AMPLIFICATION OF DNA

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AMPLIFICATION OF DNA

• WITH THE POLYMERASE CHAIN REACTION

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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

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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

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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.

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POLYMERASE CHAIN REACTION

• Requirements for PCR
• Template DNA
• Oligonucleotide primers
• Deoxyribonucleotide triphosphates (dNTPs)
• DNA polymerase

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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

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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

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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

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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

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POLYMERASE CHAIN REACTION

• The process
• Mixing of the components
• Denaturation of the template DNA
• Annealing of the primers
• Synthesis of complementary DNA
• Repeat

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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

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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

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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

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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)

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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

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POLYMERASE CHAIN REACTION
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POLYMERASE CHAIN REACTION
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POLYMERASE CHAIN REACTION

• The multiple cycles can be automated
• Requires a thermal cycler
• Repeated heating and cooling steps pre-programmed

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DNA ANALYSIS

• NUCLEAR AND MITOCHONDRIAL

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mtDNA ANALYSIS

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

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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

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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

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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

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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

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SINEs

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

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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

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Human Alu INSERTIONS

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

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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

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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

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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

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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

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Human Alu INSERTIONS

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

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Human Alu INSERTIONS

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