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					Electrophoresis made Easy



   Biotechnology
                                     Chapter 13   2
   Traditional Applications




Biotechnology is applied biology
  • Modern focus on genetic engineering,
    recombinant DNA technology, and analysis of
    biomolecules
                                      Chapter 13     3
   Traditional Applications


Traditional (historical) applications of
   biotechnology date back to over 10,000
   years ago
  • Use of yeast to produce beer and wine in Egypt
    and Near East
  • Selective breeding of plants
  • Selective breeding of animals
                                    Chapter 13    4
   Genetic Engineering

Genetic engineering refers to the modification
  of genetic material to achieve specific
  goals
  • Learn more about cellular processes,
    including inheritance and gene expression
  • Provide better understanding and treatment
    of diseases, particularly genetic disorders
  • Generate economic and social benefits
    through production of valuable biomolecules
    and improved plants and animals for
    agriculture
                                      Chapter 13   5
   Recombinant DNA

Genetic engineering utilizes recombinant DNA
  technology
  • Splicing together of genes or portions of
    genes from different organisms

Recombinant DNA can be transferred to
  plants and animals
  • Modified animals are called transgenic or
    genetically modified organisms (GMOs)
  • Most modern biotechnology includes
    manipulation of DNA
                                   Chapter 13   6
  Recombination in Nature

Many natural processes recombine DNA:


Due to crossing over during meiosis, each
  chromosome in a gamete contains a
  mixture of alleles from the two parental
  chromosomes
  • Thus, eggs and sperm contain recombinant
    DNA
                                   Chapter 13   7
  Transformation



Bacteria can naturally take up DNA from the
  environment (transformation) and integrate
  the new genes into the genome
  (recombination)
         Recombination                       Chapter 13   8
                                 Plasmid     DNA fragments
         in Bacteria           transferred    transferred
(a)        Bacterium     (b)   to new host(c) to new host


Chromosome
               Plasmid



  1 µm                     Plasmid
                         replicates in
                          cytoplasm      DNA fragment
                                         incorporated
                                             into
                                         chromosome
                                     Chapter 13   9
  Transformation


Small circular DNA molecules (plasmids) carry
  supplementary genes
  • Plasmid genes may allow bacteria to grow in
    novel environments
  • Plasmid genes may enhance virulence of
    bacteria in establishing an infection
  • Plasmid genes may confer resistance to
    antimicrobial drugs
                                       Chapter 13   10
   Viral Transfer of DNA


Viral life cycle
  1.   Viral particle invades host cell
  2.   Viral DNA is replicated
  3.   Viral protein molecules are synthesized
  4.   Offspring viruses are assembled and break
       out of the host cell
                                      Chapter 13    11
   Viral Transfer of DNA



Viral transfer of DNA
  • Viruses may package some genes from host
    cell into viral particles during assembly
  • Infection of new host cell injects genes from
    previous host, allowing for recombination
                             Chapter 13   12
Viruses May Transfer Genes
                                     Chapter 13   13
  Biotechnology and Forensics

Forensics is the science of criminal and victim
 identification

DNA technology has allowed forensic science
 to identify victims and criminals from trace
 biological samples
  • Genetic sequences of any human individual are
    unique
  • DNA analysis reveals patterns that identify
    people with a high degree of accuracy
                                     Chapter 13   14
  Polymerase Chain Reaction



Forensic technicians typically have very little
   DNA with which to perform analyses
Polymerase Chain Reaction (PCR) produces
   virtually unlimited copies of a very small
   DNA sample
                                    Chapter 13   15
  Polymerase Chain Reaction


PCR requires small pieces of DNA (called
   primers) that are complementary to the
   gene sequences targeted for copying
A PCR “run” is basically DNA replication in a
   tiny test tube
  • Template DNA, primer, nucleotides, and DNA
    polymerase are all in the reaction mix
                                              Chapter 13   16
    Polymerase Chain Reaction

Four steps of a PCR cycle
  1. Template strand separation
    –   The test tube is heated to 90-95oC to cause the
        double stranded template DNA to separate into
        single strands…

  2. Binding of the primers
    –   The temperature is lowered to 50oC to allow the
        primer DNA segments to bind to the targeted gene
        sequences through hydrogen bonding…
                                         Chapter 13     17
Polymerase Chain Reaction

3. New DNA synthesis at targeted sequences
  The temperature is raised to 70-72oC where the
     heat-stable DNA polymerase synthesizes new
     DNA of the sequences targeted by the
     primers…

4. Repetition of the cycle
  The cycle is repeated automatically (by a
     thermocycler machine) for 20-30 cycles,
     producing up to 1 billion copies of the original
     targeted DNA sequence
           Polymerase Chain Reaction:            Chapter 13   18
           (a) One PCR Cycle


            90 °C          50 °C                 72 °C



               DNA Polymerase           Primer
                                         DNA

Original        Separate        Primers &          DNA
Double-         DNA             DNA                synthesized
helix           Strands         polymerase
DNA                             bind
       Polymerase Chain Reaction:   Chapter 13   19
       (b) Multiple PCR Cycles




             2 copies    4 copies   8 copies

   DNA
fragment
  to be
amplified
                                   Chapter 13   20
  Polymerase Chain Reaction



Choice of primers determines which
   sequences are amplified (copied)
Forensic scientists focus on short tandem
   repeats (STRs) found within the human
   genome
                                  Chapter 13   21
 Polymerase Chain Reaction

STRs are repeated sequences of DNA within
   the chromosomes that do not code for
   proteins
STRs vary greatly between different human
   individuals
A match of 10 different STRs between
   suspect and crime scene DNA virtually
   proves the suspect was at the crime scene
Chapter 13   22
                                  Chapter 13   23
 Gel Electrophoresis



Mixtures of DNA fragments can be separated
   on the basis of size
Gel electrophoresis is a technique used to
   spread out different-length DNA fragments
   in a mixture
                                      Chapter 13   24
  Gel Electrophoresis

Four steps of gel electrophoresis
  1. DNA mixtures are placed into wells at one
     end of a slab of agarose gel


  2. An electric current introduced through the
     gel causes the negatively-charged DNA
     fragments to migrate towards the positive
     electrode
                                       Chapter 13   25
  Gel Electrophoresis

Four steps of gel electrophoresis
  3. Short DNA fragments move more easily
     through the three-dimensional meshwork of
     fibers between the gel
      Short DNA fragments migrate farther than
         long DNA fragments so the mixture is
         separated into bands of DNA of specific
         lengths
  4. The invisible bands of DNA are made visible
     using stains or DNA probes
Chapter 13   26
Chapter 13   27
                                     Chapter 13   28
      RFLP: Gel Electrophoresis




                                  Larger fragments
Direction                         move more slowly;
of Migration                      smaller fragments
                                  move more rapidly
                                  Chapter 13   29
  DNA Fingerprinting
DNA from a crime scene sample can be
   amplified by PCR and run on a gel with
   suspect DNAs
Short tandem repeats (STRs) in the gel DNA
   can be identified by DNA probes
Distinctive pattern of STR numbers and
   lengths are fairly unique to a specific
   individual (forming a DNA fingerprint)
DNA fingerprint from crime scene can be
   matched with DNA fingerprint of suspect
                                               Chapter 13      30
       DNA Fingerprint in Forensics
Q: Which suspect                              A: #3 is prime
should be                                     suspect
indicted?




                      1 2 3   CS   4 5 67
                              RC
                              IE
                   Suspects   MN   Suspects
                              EE
                                    Chapter 13   31
   Restriction Enzymes Cut DNA



A DNA sequence (e.g. a gene) can be removed
   from a chromosome using special enzymes

Restriction enzymes are nucleases that cut DNA
  at specific nucleotide sequences
Chapter 13   32
                                    Chapter 13   33
   Restriction Enzymes Cut DNA




Enzymes that create staggered cuts with “sticky
  ends” are the most useful in gene cloning
                                     Chapter 13   34
   Splicing of DNA Fragments

Sticky ends allow for splicing of a DNA
   fragment with another complementary
   fragment (Bt – Bacillus thurengensis)
  • Bt gene can be cut out of the Bacillus
    chromosome with the same enzyme used to
    cut open the plasmid
  • Bt gene fragment ends can base-pair with
    sticky ends of the opened plasmid, adding
    gene to the plasmid circle
                                  Chapter 13   35
   Splicing of DNA Fragments




DNA ligase enzyme used next to permanently
  bond gene into plasmid
Chapter 13   36
Chapter 13   37
Chapter 13   38
Chapter 13   39
Chapter 13   40
Chapter 13   41
                                    Chapter 13   42
   Plasmids Are Used to Insert Genes




The Ti plasmid from Agrobacterium
  tumefaciens is ideal for transferring genes
  into plant chromosomes
                                       Chapter 13    43
   Plasmids Are Used to Insert Genes


Agrobacterium infects plant cells and inserts
  its small Ti plasmid into a plant
  chromosome in the nucleus
  • Pathogenic effects of certain tumor-causing Ti
    plasmid genes can be disabled
  • A gene inserted into a Ti plasmid is therefore
    carried into the plant cell chromosomes by a
    natural process
Chapter 13   44
                                       Chapter 13    45
   The Human Genome Project

Findings
  • Human genome contains ~25,000 genes
  • New genes, including many disease-associated
    genes have been discovered
  • Has determined the nucleotide sequence of all
    the DNA in our entire set of genes, called the
    human genome
  • The genes comprise 2% of all the DNA
                                          Chapter 13    46
   The Human Genome Project


Applications
  • Improved diagnosis, treatment and cures of
    genetic disorders or predispositions
  • Comparison of our genome to those of other
    species will clarify the genetic differences that
    help to make us human
                                        Chapter 13   47
     DNA Probes


Defective alleles can also be identified using
   DNA probes
DNA probing is especially useful where there
   are many different alleles at a single gene
   locus
  • Cystic fibrosis is a disease caused by any of
    32 alleles out of 1000 total possible alleles
                                        Chapter 13   48
     DNA Probes

Arrays of single-stranded DNA complementary
   to each of the defective alleles can be bound
   to filter paper
A person’s DNA sample is cut up and separated into
    single-strands
The array is bathed in the DNA sample
Strands of DNA binding to complementary sequence
    on the paper indicate presence of a defective
    allele in person’s genome
Chapter 13   49
                                         Chapter 13   50
     DNA Probes



An expanded version of this type of DNA analysis is
   known as a microarray
A microarray contains up to thousands of probes for
   a variety of disease-related alleles
Microarray analysis has the potential to
   comprehensively identify disease susceptibility
Chapter 13   51
                                         Chapter 13   52
   Scientific Objections to GMOs
Safety issues from eating GMOs
  • Could ingestion of Bt protein in insect-
    resistant plants be dangerous to humans?
  • Are transgenic fish producing extra growth
    hormone dangerous to eat?
  • Could GM crops cause allergic reactions?
    – USDA now monitors GM foods for allergic
      potential
  • Toxicology study of GM plants (2003)
    concluded that ingestion of current transgenic
    crops pose no significant health dangers
                                          Chapter 13    53
   Scientific Objections to GMOs

Environmental hazards posed by GMOs
  • Pollen from modified plants can carry GM
    genes to the wild plant population
    – Could herbicide resistance genes be transferred
      to weed species, creating superweeds?

  • Could GM fish reduce biodiversity in the wild
    population if they escape?
    – Reduced diversity in wild fish makes them more
      susceptible to catastrophic disease outbreaks
                                      Chapter 13   54
   Scientific Objections to GMOs



Environmental hazards posed by GMOs
  • US found to lack adequate system to monitor
    changes in ecosystem wrought by GMOs
    (National Academy of Science Study 2003)
                                         Chapter 13   55
   The Human Genome
Should parents be given information about the
  genetic health of an unborn fetus?

Should parents be allowed to select the
  genomes of their offspring?
  • Embryos from in vitro fertilization are
    currently tested before implantation
  • Many unused embryos are discarded

Should parents be allowed to design or
  correct the genomes of their offspring?
                            Chapter 13   56
Hope through Gene Therapy
        Human Cloning:                                  Chapter 13   57
        Permanent Genetic Correction?
         Parents with                   Zygote with         Baby with
        genetic disease                defective gene    genetic disorder


   Viral
  vector
   with
therapeutic
   gene                 Cell Culture    Embryo with
                                       defective gene
        Treated Culture                Genetically
                                        corrected
               Enucleated Genetically embryo clone
                egg cell corrected
                           egg cell
Genetically corrected
  cell from culture                                       Healthy baby
                            Chapter 13


       The end
see also the following related videos:
          1. Plasmid Cloning
       2. DNA Fingerprinting
           3. Gene Therapy
           and many more!

				
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