SCHOOL
9/21/2018

3. Why teach Polymerase Chain Reaction (PCR) and DNA Fingerprinting?
Powerful teaching tool
Real-world connections
Link to careers and industry
Laboratory extensions
Hook Math, English and History students into Biology
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4. PCR Procedures
Day 1
Day 2
Day 3
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5. TPA-25/D17S5 Introduction Extract genomic DNA and prepare PCR samples
Cycle samples
Agarose gel analysis
Hardy-Weinberg analysis
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6. What will we learn with the TPA-25 laboratory?
Introduce the polymerase chain reaction (PCR) technique
Apply PCR to population genetics
Directly measure human diversity at the molecular level
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7. What is PCR? PCR stands for Polymerase Chain Reaction
DNA replication gone crazy in a tube!
Devised by Kary Mullis in the 1980’s it is a simple and powerful way of making unlimited numbers of copies of a DNA template.
A single DNA molecule can be replicated a billion fold in a few hours. This allows for the resurrection of DNA from fossils like a Neanderthal.
Uses Taq Polymerase heat-resistant DNA polymerase from Thermus aquaticus
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8. What are practical uses for PCR technology in society today?
A single DNA molecule can be replicated a billion fold in a few hours. This allows for the resurrection of DNA from fossils like a Neanderthal.
Small amounts of DNA left at crime scenes can be lifted and analyzed to find the criminal.
Paternity testing and Population genetics.
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9. What is TAQ Polymerase?
Taq is a nickname for Thermus aquaticus, a bacterium that survives and reproduces in hot springs
Taq polymerase is is derived from bacteria that live in hot springs and are among the few enzymes that can function at very high temperatures. Withstands temperatures up to 95°C
Unlike other polymerase Taq can survive in extreme hot temperatures and the enzyme does not denature like most.
It is thermophilic because it loves heat.
This has become a great break through in PCR because it can survive the high heat needed.
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10. DNA extraction Heat disrupts membranes Chelex binds
Cell membrane
Nuclear membrane
Mg++
Genomic
DNA
Mg++
Mg++
Heat disrupts membranes
Mg++
Mg++
Chelex binds
released cellular Mg++
Mg++
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11. Micropipette Use Twist dial to desired volume. Be careful
this is where you can break these.
Pick up pipette tip by pressing firmly on
the tip located in the box.
Press plunger to first, soft stop
Insert pipette tip into solution to be
transferred
Slowly release plunger to retrieve liquid
Move pipette tip into desired tube
Press plunger past first stop to second,
hard stop to transfer liquid
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12. LOADING DNA IN WELLS BY PIPETTING
MAKE SURE THE PIPETTE READS THE CORRECT AMOUNT TO BE ADDED TO THE WELL.
PRESS THE PLUNGER TO THE FIRST STOP AND SLOWLY RELEASE TO EXTRACT THE DNA FROM THE TUBE.
CAREFULLY ADD DNA BY INSERTING THE TIP OF THE PIPETTE INTO THE BUFFER IN THE MIDDLE OF THE WELL.
BE CAREFUL NOT TO TOUCH THE BOTTOM OF THE WELL.
PRESS THE PLUNGER TO THE FIRST STOP ONLY AND RELEASE THE DNA INTO THE WELL. DO NOT PRESS TO THE SECOND STOP. THIS WILL RELEASE AIR INTO THE WELL. KEEP THE PLUNGER DOWN AT THE FIRST STOP AND TAKE THE PIPETTOR OUT OF THE BUFFER. THIS WILL ENSURE THAT YOU DON’T SUCK THE DNA BACK INTO THE PIPETTE.
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13. Isolation of Cheek Cell DNA
Cheek cells will first be collected by rinsing out your mouth with a saline solution.
Why saline?
The salt keeps the cells in proper osmotic balance so they don’t burst. (Isotonic)
The cells are then spun in a centrifuge and boiled with a resin (CHELEX). Chelex is an ion-exchange resin.
Boiling causes the cells to disrupt and the DNA (now in single-stranded form) extracted in water.
The chelex removes impurities that would interfere with PCR.
The Chelex™ protects the sample from DNAases* that might remain after the boiling and could subsequently contaminate the samples
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14. What does CHELEX do?
The chelex removes impurities that would interfere with PCR.
The Chelex™ protects the sample from DNAases* that might remain after the boiling and could subsequently contaminate the samples.
DNAases are enzymes which occur naturally in all body tissues.
They cut DNA, rendering it unsuitable for PCR. Magnesium ions are essential cofactors for DNAases.
Chelex™ resin binds with cations including Mg+. By binding with the magnesium ions, the Chelex™ resin renders DNAases inoperable, thus protecting DNA from their action.
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15. What does the PCR mix contain?
PCR mix contains
reagents needed for PCR amplification
red and yellow loading dyes
glycerol to make samples sink
Amplified samples can be loaded directly onto agarose gels
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16. What is needed for PCR? Template (the DNA you are exploring)
Sequence-specific primers flanking the target sequence
Forward
Reverse
Nucleotides (dATP, dCTP, dGTP, dTTP)
Magnesium chloride (enzyme cofactor)
Buffer, containing salt, maintains tonicity, regulation of ions and pH.
Taq Polymerase is responsible for adding nucleotides to the newly formed DNA strand.
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17. How does PCR work? Repeat multiple cycles (30)
Heat (94o-C) to denature DNA strands
Cool (64oC) to anneal primers to template
Warm (72oC) to activate Taq Polymerase, which extends primers and replicates DNA
Repeat multiple cycles (30)
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18. Denaturing Template Heat causes DNA strands to separate5’ 3’ 3’ 5’
Denature DNA strands 94oC 3’ 5’ 5’ 3’
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19. Annealing Primers Primers bind to the template sequence
Taq Polymerase recognizes double-stranded substrate 3’ 5’ 3’ 5’
Primers anneal 58oC 5’ 3’ 3’ 5’ 3’ 5’ 5’ 3’
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20. Taq Polymerase Extends
Taq Polymerase extends primer
DNA is replicated 3’ 5’ 5’ 3’ 3’ 5’ 5’ 3’
TAQ POLYMERASE
Extend 72oC 5’ 3’ 3’ 5’ 3’ 5’ 5’ 3’
Repeat denaturing, annealing, and extending 30 cycles
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21. The target product is made in the third cycle5’ 3’ 3’ 5’
Cycle 1 3’ 5’ 5’ 3’ 5’ 3’
Cycle 2 3’ 3’ 5’
Cycle 3
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22. The target sequence Chromosome 8
Intron of tissue plasminogen activator (TPA) gene
Alu-TPA25
Exon 8
Exon 9
Exon 10 5’ 3’
Alu
Intron 8
Amplified Region
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23. Alu-TPA25
The part of your DNA that actually codes for anything is only about 5% of your total chromosomal DNA or genome.
The remaining 95% consists of stretches between genes, and interrupting sequences within genes (introns). Much of this non-coding DNA is thought to be “junk” in that it doesn’t affect phenotype.
This junk “ALU” makes up about 5% genomic DNA as much as all our genes put together.
The presence of ALU sequence in our chromosomes is thanks to an ancient retrovirus which once infected our ancestors. This virus a distant relative of the AIDS virus, copied cellular RNA sequences into DNA and stuck them in at random chromosomal locations.
9/21/2018

24. Alu-TPA25 (continued)
One such ALU sequence is called TPA-25 and is found within an intron of the gene for tissue plasminogen activator. The TPA-25 gene encodes a protein that prevents blood clotting inside tissue.)
Since it is located within a non-coding protein of a gene it doesn’t affect gene expression.
This Alu insertion seems to have happened in the past million years, in a recent human ancestor. As a result some human chromosomes have it and others don’t.
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25. PCR Results….
Alu-TPA25 is dimorphic so there are two possible PCR products:
100 bp
400 bp
No insertion: 400 bp
Alu insertion: 400 bp
330 bp each
300 bp Alu insert
Exon 8
Exon 9
Exon 10 5’ 3’
Intron 8
Amplified Region
Alu
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26. Actual Alu-PCR Results+ -
+/-
400 bp
100 bp + -
+/-
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27. Alu repeats Occurs >500,000 times in the human haploid genome
Named for the Alu I restriction site within the element
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28. Evolutionary Significance of Alu-TPA 25
Highly conserved
Inserted in the last 1,000,000 years
Dimorphic (+/+, +/-, -/- )
Used in population genetics, paternity analysis, and forensics
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29. To estimate frequency of Alu
within a population:
Amplify Alu-region from representative sample population
Calculate the expected allelic and genotypic frequencies
Perform Chi-squared Test
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30. Alu and Population Genetics
Hardy-Weinberg Equilibrium
p2 + 2pq + q2 = 1 p q pp pq p
+/+ = p2
+/- = 2pq
-/- = q2 q pq qq
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31. Calculating Observed Genotypic Frequencies
Genotype /+ (p2) /- (2pq) /- (q2) Total (N)
# of people
Observed frequency
Calculation:
+/+ genotypic frequency = # with genotype
total number of people (N)
= 9/38 =
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32. Calculating Allelic Frequencies
Frequency of p = number of p alleles = 34 = 0.45
total alleles
Number of p alleles =
+/+ = 9 with two + alleles = alleles
+/- = 16 with one + alleles = alleles
Total = alleles
Total number of alleles = 2N = 2(38) = 76
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33. Using Hardy-Weinberg Determine p2, 2pq, and q2 values=
Expected genotypic frequencies
p = 0.45 , so q = since p + q = p pq q = 1
(0.45) (0.45)(0.55) + (0.55)2 = 1
= 1
p2 = 0.20
2pq = 0.50
q2 = 0.30
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34. Calculate Expected Number of Genotypes
Genotypic frequency x population number (N)
Genotype Expected number
+/ x 38 = 8
+/ x 38 = 19
-/ x 38 = 11
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35. Chi Squared Test +/+ 9 8 0.13 +/- 16 19 0.47 -/- 13 11 0.36
Observed Expected (O-E)2 E +/ +/ -/
Total
X2 Critical Value (from statistics table) = 5.9
0.96 falls below 5.9 so the ratio is accepted.
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