Download IV. 5. Clonació ADN

UD. IV. GENÈTICA. Ll. IV. 5. Biotecnologia
2.1. ADN recombinant, enzims de restricció
Formació d’ADN recombinant.
Utilització d’enzims de restricció.
Lloc de restricció, és le punt on l’enzim pot tallar
l’ADN
Fragment de restricció.
Figure 20.3-3
Restriction site
5
3
GAATTC
CTTAAG
DNA
5
3
1 Restriction enzyme
cuts sugar-phosphate
backbones.
5
3
5
3
5 Sticky 3
3
5
end
5
2 DNA fragment added
3
3
5
from another molecule
cut by same enzyme.
Base pairing occurs.
5
3 5
3
3 DNA ligase
3 5
G AATT C
C TTAA G
G AATT C
C TTAA G
53
5 3
3
5
One possible combination
seals strands
5
3
3
Recombinant DNA molecule
5
UD. IV. GENÈTICA. Ll. IV. 5. Biotecnologia
2.2. Clonació d’un gen eucariont
LE 20-4_3
Bacterial cell
Isolate plasmid DNA
and human DNA.
Clonació usant
plàsmids
bacterians
lacZ gene
(lactose
breakdown)
Human
cell
Restriction
site
ampR gene
(ampicillin
resistance)
Cut both DNA samples with
the same restriction enzyme.
Bacterial
plasmid
Gene of
interest
Sticky
ends
Human DNA
fragments
Mix the DNAs; they join by base pairing.
The products are recombinant plasmids
and many nonrecombinant plasmids.
Recombinant DNA plasmids
Introduce the DNA into bacterial cells
that have a mutation in their own lacZ
gene.
Recombinant
bacteria
Plate the bacteria on agar
containing ampicillin and X-gal.
Incubate until colonies grow.
Colony carrying nonrecombinant plasmid
with intact lacZ gene
Colony carrying
recombinant
plasmid with
disrupted lacZ gene
Bacterial
clone
LE 20-5
Hibridació amb sonda d’àcid nucleic
Master plate
Filter
Radioactive
single-stranded
DNA
Solution
containing
probe
Master plate
Probe
DNA
Colonies
containing
gene of
interest
Gene of
interest
Single-stranded
DNA from cell
Film
Filter lifted
and flipped over
Hybridization
on filter
A special filter paper is
pressed against the
master plate,
transferring cells to the
bottom side of the filter.
The filter is treated to break open
the cells and denature their DNA;
the resulting single-stranded DNA
molecules are treated so that they
stick to the filter.
The filter is laid under
photographic film,
allowing any radioactive
areas to expose the film
(autoradiography).
After the developed
film is flipped over,
the reference marks
on the film and
master plate are
aligned to locate
colonies carrying
the gene of interest.
LE 20-6
or
Bacterial
clones
Recombinant
plasmids
Plasmid library
Foreign genome
cut up with
restriction
enzyme
Recombinant
phage DNA
Phage
clones
Phage library
UD. IV. GENÈTICA. Ll. IV. 5. Biotecnologia
2.4. Amplificació de l’ADN: reacció en cadena de la polimerasa (PCR)
Aquesta tècnica s’usa quan es disposa de quantitats molt petites d’ADN.
En poc temps s’aconsegueixen milions de còpies.
Aplicació a l’estudi de l’ADN dels neandertals
5
3
Target
sequence
Genomic DNA
Denaturation:
Heat briefly
to separate DNA
strands
Cycle 1
yields
2
molecules
Annealing:
Cool to allow
primers to form
hydrogen bonds
with ends of
target sequence
Extension:
DNA polymerase
adds nucleotides to
the 3 end of each
primer
Cycle 2
yields
4
molecules
Cycle 3
yields 8
molecules;
2 molecules
(in white boxes)
match target
sequence
3
5
5
3
3
5
Primers
New
nucleotides