Transcript Cloning

Bacterial cloning
Especially of PCR product DNA
PCR recap
PCR gel product
Gel 1 and 2
Gel 1 and 2
Gel 3 and 4
Gel 3 and 4
Cloning
• Cloning is the way in which we can take a
single molecule, and make lots of bacterial
cells that contain an identical molecule.
• These cells are clones, hence the name
• This used to be the only way to amplify
DNA. It is still by far the most accurate.
Plasmid vectors – circular, autonomous bacterial DNA
Cloning PCR products
• When we amplify DNA using PCR, it is
often necessary to “clone” this DNA
• We do this in order to replicate it without
errors
• Also, by cloning a protein coding sequence
into E. coli, we can then produce the protein
in the bacterium.
The vector is made with a “T”
overhang
Taq polymerase leaves an “A”
overhang
• Taq is the thermostable DNA polymerase from
Thermus aquaticus we used for PCR.
• When Taq synthesizes a new strand, it always puts
an extra “A” at the end
• This can be useful, but note: other polymerases
do not do this, only Taq polymerase
Restriction Endonucleases
Also called restriction enzymes
1962: “molecular scissors” discovered in in bacteria
E. coli bacteria have an enzymatic immune system that
recognizes and destroys foreign DNA
3,000 enzymes have been identified, around 200 have unique
properties, many are purified and available commercially
Named for bacterial genus, species, strain, and type
Example: EcoR1
Genus: Escherichia
Species: coli
Strain: R
Order discovered: 1
Recognition sites have symmetry (palindromic)
“Able was I, ere, I saw Elba”
5’-GGATCC-3’
Bam H1 site:
3’-CCTAGG-5’
Restriction enzymes recognize specific 4-8 bp sequences
Some enzymes cut in a staggered fashion - “sticky ends”
EcoRI
5’…GAATTC…3’
3’…CTTAAG…5’
Some enzymes cut in a direct fashion – “blunt ends”
PvuII
5’…CAGCTG…3’
3’…GTCGAC…5’
Why don’t bacteria destroy their own DNA
with their restriction enzymes?
Methylation
Uses for Restriction Enzymes
RFLP analysis (Restriction Fragment Length Polymorphism),
CAPS (Cleaved Amplified Polymorphic Sequence) markers
Cloning and construction of transgene “cassettes”
DNA storage – libraries
Genotyping by sequencing (“GBS”).
Restriction Enzymes for Cloning
Bacterial DNA cleaved with EcoRI
5’-C-G-G-T-A-C-T-A-G-OH
3’-G-C-C-A-T-G-A-T-C-T-T-A-A-PO4
Corn DNA cleaved with EcoRI
+
PO4-A-A-T-T-C-A-G-C-T-A-C-G-3’
HO-G-T-C-G-A-T-G-C-5’
Complementary base
pairing
5’-A-C-G-G-T-A-C-T-A-G A-A-T-T-C-A-G-C-T-A-C-G-3’
3’-T-G-C-C-A-T-G-A-T-C-T-T-A-A G-T-C-G-A-T-G-C-5’
+ DNA Ligase, + rATP
5’-A-C-G-G-T-A-C-T-A-G-A-A-T-T-C-A-G-C-T-A-C-G-3’
3’-T-G-C-C-A-T-G-A-T-C-T-T-A-A-G-T-C-G-A-T-G-C-5’
recombinant DNA molecule
Electroporation
i)
When the electric field is applied,
the ions move according to their
charge.
ii) Pathways are formed across the
cell membrane allowing DNA
to enter.
iii) When the electric field is
deactivated, the membrane heals.