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AMPLIFICATION OF DNA

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Amplification of very small amounts of DNA. Further analysis then possible. DNA fingerprinting. Paternity testing, crime scene analysis, etc. ... – PowerPoint PPT presentation

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Title: AMPLIFICATION OF DNA


1
AMPLIFICATION OF DNA
  • WITH THE POLYMERASE CHAIN REACTION

2
POLYMERASE CHAIN REACTION
  • Polymerase chain reaction (PCR)
  • Developed by Kary Mullis in 1985
  • 1993 Nobel Prize in Chemistry
  • Theoretical basis for Jurassic Park
  • Means of copying DNA

3
POLYMERASE CHAIN REACTION
  • Polymerase chain reaction
  • A small amount of a specific DNA molecule can be
    exponentially amplified
  • A single gene or region of DNA can be
    specifically amplified
  • Possible even if many other DNAs are present
  • A single gene can be amplified from a DNA sample
    containing all of the human chromosomes

4
POLYMERASE CHAIN REACTION
  • Uses of polymerase chain reaction
  • Amplification of very small amounts of DNA
  • Further analysis then possible
  • DNA fingerprinting
  • Paternity testing, crime scene analysis, etc.
  • Detection of hereditary or infectious diseases
  • e.g., HIV detection in newborns with HIV-positive
    mothers
  • etc.

5
POLYMERASE CHAIN REACTION
  • Requirements for PCR
  • Template DNA
  • Oligonucleotide primers
  • Deoxyribonucleotide triphosphates (dNTPs)
  • DNA polymerase

6
POLYMERASE CHAIN REACTION
  • Requirements for PCR
  • Template DNA
  • Contains the specific segment to be amplified
  • Generally contains many other segments of DNA
  • Isolation of the DNA of interest from other DNAs
    is not necessary

7
POLYMERASE CHAIN REACTION
  • Requirements for PCR
  • Oligonucleotide primers
  • Short DNA sequences
  • Complementary to the ends of the DNA segment to
    be amplified
  • Some prior knowledge of the sequence of interest
    is required
  • Present in very high concentrations

8
POLYMERASE CHAIN REACTION
  • Requirements for PCR
  • Deoxyribonucleotide triphosphates (dNTPs)
  • Monomers used to form the DNA polymer
  • Must be present in their trinucleotide form
  • Provides the energy for polymerization

9
POLYMERASE CHAIN REACTION
  • Requirements for PCR
  • DNA polymerase
  • A thermostable enzyme is used
  • Isolated from the bacterium Thermus aquaticus
  • Taq polymerase
  • Not denatured by the multiple heating steps in
    the process

10
POLYMERASE CHAIN REACTION
  • The process
  • Mixing of the components
  • Denaturation of the template DNA
  • Annealing of the primers
  • Synthesis of complementary DNA
  • Repeat

11
POLYMERASE CHAIN REACTION
  • The process
  • Mixing of the components
  • The various components are mixed in small tubes
  • Once mixed, the entire process can be completed
    without adding any additional materials

12
POLYMERASE CHAIN REACTION
  • The process
  • Denaturation of the template DNA
  • Heat treatment denatures the double-stranded
    template
  • Hydrogen bonds holding the two complementary
    strands together are easily broken
  • Covalent bonds within each strand are unaffected

13
POLYMERASE CHAIN REACTION
  • The process
  • Annealing of the primers
  • Oligonucleotide primers bind to complementary
    sequences on the template as the temperature is
    lowered
  • Excess of primers ensures efficient binding with
    template
  • Competition for binding with complementary strand
    of template DNA

14
POLYMERASE CHAIN REACTION
  • The process
  • Synthesis of complementary DNA
  • Taq polymerase catalyzes the synthesis of
    complementary DNA strands
  • Thermostabile enzyme is not irreversibly
    denatured by earlier heat treatment(s)

15
POLYMERASE CHAIN REACTION
  • The process
  • Repeat
  • A single cycle can double the amount of the
    amplified DNA
  • Successive cycles can continue this doubling
  • The DNA of interest is exponentially amplified

16
POLYMERASE CHAIN REACTION
17
POLYMERASE CHAIN REACTION
18
POLYMERASE CHAIN REACTION
  • The multiple cycles can be automated
  • Requires a thermal cycler
  • Repeated heating and cooling steps pre-programmed

19
DNA ANALYSIS
  • NUCLEAR AND MITOCHONDRIAL

20
mtDNA ANALYSIS
  • Mitochondria
  • Endosymbiotic organelles
  • Possess their own DNA
  • Present in multiple copies
  • Maternally inherited

21
mtDNA ANALYSIS
  • Human mitochondrial DNA
  • Approximately 16,569 bp in length
  • Sequence determined in 1981
  • Two general regions
  • Coding region
  • Responsible for the production of proteins and
    functional RNAs
  • Control region
  • Responsible for the regulation of the mtDNA

22
mtDNA ANALYSIS
  • Human mitochondrial DNA
  • Two regions within the control region are highly
    variable within the human population
  • Hypervariable region I (HV1)
  • 342 bp in length
  • Hypervariable region II (HV2)
  • 268 bp in length

23
mtDNA ANALYSIS
  • Human mitochondrial DNA
  • Hypervariable regions accumulate mutations at
    approximately 10 times the rate of nuclear DNA
  • Results in unique patterns of single nucleotide
    polymorphisms (SNPs)
  • Inherited through generations
  • Forensic examination are performed using these
    two regions
  • Regions compared to reference sequence

24
mtDNA ANALYSIS
  • Human mitochondrial DNA
  • Can be used to construct a human family tree
  • Shows ancestral relationships between modern
    populations
  • Humans arose in Africa perhaps 200,000 years ago
  • Mitochondrial Eve
  • Named after the mythical figure
  • Various migrations populated Africa and the rest
    of the world

25
SINEs
  • Short interspersed elements (SINEs)
  • Comprise roughly 45 of the human genome
  • Alu elements are the most prevalent

26
Human Alu INSERTIONS
  • DNA between individuals is identical or very
    similar in many regions
  • e.g., Highly conserved genes
  • Many regions of human chromosomes display great
    diversity
  • Such regions are termed polymorphic
  • Many polymorphisms exist in the estimated 95
    non-coding DNA

27
Human Alu INSERTIONS
  • Polymorphisms are useful in many ways
  • Diagnosis of genetic disease
  • Forensic identification
  • Paternity testing
  • etc.

28
Human Alu INSERTIONS
  • Alu elements
  • Components of primate non-coding DNA
  • Approximately 300 bp in length
  • Named for the single Alu I endonuclease
    recognition site near the center of the element
  • Likely derived from a gene encoding the RNA
    component of the signal recognition particle
  • Labels proteins for export from the cell

29
Human Alu INSERTIONS
  • Alu elements
  • Examples of jumping genes
  • Transposable genetic elements
  • Copy self and insert in new location in the
    genome
  • Selfish DNA
  • Encodes no protein
  • Appears to exist solely for its own replication

30
Human Alu INSERTIONS
  • Alu elements
  • Present in an estimated 1,000,000 copies in the
    human genome
  • Comprises 10 of the human genome
  • Each Alu insertion is the fossil of a unique
    transposition event occurring once in primate
    evolution
  • All primates sharing an Alu allele are descended
    from a common ancestor in which the transposition
    first occurred

31
Human Alu INSERTIONS
  • Alu elements
  • 500 2,000 Alu elements are restricted to the
    human genome
  • Inserted after the human-chimpanzee split
  • Most Alu mutations are fixed
  • Both paired chromosomes have an insertion at the
    same position
  • About 25 of the human-specific Alus are not
    fixed
  • Dimorphic
  • May be present or absent on each chromosome of a
    pair

32
Human Alu INSERTIONS
  • Dimorphic Alu elements
  • Useful DNA markers
  • Human population studies
  • Forensic studies
  • Paternity analysis
  • etc.

33
Human Alu INSERTIONS
  • Alu elements
  • Specific loci can be amplified via PCR
  • e.g., A short DNA sequence from human chromosome
    16
  • PV92 sequence
  • Amplified DNA is separated via gel
    electrophoresis
  • Presence or absence of Alu element can be
    detected
  • Personal DNA fingerprint is created
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