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Gene Transfer - Coggle Diagram
Gene Transfer
1. Obtaining Required Section of DNA
RESTRICTION
ENDONUCLEASE
(enzymes)
role
cuts DNA double strand
by hydrolysis reaction
@ specific bases - recognition sequence
occur naturally
defensive bacterial enzymes
cut up foreign DNA injected
by bacteriophages
types of DNA cuts
BLUNT CUT
e.g. Hae111
straight across 2 strands
between opposite adjacent bases
STAGGERED CUT
e.g. EcoR1
cuts in DNA strand
between adjacent bases do not lie opposite each other
produces
sticky ends
D
: short sections of DNA where only 1 strand + bases in section unpaired + exposed
useful
= readily join w/ other piece DNA
(cut by same RE enzyme)
advantage
produces DNA w/ sticky ends
easily join with another section DNA
w/ complementary sticky ends
different types of RE enzyme
cut DNA @ specific nucleotide sequence (recognition site)
by hydrolysis reaction
GENES WILL CONTAIN INTRONS
need to be removed before inserting into plasmid
questions
why single type RE enzyme not used cut out all genes on chromosome?
single RE enzyme cut @ base sequence
in gene
OR
either side of several genes
why different RE enzyme have different recognition sites?
RE enzymes = specific active site
complementary to particular base sequence
∴ different RE cut DNA @ different places
REVERSE
TRANSCRIPTASE
(enzyme)
occur naturally
in retroviruses
role
make desired section of DNA
from gene mRNA
method
ISOLATE + EXTRACT mRNA
of desired gene
(cells where desired gene v active = contain a lot of relevant mRNA)
REVERSE TRANSCRIPTASE
- makes single strand DNA (cDNA)
using the mRNA as a template
DNA POLYMERASE
enzyme - makes double stranded DNA
(from single strand of cDNA)
GENES WILL NOT CONTAIN INTRONS
can be directly inserted into plasmid
& carry out protein synthesis
advantage
- of manufacturing rather than cutting out DNA
many copies of mRNA in cell
no need to remove introns
DNA (gene)
PROBES
role
used identify desired sections of DNA
that contain specific base sequence
D: short single strand of DNA w/ known base sequence
complementary to target section of DNA
overview
base sequence
complementary
to base sequence of target DNA
labelled with
Fluorescent/Radioactive
label
bases of probe
hybridise
to bases of target DNA
detect probe using -
UV light/X-ray Film
method
DNA with target sequence = hydrolysed into sections
using
RE enzymes
DNA fragments separated by
gel electrophoresis
separated DNA sections - transferred to
NYLON membrane
fluorescently labelled DNA probe
added
if target sequence present
-
DNA PROBE HYBRIDISE W/ IT
if not =
washed off + removed
(fluorescently labelled probe show up whether hybridised or not so need to remove if not)
nylon membrane exposed to UV light
DNA probe +
target sequence
=
fluorescent band/fogging on X ray film
2. Inserting Gene Into Vector
(transfer of donor gene)
PLASMIDS
D: small circular loops of DNA
found in bacteria (separate from
main loop of DNA)
USE R-PLASMID
contain
genes
that provide
antibiotic resistance
TETRACYCLINE & AMPICILLIN
method
1. P cut open within 1 gene for
AB resistance
RE enzyme
same RE used cut gene
comple. sticky ends
bp gene = match perfectly w/ exposed base on plasmid
if donor gene inserted in T resistant gene
plasmid no longer resistant to T
2. DNA ligase
enzyme = anneal DNA backbone
phosphodiester bonds
(between sugar-phosphate backbone gene + plasmid)
3. Donor DNA spliced into plasmid
becomes closed loop again
final forms = recombinant plasmid/recombinant DNA
why donor genes no
sticky ends?
formed using
reverse transcriptase
blunt cuts from
RE enzyme
if not present - can be added
must have comple. bases
to sticky ends of plasmid
VIRUSES
advantage
naturally adapted to shoot genetic material into host cell
donor gene spliced into bacteriophage virus (BV)
BV fires viral DNA into host
bacterial DNA = contains donor gene
3. Inserting Donor Gene into Host Cell
(transfer of donor gene)
bacterial cells encouraged
to take up recombinant plasmid
why bacteria?
easily cloned
used for
rapid production
of desired gene
conditions
incubated w
calcium ions
heat shock
rapid temp rise - 0-40°C
makes bacteria more permeable
v
small proportion
of bacterial cells actually take up recombinant plasmid
3 possible
outcomes
taken up
UNCHANGED
plasmid
resistant
to both A + T
taken up
MODIFIED
plasmid
resistant
to
intact gene
(eg A)
sensitive
to
gene w/ donor gene between
(T)
FAILED
to take up any plasmid
sensitive
to both A + T
4. Identifying Transformed Bacteria
Containing Desired Gene
MARKER GENES
method
1.
bacteria transferred to
agar plate w/ AMPICILLIN
colonies only develop from bacterial cells
RESISTANT TO A
2. need to check if contain donor gene or not?
could have taken up unchanged plasmid
3. 2 replica plates made
blotting orig plate carefully w/ absorbent pad
pressing against surface fresh agar plate with T
allows some cells of colonies be transferred
colonies should form on same position of new plate
4. some colonies will be missing
do
NOT HAVE RESISTANCE to T
∴ bacteria contain
recombinant plasmid
position located on A agar plate
DNA PROBES
incorporate gene into P = produces fluorescent protein
RE cut through this gene
& donor gene added
bacteria do not fluoresce under UV light = contain recombinant P + desired gene
5. Clone Bacteria to
Produce Large Numbers
occur in large fermenters
where conditions ideal for:
rapid growth & production of desired product