P1252109267mPENy

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DNA Technology and Genomics

• Chapter 12

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Gene Cloning

• Genetic engineering involves manipulating genes
  for practical purposes
• Gene cloning leads to the production of multiple
  identical copies of a gene-carrying piece of DNA
• Recombinant DNA is formed by joining DNA
  sequences from two different sources
• One source contains the gene that will be cloned
• Another source is a gene carrier, called a vector
• Plasmids (small, circular DNA molecules
  independent of the bacterial chromosome) are
  often used as vectors

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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

1. Conjugation, the union of cells and the DNA
   transfer between them

1. 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

• Steps in cloning a gene
• Plasmid DNA is isolated
• DNA containing the gene of interest is isolated
• Plasmid DNA is treated with restriction enzyme
  that cuts in one place, opening the circle
• DNA with the target gene is treated with the same
  enzyme and many fragments are produced
• Plasmid and target DNA are mixed and associate
  with each other

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Plasmids are used to customize bacteria

1. Recombinant DNA molecules are produced when DNA
   ligase joins plasmid and target segments together
2. The recombinant DNA is taken up by a bacterial
   cell
3. The bacterial cell reproduces to form a clone of
   cells

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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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1. Colonies are blotted on filter paper
2. Paper is treated to break up DNA
3. Probe is added
4. Paper laid on photographic film
5. Film is compared to colonies
6. 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