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Chapter 10
Biotechnology
Biotechnology was used to create the pGLO cloning
plasmid (chapter 9 notes)
A jelly fish gene was combined with bacterial DNA ultimately
resulting in fluorescent bacteria
Biotechnology
Biotechnology is possible due to
The central dogma
All living organisms genetic information is encoded in DNA which is
transcribed and translated to create a protein resulting in a particular
trait
The DNA and process is so similar that a gene from one organism can be
used by other organisms
Transcriptio
n
Translation
Biotechnology
Biotechnology is possible due to
Knowing the structure of DNA as well as the enzymes involved
in its function
Restriction enzymes
Each type of restriction enzyme cuts DNA at very specific nucleotide
sequences
EcoR1 cuts at the sequence GAATTC
Biotechnology
Biotechnology is possible due to
Knowing the structure of DNA as well as the enzymes involved
in its function
Complementary base pairing or hybridization
Any nucleotide sequence that is complementary to another sequence can
bind forming double-stranded DNA
Biotechnology
Biotechnology is possible due to
Knowing the structure of DNA as well as the enzymes involved
in its function
Ligase
The enzyme used to bind the phosphate-sugar backbone between
nucleotides
The combination of restriction enzymes and ligase allows for cutting and
pasting of any two pieces of DNA
Forms recombinant DNA
DNA Cloning
A set of methods that uses living cells to
make many identical copies of a DNA
fragment
Allows manipulation and analysis of a gene from
one organism in a second organism
Example using pGLO
DNA Cloning
Example using pGLO
Jellyfish (Aequorea victoria) express the
trait of fluorescence
Gene transcribed to mRNA translated to
a polypeptide trait is expressed
DNA Cloning
Example using pGLO
Plasmids are manipulated to work as cloning
vectors
Plasmids are small circles of DNA with just a few genes
Non-essential for survival and not part of the major
bacterial chromosome
Plasmids are copied and distributed to daughter cells when
Bacterium
Plasmid
the bacterium divides
Chromosome
DNA Cloning
Example using pGLO
Plasmids are manipulated to work as cloning vectors
Addition of restriction enzyme sites
To create recombinant plasmids with foreign DNA fragments
DNA Cloning
Example using pGLO
Cut the cloning plasmid with the same specific
restriction enzyme used on the jelly fish DNA
Creates complimentary sticky ends
DNA Cloning
Example using pGLO
The cut plasmid and jelly fish DNA fragments are
mixed with DNA ligase
Because the plasmid and fragments have the same
sticky ends, they will base pair
DNA ligase glues the back-bone of the two DNAs
together forming recombinant plasmids
Some plasmids will close back up without any foreign
DNA
DNA Cloning
Example using pGLO
The recombinant plasmids are mixed with host bacterial cells
A few bacteria take up recombinants plasmids
Referred to as transformed cells
DNA Cloning
Example using pGLO
The bacteria are plated on nutrient agar with ampicillin (an
antibiotic)
Only transformed cells containing the plasmid will survive
Due to the antibiotic resistance gene in the plasmid
X
X
DNA Cloning
Example using pGLO
Each surviving, transformed bacterium will divide multiple
times creating a colony of identical bacterial cells or clones
Each clone contains a copy of the vector with the foreign DNA
Selection of clones
Look for the trait coded by the foreign gene
pGLO = fluorescence
DNA Cloning
Example using pGLO
The only changes in the bacteria are
Fluorescence
Ampicillin resistance
Replicate the new single strands of DNA to form doublestranded fragments of DNA
Using the enzyme DNA polymerase
PCR Method
Start with a sample of DNA that will have the target sequence
Could be DNA from different clones, one sperm, a hair left at a crime
scene, or a mummy
Any sample that has DNA in it can be used
PCR
DNA
DNA is negatively charged (due to the phosphate groups)
Will migrate toward the positive electrode
The longer the fragment of DNA, the slower it migrates
The shorter the fragment of DNA, the faster it migrates
Gel electrophoresis
Boyfriend
Control DNA
Control DNA
Size Reference
Size Reference
Size Reference
Control DNA
Size Reference
Victim
Suspect 1
Suspect 2
Female Cells
Semen
Evidence from
Crime Scene
Forensic Evidence
Determine guilt
Determine paternity
Determine identity
Environmental Cleanup
Extract heavy metals
Clean up oil spills
Degrade chlorinated hydrocarbons in wastewater treatment plants
Agricultural Applications