DNA Technology and the Human Genome

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DNA Technology and the Human Genome

• Chapter 12

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Bacteria as Tools for Manipulating DNA
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DNA Technology

• The Human Genome Project
• Better understand diseases and their causes
• Gene therapy
• The production of vaccines, cancer drugs, and
  pesticides
• Engineered bacteria that can clean up toxic
  wastes
• Restore damaged ecosystems

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Bacteria as a Tool for Manipulating DNA

• In nature, bacteria can transfer DNA in three ways

• Transformation, the taking up of DNA from the
  fluid surrounding the cell
• Can even take up DNA from dead cells

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Bacteria as a Tool for Manipulating DNA

• Conjugation, the union of cells and the DNA
  transfer between them

• Transduction, the transfer of bacterial genes by
  a phage

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• The transferred DNA is then integrated into the
  recipient cells chromosome

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Bacterial Phages as Carriers

• Bacterial plasmids can serve as carriers for gene
  transfer
• An F factor is a DNA segment in bacteria that
  enables conjugation and contains an origin of
  replication
• The F factor starts replication and transfers
  part of the chromosome

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• An F factor can exist as a plasmid, a small
  circular DNA molecule separate from the bacterial
  chromosome
• R plasmids carry genes for resistance of
  antibiotics and that is how bacteria can become
  resistant

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Plasmids are used to customize bacteria

• Plasmids are key tools for DNA technology
• Researchers use plasmids to insert genes into
  bacteria
• Plasmids are obtained from other bacteria
• Desired DNA inserted into plasmid
• Bacteria takes up DNA
• Can be used for several applications

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How they insert DNA into plasmids

• Enzymes are used to cut and paste DNA
• Restriction enzymes cut DNA at specific points
• Recognize specific sequences
• Make sticky ends
• DNA ligase pastes the DNA fragments together
• Catalyst for hydrogen bonds
• The result is recombinant DNA

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Cloning genes using recombinant plasmids

• Bacteria take the recombinant plasmids and
  reproduce
• This clones the plasmids and the genes they carry
• Products of the gene can then be harvested
• Make large amounts of a desired gene so that its
  protein can be manufactured

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Genomic Libraries

• Recombinant DNA
• technology allows the construction of genomic
  libraries
• Genomic libraries
• are sets of DNA
• fragments containing
• all of an organisms genes
• Copies of DNA fragments can be stored in a cloned
  bacterial plasmid or phage

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Other Tools of DNA Technology
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Using mRNA as a Template

• Researchers can use mRNA as a template to isolate
  the gene that makes that mRNA
• Reverse transcriptase can be used to make smaller
  cDNA libraries
• These contain only the genes that are transcribed
  by a particular type of cell

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Nucleic Acid Probes

• In order to find the bacteria or phage that
  contains the desired gene in a library
• Radioactive probes of complimentary DNA sequences
  to the desired gene can be used to find the
  desired gene

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• Colonies are blotted on filter paper
• Paper is treated to break up DNA
• Probe is added
• Paper laid on photographic film
• Film is compared to colonies
• Gene is identified

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Gel Electrophoresis

• Physically sorts out macromolecules (DNA, RNA) on
  the basis of their charge and size
• Current is run through the gel and since DNA is
  negatively charged it moves through the gel
• The longer the DNA molecules are. The slower they
  move
• Bands are made, each consisting of DNA molecules
  of one size

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Gel Electrophoresis

• Restriction fragments of DNA can be sorted by size

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Restriction Fragment Analysis

• Everyones DNA sequence is different
• Scientists can compare DNA sequences of different
  individuals based on the size of the fragments
  created by restriction enzymes
• They can only use DNA that varies from person to
  person
• When run on a gel it makes a distinct pattern

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Restriction Fragment Analysis
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Detecting Harmful Alleles

• Radioactive single-stranded DNA complimentary
  strands are used to verify the presence of
  certain nucleic acid sequences known to code for
  harmful alleles

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PCR

• The polymerase chain reaction (PCR) can quickly
  clone a small sample of DNA in a test tube
• DNA sample mixed with DNA polymerase, nucleotides
  and other things and it replicates exponentially
• Can replicate billions of clones within a few
  hours

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PCR

• Can copy a specific segment of DNA in a mass of
  DNA
• Needs only minute amounts of DNA
• Cannot produce large amounts of DNA
• Scientists are using it to identify missing cases
  with bone marrow, prehistoric beasts etc

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The Challenge of the Human Genome
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The Human Genome

• The 23 chromosomes in the haploid human genome
  contain about 3 billion nucleotide pairs
• This DNA is believed to include about 35,000
  genes and a huge amount of noncoding DNA (do not
  code for proteins)
• About 97 of the total human genome
• Much of the DNA between genes consists of
  repetitive DNA
• Small sequences repeated over and over again on
  the same chromosome
• Large sequences that are scattered throughout the
  genome

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The Human Genome Project

• The Human Genome Project involves
• Genetic and physical mapping of chromosomes
• Gene mapping and fragment analysis
• DNA sequencing
• Nucleotide sequence of the genes
• Comparison of human genes with those of other
  species
• Help the scientists interpret the human data

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Other Applications of DNA Technology
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DNA Technology and The Criminal Court

• Restriction fragment analysis using gel
  electrophoresis
• Compare DNA from a crime
• scene to a
• sample from
• a suspect
• Paternity

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Mass Production of Gene Products

• Most are grown in cell culture using bacteria
• E. coli can host the most plasmids, that is why
  it is used the most
• Yeast is often better for manufacture of
  eukaryotic gene products
• Study into using whole animals in gene product
  manufacture is underway

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Mass Production of Gene Products
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Genetically Modified Organisms (GMOs)

• New genetic varieties of animals and plants are
  being produced
• A plant with a new trait can be created using the
  Ti plasmid

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GMOs

• Golden rice has been genetically modified to
  contain beta-carotene
• This rice could help prevent vitamin A deficiency

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GMOs and the Environment

• Genetic engineering involves some risks
• Possible ecological damage from pollen transfer
  between GM and wild crops
• Pollen from a transgenic variety of corn that
  contains a pesticide may stunt or kill monarch
  caterpillars