DNA Technology

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DNA Technology Genomics

• CHAPTER 20

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DNA Cloning (gene cloning)

• methods
• insert gene of interest into a bacterial plasmid
  allow the recombinant bacterium to reproduce
• PCR (polymerase chain reaction)
• uses
• make multiple copies of a particular gene for
  use in basic research or insertion into another
  organisms genome
• harvest large quantities of the encoded protein
  from bacterial cultures carrying the cloned gene

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Restriction Enzymes

• enzymes that cut DNA at specific locations
  (restriction sites) yielding restriction
  fragments
• used to insert genes into bacterial plasmids
• the sticky ends of the restriction fragments can
  base-pair with the sticky ends of a cut plasmid
  then be sealed together by DNA ligase

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Genomic Libraries

• the process of DNA cloning usually results in
  thousands of different types of recombinant
  plasmids
• the complete set of these recombinant plasmids is
  called a genomic library
• one method for identifying bacteria with the
  recombinant plasmid containing the gene of
  interest is nucleic acid probe hybridization

white colonies
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Nucleic Acid Hybridization
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Expressing Eukaryotic Genes in Prokaryotic Cells

• Problem 1 bacterial cell doesnt recognize the
  eukaryotic genes promoter
• solution link the gene of interest to a highly
  active prokaryotic promoter that the bacterial
  cell will recognize
• this is accomplished by inserting the gene of
  interest into a cloning vector (plasmid) that
  contains a this promoter
• Problem 2 eukaryotic genes contain introns
  bacterial cells lack RNA-splicing machinery
• solution insert the cDNA (complimentary DNA)
  form of the gene

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Creating cDNA

• mRNA transcript of gene of interest is combined
  with reverse transcriptase to create a
  complimentary DNA strand
• the mRNA strand is then degraded and the
  complimentary, second strand of DNA is created by
  adding DNA polymerase
• the resulting cDNA molecule is modified by adding
  sticky ends so that it can be inserted into a
  cloning vector

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Eukaryotic Hosts for DNA Cloning

• yeast cells (which also contain plasmids) can be
  used as hosts for DNA cloning to avoid
  eukaryotic-prokaryotic incompatibility

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PCR (Polymerase Chain Reaction)

• alternative method for cloning a gene
• used primarily when the source of DNA is scanty
  or impure
• applications
• amplify fragments of ancient DNA
• amplify DNA from fingerprints or tiny amounts of
  blood, tissue, or semen found at crime scenes
• amplify DNA from embryonic cells to test for
  genetic disorders

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Restriction Fragment Analysis

• the DNA fragments produced by restriction enzyme
  digestion can be sorted by gel electrophoresis
• uses a gel an electric field to separate
  nucleic acids or proteins on the basis of size
  and charge
• the bands produced by gel electrophoresis can be
  analyzed for a particular gene using nucleic acid
  hybridization (this technique is called Southern
  blotting)

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Genome Mapping

• 3 stages
• linkage mapping
• physical mapping
• DNA sequencing

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Cytogenetic Maps

• the starting point for DNA mapping
• shows a chromosomes banding pattern and the
  location of specific genes on the chromosome
• the genes were located using a technique called
  FISH (fluorescence in situ hybridization) in
  which fluorescently labeled probes are allowed to
  hybridize to an immobilized array of whole
  chromosomes

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Linkage Maps

• a map of genetic markers based on recombination
  frequencies
• the markers can be
• genes
• RFLPs (restriction fragment length polymorphisms)
  different restriction fragment patterns on
  homologous chromosomes due to differences in
  their restriction sites
• simple sequence DNA sections of DNA that
  contain repeated short sequences

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Physical Maps

• express the distance (number of base-pairs)
  between markers
• made by cutting the DNA of a chromosome into a
  number of restriction fragments and then
  determining the original order of the fragments
  in the chromosome by looking for areas where the
  fragments overlap

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DNA Sequencing

• determining the complete nucleotide sequence of
  each chromosome
• techniques for DNA sequencing include
• dideoxy chain-termination method
• shotgun approach developed by Celera Genomics
• skips the linkage physical mapping stages
• takes random DNA fragments uses computer
  programs to sequence and order them

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shotgun approach
dideoxy chain- termination method
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Human Genome Project

• sequencing of the 22 autosomes and the sex
  chromosomes (largely completed in 2003)
• also involves mapping the genomes of other
  species important to biological research
• allows scientists to study whole sets of genes
  their interactions

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Searching for Protein-Coding Genes

• scientists used software to scan DNA sequences
  for
• transcriptional translational start and stop
  sequences
• RNA-splicing sites
• short coding sequences (expressed sequence tags)
  similar to those in known genes

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Determining Gene Function

• disable gene and observe the consequences
• in vitro mutagenesis a mutation is introduced
  in a cloned gene then returned to the cell
• RNA interference (RNAi) synthetic,
  double-stranded RNA molecules matching the
  sequence of a particular gene are used to trigger
  the breakdown or block the translation of the
  genes mRNA

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Studying Gene Expression

• to determine which genes are expressed in a
  particular cell, scientists can use DNA
  microarray assays
• isolate all the mRNAs made in a particular cell
• use them to make fluorescently-labeled cDNAs by
  reverse transcription
• apply the cDNA to a microarray containing all
  the organisms genes

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Comparing Genomes of Different Species

• allows scientists to determine evolutionary
  relationships between species
• helps scientists better understand the human
  genome
• provides scientists with a scaffold for
  organizing the DNA sequences of closely related
  species
• makes it easier for scientists to correlate
  phenotypic differences between species with
  particular genetic differences
• can be used to help study certain genetic diseases

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Future Areas of Research

• Proteomics studying the full protein sets
  encoded by genomes
• studying human evolution the history of human
  populations through the identification of SNP
  sites
• SNPs (single nucleotide polymorphisms) are single
  base-pair variations that account for the genetic
  differences between individuals (which is only
  about 0.1)

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Applications of DNA Technology

• Medicine
• Pharmaceuticals
• Forensics
• Environmental Cleanup
• Agriculture

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Medical Applications

• diagnosing diseases
• detecting susceptibility of diseases
• treating diseases
• gene therapy alteration of an afflicted
  individuals genes

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Pharmaceutical Applications

• manufacture large quantities of human proteins
  (ex insulin)
• production of vaccines
• pharmacogenomics

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Forensic Applications

• DNA fingerprinting

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Environmental Cleanup

• genetically engineered microbes are being used or
  created to
• remove heavy metals from the environment
• cleanup highly toxic mining wastes
• degrade chlorinated hydrocarbons in wastewater
• breakdown chemical released during oil spills
• breakdown toxic wastes in waste dumps

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Agricultural Applications

• make vaccines growth hormones for treating farm
  animals
• creating transgenic animals plants