dna technology - bio resource site | stuff from ma'am...
TRANSCRIPT
Biotechnology alteration of organisms or their components with
specific applications in mind
Breeding strategies
Artificial selection
Mutations
Genetic engineering - Direct manipulation of genes
- Recombinant DNA technology
https://sites.google.com/site/asoftsb/home/methods-of-selective-breeding/examples
Basic DNA Techniques
Identification and isolation of GENE
OF INTEREST
Purification and fragmentation
using RESTRICTION
ENZYMES
Incorporation of DNA into CLONING
VECTORS (i.e. plasmids)
Incorporation of plasmids into bacterial host
through TRANSFORMATION
SELECTION of hosts carrying
desired recombinant gene
GROWING of selected host cells in culture medium
Gene cloning
• amplification of genes
• production of protein products
Restriction
Enzymes
• aka restriction
endonucleases
• produced by
Bacteria/Archaea
• cut at (restriction
sites) that are 4-8
base pairs long
• long DNA fragments
can be cut to
produce restriction
fragments
• e.g. EcoRI
(G*AATTC)
Constructing
recombinant
DNA with the
aid of
restriction
enzymes
A comprehensive list is available at
http://www.sciencegateway.org/RES
/index.html
DNA Insertion
• conditions that
allow host cells to
take up the vector
– high salt
concentration
– high temperature
(~42oC)
– electric pulse
– microinjection
Selection of transgenic host cells • recombinant molecules
must be separated from molecules consisting of just donor or just plasmid DNA
• plasmid used contains genes for resistance against certain antibiotics
• large number of cells grown from single cell carrying recombinant plasmid
• w/in hrs, the a whole culture of cells containing the recombinant DNA
http://filebox.vt.edu/users/chagedor/biol_4684/Methods/vector.gif
Using cDNA
• difference
between
prokaryotes &
eukaryotic DNA
• DNA copied from
mature mRNA
transcript by
reverse
transcription
• cDNA library
Basic DNA Techniques
• DNA Amplification
– method of creating multiple copies
of a particular segment of DNA
– e.g. growing recombinant cells
polymerase chain reaction
(PCR)
Polymerase
Chain Reaction
Makes use of Taq
polymerase
Steps:
1. Denaturation
2. Annealing
3. Extension
Each PCR cycle of
heating and cooling
the DNA mixture
doubles the number
of DNA molecules.
Basic DNA Techniques
Gel Electrophoresis
technique used to
distinguish DNA
molecules
applied electric field
forces DNA to migrate
through a medium and
distance themselves
from one another by
length
<16.2-2>
Basic DNA Techniques
• DNA Sequencing
– Sanger method -
for determining
the nucleotide
sequence of a
piece of DNA
– many copies are
needed (by cloning
or PCR)
QUIZ 2 (by pair)
1. Describe 1 natural way to manipulate DNA.
2. Differentiate between biotechnology and
recombinant DNA technology.
3. What are restrictzion enzymes?
4. What is the role of ligase in DNA
recombination?
5. How can we ensure that only the host cell with
the gene of interest will be cloned?
6. What enzyme is needed in creating cDNA?
7. What are the three steps in PCR?
8. How does gel electrophoresis separate DNA of
different lengths?
9. What is the use of fluorescently-labeled bases
in DNA sequencing?