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Genetic Engineering (Gene Transfer) - Coggle Diagram
Genetic Engineering
(Gene Transfer)
1. Obtaining Required Section
of DNA (gene)
correct sequences
have to be identified
DNA probes - identify desired section of DNA
restriction endonuclease - cut out desired sections
Restriction Endonuclease Enzymes
enzymes naturally occur in bacteria
as defence against DNA injected by invading viruses
role
cut virus DNA into small fragments
to stop the infection process
different types
of restriction endonuclease
each one cuts DNA at specific nucleotide
sequence (recognition sequence)
by hydrolysis reaction
types of DNA cuts
BLUNT cut
STAGGERED cut
produces
sticky ends
D: short sections of DNA where
only 1 strand and bases in section
are unpaired and exposed
more useful
will readily join with another piece of DNA
that was cut w/ same enzyme
why different restriction
enzymes have different
recognition (restriction) sites?
restriction enzyme has specific active site,
complementary to particular sequence bases
∴ different restriction enzymes cut DNA in different specific places
why single type of restriction endonuclease enzyme cannot be used to cut out all genes on a chromosome?
single restriction enzyme may cut at base sequence:
in gene
or cut either side of several genes
Reverse Transcriptase
D: enzyme used to make a desired section of DNA (gene) from the gene’s mRNA
method
ISOLATE + EXTRACT mRNA
of the desired gene
(cells where desired gene v active = contain a lot of relevant mRNA)
REVERSE TRANSCRIPTASE
- used to make single strand of DNA (cDNA)
using the mRNA as a template
DNA POLYMERASE
enzyme - makes double stranded DNA (from single strand of cDNA)
found naturally
in retroviruses
human genes synthesised from mRNA
NOT contain INTRONS
can direct protein synthesis when inserted into bacteria
genes made from restriction endonuclease
WILL contain INTRONS
need be removed before inserted into bacteria
advantage of manufacturing DNA rather than cutting out of gene?
many copies of mRNA in cell
no need to remove introns
DNA (Gene) Probes
D: short single strand of DNA
w/ known base sequence
role
used to identify sections of DNA that
contain specific sequence of bases
base pair + hybridise w/ complementary bases of target section
can be labelled w/
FLUORESCENT
RADIOACTIVE LABEL
help identify exactly where
target section is on DNA
fluorescent
probes detected =
UV light
radioactive
probes detected =
X ray film
also used in Gel Electrophoresis +
Genetic Fingerprinting
2. Transferring Donor Gene
into Recipient Cell
desired gene transferred into vector BEFORE
transferred into donor cell/organism
role
transfer donor gene into host cell
1. inserting gene
into VECTOR
VECTORS =
VIRUSES
PLASMIDS
- small circular loops of DNA found in bacteria
(usually contain genes that provide antibiotic resistance)
PLASMIDS
P cut open using
restriction endonucleases
same RE used to cut plasmid + donor gene = will have
COMPLEMENTARY STICKY ENDS
base pairs
of donor gene = perfectly join with exposed base pairs of plasmid
DNA ligase
- enzyme joins (anneal) DNA backbones
Phosphodiester bonds
formed between
sugar-phosphate backbones of donor gene & plasmid DNA
donor DNA =
spliced
into plasmid = becomes closed loop again
plasmid DNA now =
recombinant DNA
(plasmid = recombinant plasmid)
why may the donor gene
not have sticky ends?
donor gene may have been formed
using reverse transcriptase
(converting mRNA into cDNA)
restriction endonuclease was used
that produced blunt ends
if not present -
can be added
must have complementary
bases to the bases of the sticky
ends of the cut plasmid
VIRUSES
donor gene spliced into a
bacteriophage virus
bacteriophage fires viral DNA into host
bacterial DNA - also contain donor gene
2. inserting gene
into HOST CELL
host cell =
BACTERIAL
cell
(can be cloned easily)
how bacterial cells encouraged to take up recombinant plasmids?
incubated with
calcium ions
heat shock
(rapid temperature rise from 0°C to 40°C)
makes bacteria more
permeable
uptake v
slow
- need to identify transformed bacteria (only ones w/ desired product wanted for further culture)
R-plasmid which has genes for resistance to antibiotics:
tetracycline (T)
ampicillin (A)
method
RE enzyme
chosen - that will cut 1 of genes that provides antibiotic resistance (eg. T)
donor gene inserted
within here
recombinant plasmid no longer resistant to T (cut in two)
but still resistant to A
plasmid
annealed
- DNA ligase
when bacteria encouraged take
up recombinant plasmid...
3 possible outcomes
bacteria failed to take up plasmids
sensitive to both A + T
bacteria take up unchanged plasmid
resistant to both A + T
bacteria took up modified plasmid
resistant to A but not to T (sensitive)
3. Identifying the Transformed
Bacteria that Contain the
Desired Gene
Marker genes
used identify the transformed bacteria
Marker Genes - method
bacteria (orig P+ recom. P) cultured on agar plates w/ A
colonies allowed to develop
bacteria replica plated
involved blotting original plate w/ pad
pressing against surface 2 fresh agar plates
1 WITH T
1 ordinary plate
results
colonies on T plate - do not have resistance to T:
will be killed
appear as missing
colonies contained transformed bacteria with desired gene
position of missing colonies be located on the ordinary replica plate
further cultured for use
DNA probes
adding a florescent marker to
the desired gene
DNA Probes - method
incorporate gene that produces a florescent protein into P
RE cut through this gene + donor gene added in here... gene for fluorescent protein not work
results
bacteria that do NOT fluoresce under UV light
contain recombinant P + desired gene
4. Clone Bacteria to
Produce Large Numbers
occur in large fermenters
where conditions ideal for:
rapid growth & production
of desired product