Download Introduction to Biotechnology

Recombinant DNA
Technology………..
BTEC3301
DNA Libraries
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How do you identify the gene of interest
and clone only the DNA sequence you are
interested? Read Pg 63
DNA Libraries
Library screening of gene of
interest
• Libraries are collections of cloned
DNA fragments from a particular
organisms contained within
bacteria or virus as a host.
DNA Libraries
Library screening of gene of
interest
•
Libraries can be kept or saved
as such for a long time and
screened to pick up the gene of
interest whenever required.
DNA Libraries
Library screening of gene of
interest
•
Two types of libraries which are
typically used for cloning are
genomic DNA libraries and
complimentary DNA libraries cDNA
libraries.
DNA Libraries
Human genomic DNA
libraries
•
Human DNA is cleaved with a
restriction enzyme to several
fragments.
•
These fragments are cloned into
plasmids
DNA Libraries
Human genomic DNA
libraries
•
Hence a human genomic library
consists of a collection of bacteria
each containing a different
fragment of human DNA.
DNA Libraries
Library screening
•
Once the genomic library or
cDNA library is created, it must
be screened for gene of interest.
DNA Libraries
Library screening
•
Although libraries are used for
cloning and identifying a gene of
interest, Polymerase chain reaction
(PCR) is a much more rapid approach
to cloning than building a library and
screening for a gene of interest
Hybridization
DNA hybridization
•
The pairing of two DNA
molecules, often from different
sources, by hydrogen bonding
between complementary
nucleotides.
Hybridization
DNA hybridization
•
This technique is frequently used to
detect the presence of a specific
nucleotide sequence in a DNA sample.
•
Nucleic acid hybridization on membrane
filters is a simple, sensitive, and specific
means of detecting nucleic acid
sequences of interest.
Hybridization
DNA hybridization
•
Western Blotting is used to
analyze proteins which have
been immobilized on
nitrocellulose/nylon filters.
Hybridization
DNA hybridization
•
•
Southern blotting (pg75)
Southern Blot (hybridization) was
devised by Ed Southern in 1975 for
the identification of DNA fragments
that are complementary to a known
DNA sequence.
•
The Polymerase Chain Reaction
(PCR) provides an extremely
sensitive means of amplifying small
quantities of DNA.
Polymerase Chain Reaction (PCR)
Diagrammatic steps in PCR process
(Review):
•
Three major steps in PCR:
1. Template denaturation
2. Primer annealing
3. Primer extension
Fig. 3.8 The Polymerase Chain
Reaction
Go to Animation courtesy of the
following websites:
References
http://faculty.plattsburgh.edu/donald.slish/PCRmov.html (Animation)
http://www.accessexcellence.org/RC/AB/IE/PCR_Xeroxing_DNA.html
 http://www.people.virginia.edu/~rjh9u/pcranim.html ( PCR Animation)
 http://www.escience.ws/b572/L3/L3.htm (PCR Animation)
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http://homepages.strath.ac.uk/~dfs99109/BB211/RecombDNAtechlect4.html
http://en.wikipedia.org/wiki/Polymerase_chain_reaction
http://www.escience.ws/b572/L3/L3.htm
 http://allserv.rug.ac.be/~avierstr/principles/pcrani.html
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