DNA TECHNOLOGY the new genetics

1
DNA TECHNOLOGYthe new genetics

• Chapter 13

2
Ch 13 VOCABULARYput a by the terms you know
and by the ones you dont.
GMO Clone Vaccine biotechnology Genetic engineering
GFP ligation plasmid endonuclease technology
PCR insulin Gene expression Transgenic Gel electrophoresis
DNA Gene therapy Restriction enzyme Somatic cell Nuclear transfer DNA Fingerprint
HGH Dolly vector Biomedical agriculture
RFLP interleukin ethical interferon transformation
GENERATE YOUR OWN QUESTIONS 1pt question words
Who? What? Where? When? 2pt question words
Which? How? 3pt question words Why?
3
Transgenic/Recombinant organisms contain DNA that
was not part of their original genome.

• Green fluorescent protein (GFP) is responsible
  for the green bioluminescence of the jellyfish
  Aequorea victoria.
• This is a GM mouse!

4
5. The genetic composition of cells can be
altered by incorporation of exogenous DNA into
the cells. As a basis for understanding this
concept

• a.Students know the general structures and
  functions of DNA, RNA, and protein.
• b. Students know how to apply base-pairing rules
  to explain precise copying of DNA during
  semiconservative replication and transcription of
  information from DNA into mRNA.

5
5. The genetic composition of cells can be
altered by incorporation of exogenous DNA into
the cells. As a basis for understanding this
concept

• c. Students know how genetic engineering
  (biotechnology) is used to produce novel
  biomedical and agricultural products.
• d. Students know how basic DNA technology
  (restriction digestion by endonucleases, gel
  electrophoresis, ligation, and transformation) is
  used to construct recombinant DNA molecules.
• e. Students know how exogenous DNA can be
  inserted into bacterial cells to alter their
  genetic makeup and support expression of new
  protein products.

6
(No Transcript)
7

• Genetic Engineering is the application of
  molecular genetics for practical purposes.
• Uses
• Identify genes for specific traits
• Transfer genes for a specific trait from one
  organism to another.
• Tools for manipulating genes
• Restriction enzymes (endonucleases)
• Cloning vector (bacterial plasmid)

8
The Human Genome Project
9
Goals of the Human Genome Project

• Determine the nucleotide sequence of the entire
  human genome.
• Map the location of every gene on each
  chromosome.
• Compare the genomes of other organisms to the
  human genome to understand
• How genomes are organized.
• How gene expression is controlled.
• How cellular growth and differentiation are under
  genetic control.
• How evolution occurs.

10
GENOMIC LIBRARYis a catalog of the DNA of a
species

• Cut up the DNA of the species into tiny pieces
  using restriction enzyme.
• Put each DNA fragment into a different cloning
  vector- ex. plasmid.
• Put each recombinant plasmid into a separate
  bacterium.
• FREEZE until needed.

11
Gene Therapy

• Treating a genetic disorder by introducing a gene
  into a cell or by correcting a gen defect in a
  cells genome.
• 1990s.
• Cystic Fibrosis, AIDS, Ovarian Cancer.

12
Practical Uses of DNA Technology

• Pharmaceuticals- HGH, Interferons, Interleukins
  etc.
• Vaccines- solution that contains a harmless
  version of a virus or bacterium to stimulate an
  immune response formation of memory cells.
• Increased Agricultural Yields- ex. crops that
  dont need fertilizer.

13
Ethical Issues

• Describe two potential safety and environmental
  problems that could result from genetic
  engineering.

14
DNA technology can be used to

• Cure diseases
• Treat genetic disorders
• Improve food crops

15
Golden rice contains beta-carotene, which our
bodies use to make vitamin A.
16
Figure 20.18 Pharm animals secrete spider silk
in their milk
Could use this Technology to Make insulin or
Human growth Hormone
17
Figure 20.16 One type of gene therapy procedure
18
(No Transcript)
19
transgenic organism

• Contains new DNA.
• Ex. Bacterium with plasmid containing insulin
  gene.
• Grow bacteria (beaker or petri dish)
• Bacteria express (transcribe/translate) the
  cloned gene to make insulin.
• Insulin is extracted (purified) from the medium.
• Treatment for Diabetes.

20
(No Transcript)
21
Injecting DNA into an embryo how you create a
cloned organismSCNT Somatic Cell Nuclear
Transfer
22
Tools for manipulating genes
23
Restriction Enzymes (endonucleases)

• Molecular scissors that cut DNA at specific
  sequences.
• Provide protection for bacteria against viruses.

24
3 examples of restriction enzymesEcoRI, BamHI,
HindIII
25
Restriction Enzymes or Restriction Endonucleases

• Are bacterial enzymes that cut DNA molecules into
  smaller pieces
• They cut the DNA at a specific site, a known
  sequence of DNA .
• for example CTTAAG
• GAATTC
• EcoRI cuts between the G and A, leaving two open
  ends with single-chain tails called sticky
  ends
• Ex. CTTAA and G
• G AATTC

26

• Sticky ends readily bind to complementary chains
  of DNA.
• Thus, pieces of DNA that have been cut with the
  same restriction enzyme can bind together to form
  a new sequence of nucleotides.

27
Restriction Enzymes
28
CLONING VECTORS

• Restriction enzymes can be used to isolate a
  specific gene of interest from a donor called the
  donor gene.
• A plasmid is a ring of DNA found in a bacterium
  in addition to its main chromosome.
• Cut the plasmid with the same restriction enzyme
  as the donor gene to splice it into the
  plasmid.
• Insert this recombinant DNA plasmid into the
  bacterium.
• When the bacteria reproduces by binary fission
  the recombinant plasmid does too, we call this
  cloning a gene.
• When a virus is used as the vector for gene
  transmission, this is called transduction.

29
Plasmids small circular pieces of DNA.

• Plasmids often contain genes for antibiotic
  resistance.

30
Conjugation- when bacteria exchange plasmids.
31
Transformation

• bacteria can incorporate new DNA into their
  genome.
• They do it all the time naturally.
• Pick up plasmids from their environment.

32
Transplanting Genes

• Plasmids are used to transfer a gene to bacteria
  so the bacteria will produce a specific protein.
• Ex. Human insulin
• Human Growth Hormone
• Just give the bacteria food and they will
  reproduce and produce your protein. You have a
  protein factory!!!! (clean up your protein,
  separate it and purify it)

33

1. Plasmid Donor gene (cut by same RE)
2. Spliced w/ ligase
3. Recombinant plasmid inserted into bacteria
   (transformed)
4. Bacteria replicates produces
5. Gene clone- exact copy of the gene.

34
Cloning A Gene
35
Recombinant Organisms

• Organisms that receive the recombinant plasmid.
• Ex. Glo fish, glowing cat, pharm animals, golden
  rice, roundup ready soybeans,

36
How to create a recombinant plasmid

• Treat plasmid and donor gene with the same
  restriction endonuclease (they used EcoRI)
• 2) This creates the same sticky ends on plasmid
  and donor gene DNA.

37

• 3) Place both together with DNA ligase to join
  the donor gene with the plasmid.
• Phosphodiester bonds link sugar-phosphates of
  nucleotides hydrogen bonds form/break
  spontaneously.

38
Figure 20.19 Using the Ti plasmid as a vector
for genetic engineering in plants
39
DNA Technology Techniques

• PCR
• Gel Electrophoresis

40
Polymerase Chain Reaction a way to make millions
of copies of DNA!!!

• What you need
• DNA sample
• Free nucleotides
• A heat resistant DNA polymerase
• Example Taq polymerase
• Primers short segments(20-30bases) of DNA
  complementary to the ends of the DNA being copied.

41

• Polymerase Chain Reaction
• Denature the original strand of DNA with heat.
• Cool the mixture, allowing the primers to bind
  (anneal) to the DNA.
• The DNA polymerase binds free nucleotides to the
  primer using the original DNA strand as a
  template. This creates two copies of the DNA
  sample.
• Repeat.

42
(No Transcript)
43
Gel Electrophoresis

• Technique used to separate restriction fragments.
• DNA fragments of different lengths are separated
  as they diffuse through a gelatinous material
  under the influence of an electric field.
• Since DNA is negatively charged (phosphate
  groups), it moves toward the positive electrode.
• Shorter fragments move further than longer ones.

44
(No Transcript)
45
Figure 20.x1a Laboratory worker reviewing DNA
band pattern
46
DNA TECHNOLOGY TECHNIQUES USED TO ANALYZE DNA
sequences

• DNA Fingerprint
• Pattern of bands, arranged in colums, made up of
  specific fragments from an individuals DNA.
• Can be used to
• compare samples of blood or tissue left at a
  crime scene
• determine how closely related species are
• Paternity testing.

47
Making a DNA FingerprintRFLP analysis

• A DNA sample is extracted from nucleated cells.
• The DNA is amplified using P.C.R.
• The DNA is cut into fragments by restriction
  enzymes.
• The stained fragments are placed into a gel, and
  are moved by an electrical current.
• Smaller fragments migrate the furthest and the
  result is a column of dark DNA bands that extend
  across the gel.
• The amount of DNA between restriction sites
  varies from individual to individual of the same
  species. The differences are called restriction
  fragment length polymorphisms or RFLPs. RFLPs
  result in unique restriction fragment patterns on
  a gel.

48
APPLICATIONS Gel Electrophoresis

• Compare DNA fragments of closely related species
  to determine evolutionary relationships.
• CSI. Compare restriction fragments between
• individuals of the same species!
• Fragments differ in length because of
  polymorphisms, slight differences in DNA
  sequences. These fragments are called restriction
  fragment length polymorphisms, or RFLPs.

49
Figure 20.17 DNA fingerprints from a murder case