AluTPA PCR Kit - PowerPoint PPT Presentation

About This Presentation
Title:

AluTPA PCR Kit

Description:

DNA replication gone crazy in a tube! ... single DNA molecule can be replicated a billion fold in a few hours. This allows for the resurrection of DNA from ... – PowerPoint PPT presentation

Number of Views:448
Avg rating:3.0/5.0
Slides: 36
Provided by: valerieam2
Category:
Tags: alutpa | pcr | dna | kit

less

Transcript and Presenter's Notes

Title: AluTPA PCR Kit


1
DNA Van Program
University of Missouri Columbia
Thanks to Dr. Miriam Golomb AND NATIONAL STARCH
COMPANY
2
TPA-25 ALU DNA Fingerprinting and PCRTM VAN
PROGRAM
  • INSTRUCTOR
  • SUSIE HELWIG
  • NORTH KANSAS CITY HIGH SCHOOL

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

4
PCR Procedures
Day 1
Day 2
Day 3
5
TPA-25/D17S5
  • Introduction
  • Extract genomic DNA and prepare PCR samples
  • Cycle samples
  • Agarose gel analysis
  • Hardy-Weinberg analysis

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

7
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

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.

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 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.

10
DNA extraction
Cell membrane
Nuclear membrane
Mg
Genomic DNA
Mg
Mg
Heat disrupts membranes
Mg
Mg
Chelex binds released cellular Mg
Mg
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

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 DONT SUCK THE DNA BACK INTO THE
    PIPETTE.

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 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

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.

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

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.

17
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)

18
Denaturing Template
Heat causes DNA strands to separate
Denature DNA strands 94oC
19
Annealing Primers
  • Primers bind to the template sequence
  • Taq Polymerase recognizes double-stranded
    substrate

3
5
Primers anneal 58oC
5
3
5
3
20
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
21
The target product is made in the third cycle
5
3
Cycle 1
5
3
Cycle 2
3
Cycle 3
22
The target sequence
  • Chromosome 8
  • Intron of tissue plasminogen activator (TPA) gene
  • Alu-TPA25

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 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.

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 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.

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
26
Actual Alu-PCR Results
-
/-

400 bp
100 bp

-
/-
27
Alu repeats
  • Occurs 500,000 times in the human haploid genome
  • Named for the Alu I restriction site within the
    element

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

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

30
Alu and Population Genetics
Hardy-Weinberg Equilibrium
p2 2pq q2 1
p
q
pp
pq
p
/ p2 /- 2pq -/- q2
q
pq
qq
31
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
32
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

33
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

34
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

35
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.
Write a Comment
User Comments (0)
About PowerShow.com