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TECHNOLOGY
D Newby
Recombinant DNA
technology
Recombinant DNAare:
DNA sequences that result from the use of laboratory methods
(molecular cloning) to bring together genetic material from
multiple sources, creatingsequences that would not otherwise be
found in biological organisms.
GGACGCTAGCTGATGAATTCGCATCGGATCCGAATCCGCTCTTTCAA
CCTGCGATCGACTACTTAAGCGTAGCCTAGGCTTAGGCGAGAAAGTT
Recognition Sequence
GGACGCTAGCTGATGAATTCGCATCGGATCCGAATCCGCTCTTTCAA
CCTGCGATCGACTACTTAAGCGTAGCCTAGGCTTAGGCGAGAAAGTT
Cleavage
GGACGCTAGCTGATG AATTCGCATCGGATCCGAATCCGCTCTTTCAA
CCTGCGATCGACTACTTAA GCGTAGCCTAGGCTTAGGCGAGAAAGTT
Classification
Evolved independently rather than
diverging form a common ancestor
EcoRI
Blunt End Cutters
Some restriction enzymes cut DNA at
opposite base
They leave blunt ended DNA fragments
These are called blunt end cutters
AluI
HaeIII
Use of Restriction Enzymes in
recombinant DNA Technology
How Restriction Enzymes are named
sequences
Symmetrical sequences which read in the same order of nucleotide bases on
Examples of Hosts
bacteria (E. coli)
yeast (S. cerevisiae)
insects (D. melanogaster)
plants (Arabadopsis thaliana)
mammals (Mus musculus) mouse
Characteristics of cloning vectors:
Must be small in size
Have a origin of replication
Have a selectable marker
Have unique cloning sites (polylinker)
Steps in creating recombinant DNA
DNA
4.
Steps in creating recombinant DNA
The methods used to get DNA into cells are varied, and the name
.
applied to this step in the molecular cloning process will often depend
upon the experimental method that is chosen
(e.g.transformation,transduction,transfection,electroporation).
TypicallyAmpicillin is used
.
No ATP requirement.
Recognition sites in double stranded DNA have a
2-fold axis of symmetry a palindrome.
Cleavage can leave staggered or "sticky" ends or
can produce "blunt ends.
DNA cloning requires
restriction endonuclease
and DNA ligase
Consider a plasmid with a unique EcoRI
site:
5'NNNNGAATTCNNNN3'
3NNNNCTTAAGNNNN5'
5'NNNNGAATTCNNNN3'
3'NNNNCTTAAGNNNN5