AluTPA PCR Kit

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DNA Van Program
University of Missouri Columbia
Thanks to Dr. Miriam Golomb AND NATIONAL STARCH
COMPANY
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TPA-25 ALU DNA Fingerprinting and PCRTM VAN
PROGRAM

• INSTRUCTOR
• SUSIE HELWIG
• NORTH KANSAS CITY HIGH SCHOOL

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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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PCR Procedures
Day 1
Day 2
Day 3
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TPA-25/D17S5

• Introduction
• Extract genomic DNA and prepare PCR samples
• Cycle samples
• Agarose gel analysis
• Hardy-Weinberg analysis

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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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What is PCR?

• PCR stands for Polymerase Chain Reaction
• DNA replication gone crazy in a tube!
• Devised by Kary Mullis in the 1980s 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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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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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 95C
• 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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DNA extraction
Cell membrane
Nuclear membrane
Mg
Genomic DNA
Mg
Mg
Heat disrupts membranes
Mg
Mg
Chelex binds released cellular Mg
Mg
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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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• 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 DONT SUCK THE DNA BACK INTO THE
  PIPETTE.

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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 dont 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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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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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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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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How does PCR work?

• 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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Denaturing Template
Heat causes DNA strands to separate
Denature DNA strands 94oC
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Annealing Primers

• Primers bind to the template sequence
• Taq Polymerase recognizes double-stranded
  substrate

3
5
Primers anneal 58oC
5
3
5
3
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Taq Polymerase Extends

• Taq Polymerase extends primer
• DNA is replicated

5
3
5
3
TAQ POLYMERASE
Extend 72oC
5
3
5
3
Repeat denaturing, annealing, and extending 30
cycles
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The target product is made in the third cycle
5
3
Cycle 1
5
3
Cycle 2
3
Cycle 3
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The target sequence

• Chromosome 8
• Intron of tissue plasminogen activator (TPA) gene
• Alu-TPA25

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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 doesnt 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.

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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 doesnt 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 dont.

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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
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Actual Alu-PCR Results
-
/-

400 bp
100 bp

-
/-
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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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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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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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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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Calculating Observed Genotypic Frequencies
Genotype / (p2) /-
(2pq) -/- (q2) Total (N)
of people 9
16 13
38 Observed frequency 0.24 0.42
0.34 1.00
Calculation / genotypic frequency
with genotype total number of
people (N) 9/38
0.24
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Calculating Allelic Frequencies

• Frequency of p number of p alleles 34
  0.45
• total alleles
  76
• Number of p alleles
• / 9 with two alleles 18
  alleles
• /- 16 with one alleles 16
  alleles
• Total 34
  alleles
• Total number of alleles 2N 2(38) 76

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Using Hardy-Weinberg

• Determine p2, 2pq, and q2 values
• Expected genotypic frequencies
• p 0.45 , so q 0.55 since p q 1
  p2 2pq
  q2 1
• (0.45)2 2 (0.45)(0.55) (0.55)2
  1
• 0.20 0.50 0.30
  1
• p2 0.20
• 2pq 0.50
• q2 0.30

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Calculate Expected Number of Genotypes

• Expected number of genotype
• Genotypic frequency x population number (N)
• Genotype Expected number
• / 0.20 x 38 8
• /- 0.50 x 38 19
• -/- 0.30 x 38 11

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Chi Squared Test

Observed
Expected (O-E)2
E /
9 8 0.13 /-
16 19 0.47 -/-
13 11 0.36
Total 0.96 X2 Critical Value
(from statistics table) 5.9 0.96 falls below
5.9 so the ratio is accepted.