Biotechnology and Recombinant DNA

1
Biotechnology and Recombinant DNA

• Chapter 9

2
Biotechnology and Recombinant DNA

• Biotechnology
• The use of microbiological and biochemical
  techniques to solve problems and produce product
• Recombinant DNA techniques
• Methods used to manipulated DNA to intentionally
  genetically alter organisms through genetic
  engineering
• Often to give them more useful traits

3
Fundamental Tools of Biotechnology

• Basic components of molecular biologists
  toolkit
• Restriction enzymes
• Gel electrophoresis
• DNA probes
• Primers

4
Fundamental Tools of Biotechnology

• Restriction enzymes
• Naturally occurring enzymes that cut DNA into
  fragments
• Cut in predictable and controllable manner
• Generates pieces of DNA called restriction
  fragments
• These fragments can be joined to new fragments
• Enzymes produce jagged cuts called sticky ends
• Ends anneal together to form new strand
• DNA ligase covalently joins fragments

5
Fundamental Tools of Biotechnology

• Gel electrophoresis
• Used to separate DNA fragments according to size
• DNA is put into wells in gel
• Gel subjected to current
• DNA moves through the gel
• Fragments are separated according to size
• Large fragments remain high in the gel
• Small fragments migrate lower
• Gel must be stained to view DNA
• Stained with ethidium bromide solution

6
Fundamental Tools of Biotechnology

• DNA probes
• Used to locate nucleotide sequences in DNA or RNA
• Probe is single-stranded piece of DNA tagged with
  detectable marker
• Location can be easily determined
• Probe will hybridize to complementary fragment of
  interest

7
Fundamental Tools of Biotechnology

• Primers
• Single stranded DNA fragments the bind sequences
  of DNA
• Used in in vitro DNA synthesis
• Primer serves fragment for addition of DNA
  nucleotides

8
Applications of Genetic Engineering

• Genetically engineered bacteria
• Genetic engineering relies on DNA cloning
• Process of producing copies of DNA
• Cloned DNA generally combined with carrier
  molecule called cloning vector
• Insures replication of target DNA

9
Applications of Genetic Engineering

• Genetically engineered organisms have variety of
  uses
• Protein production
• DNA production
• Researching gene function and regulation

10
Applications of Genetic Engineering

• Protein production
• Produce commercially important proteins
• Pharmaceutical proteins
• Human insulin
• Vaccines
• Hepatitis B vaccine
• Commercially valuable proteins
• Chymosin used in the production of cheese

11
Applications of Genetic Engineering

• DNA production
• Researches interested in acquiring available
  sources of specific DNA fragments
• Fragments used for
• DNA study
• Looking genomic characteristics
• DNA vaccines
• Looking at injecting DNA of pathogen to produce
  immune response

12
Applications of Genetic Engineering

• Researching gene function and regulation
• Function and regulation can be more easily study
  in certain bacteria
• E. coli used often due to established protocols
• Gene expression can be study by gene fusion
• Joining gene being study to reporter gene
• Reporter gene encodes observable trait
• Trait makes it possible to determine changes in
  gene

13
Applications of Genetic Engineering

• Genetically engineered eukaryotes
• Yeast serve as important eukaryotic model for
  gene function and regulation
• Plant or animal that receive engineered gene
  termed transgenic organism
• Examples of genetically altered plants include
• Pest resistant plants
• Corn, cotton and potatoes
• Herbicide resistant plants
• Soybeans, cotton and corn
• Plants with improved nutrient value
• Rice
• Plants as edible vaccines
• Bananas and potatoes

14
Applications of Probe Technologies

• Variety of technology employ DNA probes
• Colony blotting
• Southern blotting
• Fluorescence in situ hybridization (FISH)
• DNA microarray

15
Applications of Probe Technologies

• Colony blotting
• Used to detect specific DNA sequences in colonies
  grown in agar plates
• Colonies are transferred in place on nylon
  membrane
• Colony blots are used to determine which cell
  contain gene of interest

16
Applications of Probe Technologies

• Southern blotting
• Uses probes to detect DNA sequences in
  restriction fragments separated using gel
  electrophoresis
• Application of Southern blotting is locating DNA
  sequences similar to ones being studied

17
Applications of Probe Technologies

• Fluorescence in situ hybridization (FISH)
• Uses fluorescently labeled probed to detect
  certain nucleotide sequences
• Detects sequences inside intact cell
• Specimen is view using fluorescence microscope
• FISH can be used to identify specific properties
  of bacteria
• Mycobacterium tuberculosis in sputum sample

18
Applications of Probe Technologies

• DNA microarray technologies
• DNA arrays are solid supports with fixed patterns
  of different single stranded DNA fragments
  attached
• Enables researches to screen sample for numerous
  sequences simultaneously

19
Applications for DNA Sequencing

• Knowing DNA sequence of particular cell helps
  identify genetic alterations
• Alterations that may result in disease
• Sickle cell anemia
• Due to single base-pair change in gene
• Cystic fibrosis
• Caused by three base-pair deletion
• DNA sequence analysis assists in studying
  evolutionary relatedness

20
Applications of Polymerase Chain Reaction

• Creates millions of copies of given region of DNA
  in matter of hours
• Technique exploits specificity of primers
• Allows for selective replication of chosen
  regions
• Termed target DNA
• Large amounts of DNA can be produced from very
  small sample

• Care must be taken to prevent contamination with
  external source of target DNA
• Basis for false-positive test results

21
Techniques Used in Genetic Engineering

• Obtaining DNA to be cloned
• Generally through cell lysis
• Generating a recombinant molecule
• Restriction enzymes and ligases used to create
  recombinant molecule
• Introducing recombinant molecule into new host
• Host acts incubator for DNA replication
• DNA-mediated transformation often used to get DNA
  into host

22
Techniques used in Probe Technologies

• Probe technologies include
• Colony blotting
• Southern blotting
• FISH
• Microarray technology

23
Techniques used in Probe Technologies

• Techniques in colony and Southern blotting
• Blotting steps transfer sample to nylon membrane
• Probe is added
• Probe hybridizes with complementary sequence
• Process is used to locate positions of hybridized
  probe

24
Techniques used in Probe Technologies

• Techniques used in FISH
• Sample preparation is critical
• Methods used depend on type of organism
• Specimen is applied to glass slide
• Fluorescent label is applied and incubated
• Incubation allows for hybridization
• Specimen is view with fluorescence microscope

25
Techniques Used inDNA Sequencing

• Dideoxychain termination
• Elements for termination reaction include
• Single-stranded DNA template
• Primer that anneal to template
• DNA polymerase
• Each of the nucleotide bases
• One of these bases is labeled with marker for
  detection
• Dideoxynucleotides
• Like deoxynucleotide counterparts but lack 3 OH
• Incorporation causes chain termination
• Special gel electrophoresis used to separate DNA
  fragments by size

26
Techniques Used inDNA Sequencing

• Automated DNA sequencing
• Most automated systems use fluorescent dyes to
  detect newly synthesized DNA
• Gel electrophoresis used to separate fragments
  into colored bands
• Laser used to detect color differences
• Order of color reflects nucleotide sequence

27
Techniques Used inPolymerase Chain Reaction

• Starting with double stranded DNA molecule,
  process involves number of amplification cycles
• PCR requires three step amplification cycle
• Step 1 double stranded DNA denatured by heat
• Step 2 primers anneal to complementary sequence
  of target DNA and DNA synthesis occurs with heat
  stable DNA polymerase
• Step 3 duplication of target DNA
• DNA is amplified exponentially