Research Papers: Outlines due Oct' 27'

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Research PapersOutlines due Oct. 27.
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Cloning a gene into a plasmid for replication in
E. coli
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HindIII BamH1
CAT
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Restriction enzymes used in cloning experiments
Blunt ends are compatible with any other Blunt
end Sticky ends are only compatible with an
end that leaves the complementary
single- stranded overhang. Because sites are
palindromic, sites cut with the same enzyme are
compatible Single stranded overhang aids
ligation preligation complex lasts longer.
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B
CH2
O
PO2
O
O
O
CH
CH
B
CH
CH2
OH
CH2
O
PO2
HO
O
O
CH
CH
PPP-adenosine
CH
CH
OH
H
B
CH2
O
PO2
O
O
O
CH
CH
Approximation of ligase reaction
CH
CH2
H2O
Pi PP-adenosine
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HindIII BamH1
CAT
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Libraries of genome sequences or transcripts
(cDNA)
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Summary
Cloning involves the action of enzymes for DNA
repair and replication Libraries Restriction
enzymes cut DNA at predictable sites. Ligase
enzymes repair phosphate bonds to create a
single DNA molecule from fragments. Transformatio
n Once inside E. coli, plasmids are replicated
by natural DNA polymerases. Identify desired
clones Disruption of a genes function can be
used to identify bacterial colonies with
plasmids that have taken up an insert in that
gene.
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Analysis methods
Protein isolated, sequence of peptide determined.
cDNA clone isolated and sequenced. DNA
sequences that could encode fragments predicted
based on related genes. Make DNA oligonucleotide
that matches, Make a radioactive probe, Use to
screen phage, YAC, BAC, or plasmid library
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One method to identify cloned inserts with a
chosen DNA sequence
Transfer bacteria (phage) onto nitrocellulose Filt
ers. Make the filter a replica of the
plate Lyse the bacteria so the DNA is fixed in
place Probe with a radioactive probe Isolate
positive clones- Sequence insert Express
protein if possible.