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In vitro
DNA recombination.
Recombinant DNA. Introduction of genetic information in bacteria
(transformation, conjugation and transduction)
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A single-strand specific DNA and RNA endonuclease which will degrade single-stranded
extensions from the ends of DNA and RNA molecules, leaving blunt, ligatable ends.
S1 nuclease
S1 Nuclease degrades single-stranded DNA and RNA endonucleolytically to yield 5phosphoryl-terminated products. Double-stranded nucleic acids (DNA:DNA, DNA:RNA or
RNA:RNA) are resistant to degradation except with extremely high concentrations of enzyme.
DNA POLYMERASES
Enzyme that synthesizes DNA is called DNA polymerase and one that copies an existing DNA or
RNA molecule is called template-dependent DNA polymerase.
The mode of action of a template dependent DNApolymerase
A template-dependent DNA polymerase makes a
new DNA polynucleotide whose sequence is
dictated, via the base-paired rules, by the sequence
of nucleotides in the DNA molecule that is being
copied.
New polynucleotide are synthesized in 53
direction.
DNA polymerase is unable to use an entirely single-stranded molecule as the template so in
order to start the synthesis there must be a short double stranded region to provide a 3 end
onto which the enzyme will add nucleotides.
In the test tube, a DNA copying reaction is
iniciated by attaching to the template a short
synthetic oligonucleotide (usually a about 20
nucleotides in length) that act as a primer for
DNA synthesis.
The position within the template molecule at
with DNA copying is initiated can be specified
by synthesizing a primer with the apropiate
nucleotide sequence.
(Apreciatte then the importance of priming in
PCR)
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35 EXONUCLEASE. Nucleotides
removal from the 3 end of the strand
that has just been synthesized. This is
called PROOF-READING activity
because allow to correct errors by
removind incorrectly inserted
nucleotides.
53 EXONUCLEASE. Possessed by
DNA polymerases whose function in
genome replication requires that must
be able to remove at least part of
polynucleotide that is already attached
to the template strand that the
polymerase is copying.
DNA LIGASES.
DNA fragments that has been generated by treatment with a restriction endonuclease can be
joined back together again or attached to a new partner by a
DNA ligase.
The reaction requires energy (ATP or NAD).
The most used DNA ligase is obtained from
E.Coli cells infected with T4 bacteriophage.
It is involved in replication of the phage
DNA an is encoded by the T4 genome. Its
natural role us to synthesize
phosphodiester polynucleotide of a
double-stranded molecule.
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The reaction can ocure only if the ends to be joined come close enough to one another by
chance. If the who molecules have complementary sticky ends and the ends come together by
random diffusion, then transient base pair might form between the two overhangs. These base
pairs are not particulary stable but they may persist for sufficient time for ligase enzyme to
attach to the junction and synthesizes phosphodiester bonds to fuese the ends together.
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Another way to create a sticky end is by HOMOPOLYMER TAILING, in which nucleotides are
added one after the other to the 3 terminus at a blunt end. The enzume involved is called
terminal deoxynucleotidyl transferase.
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END-MODIFICATION ENZYMES.
Terminal deoxynucleotidyl transferase is a template-independent DNA polymerase because it
is able to synthesize a new DNA polynucleotide without basepairing of the incoming
nucleotides to an existing strand of DNA or RNA.
Alkaline phosphatas. Removes phosphate goups from the 5 phosphates can be ligated to one
another and a phosphatased end can ligate to a non phosphatased end but a link cannot be
formed between a pair of ends if neither carries a 5 phosphate. T4 polynucleotide kinase
T4 polynucleotide kinase
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Disadvantages:
1. Produces an endotoxine
2. Does not have splicing machinery
3. Post-translational modifications of proteins is different from that in eukaryotic cells.
Shuttle vectors have been developped
Different strains for different purposes
Easy to transform
Replicates vectors faithfully
Selectable markers available
DISCRIMINATION BETWEEN TRANSFORMED AND UNTRANSFORMED-CELLS.
1. DOMINANT SELECTION MARKERS:
AmpR: ampicillin acts on bacterial membrane
enzymes.
CmR:
(subunit 50S)
TetR:
acetylated antibiotic-derivates
30S)
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PLASMIDS
Plasmids are covalently closed double-stranded DNA molecules. Are circular molecules of DNA
that lead an independent existence in the bacterial cell. Plasmids almost always carry one or
more genes and often these genes are responsible for a useful characteristic displayed by the
host bacterium.
They behave as accessory genetic units; they replicate and inherit independently from the
bacterial chromosome.
They need the host cell for replication and transcription.
Often they provide genes that encode for enzymes which give some advantage to the host cell
under stressful environmental conditions.
In the laboratory antibiotic resistance is often used as a selectable marker to ensure that the
bacteria in a culture contain a particular plasmid.
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Todos los plsmidos contienen un adaptador de policlonaje (polilynker) con un amplio abanico
de dianas de insercin que se pueden usar individualmente o combinadas. En este ltimo caso
se puede definir la orientacin del insert. En muchos casos el fragmento de policlonaje est
situado en medio de un segmento derivado del opern lac de E.coli.
Selection marker are genes that add a new trait to cells.
Origin of replication, selection marker, MCS o Polylinker.........
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(See also
pag:34)
The pUC series is derived from an earlier cloning
vector, Pbr322, which was originally constructed
by ligating together restriction fragments from
three naturally occurring E.coli plasmids.
pUC18 contanins an ampicillin resistance gene, a
regulatable segment of the B-galactosidae gene
(lacZ) of the lactose operon of E.coli. Ligation of a
new DNA into any one of these sites results in
insertional inactivation of the gene and hence loss
of b-galactosidase activity. This is the key to
distinguish a recombinant plasmid from a
nonrecombinant plasmid that has no new DNA.
Screening for B-galactosidase presence or absence
is in fact quite easy. (See Lac Selection)
LAC SELECTION:
Rather than assay for lactose being splite to
glucose and galactose, the presence of functional B-galactosidase molecules in the cell is
checked by a histochemical test with a compound called X-gal (a-complementation of bgalactosidase (X-gal: 5 bromo-4 chloro-3 indolyl-b-D-galactoside IPTG: isopropyl thio b-Dgalactoside)), which the enzyme converts into blue producuct. If x-gal (plis IPTG) is added to
the agar, along with ampicillin, then nonrecombinant colonies, the cells of which syntehize bgalactosidase will be colored blue whereas recombinant with a disrupted lacZ gene which are
unable to make b-galactosidase will be white.
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Substrain 71-18:
There is a lac operon deletion. Notwithstanding there is a F factor with a modified lac operon
(delta M15) allowing alpha-complementation.
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cell.
If they share the ORI they are incompatible
There are several elements that works as replication origin in E.coli. You can have two
different plasmids in a single cell and grow them both if they contain selection markers. The
small transcript can join to the big one and the transcription turns off. So when we have hight
concentration of plasmid, there are acomulation of RNA and transcription stops.
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Bacteriophage lambda.
PROPERTIES, CYCLES LYTIC AND LYSOGENIC
Los bacterifagos son parasites bacterianos que solo pueden replicarse en el interior de una
bacteria. Sus funciones bsicas i de supervivencia son:
-
Esta serie de sucesos constituyen el ciclo ltico. Los fagos que solo son capaces de llevar a cabo
este ciclo son conocidos como virulentos.
Los fagos atemperados (temperate) tienen como alternativa la integracin en el genoma del
husped y permanecen en lisogenia, es decir, replicndose con el cromosoma del husped y
expreando slo el gen represor, imprescindible para impedir que se inicie el ciclo titico.
El DNA de lambda es una molcula lineal de doble banda de unas 48,5KB. Se conoce su
secuencia totamente i en los extremos de la cadena posee un corto fragmento de 12 bases.
Estos expremos son llamados COS (Extremos cohesivos) son complementarios y permiten la
circularizacin del profago antes de la integracin en el genoma del husped.
Los genes localizados en la aparte izquierda del mapa lineal codifican las protenas de la cabeza
y cola, los de la aprte centrak estn implicados en la recombinacin y los procesos de
lisogenizacin, estos genes no son esenciales para la infeccin i crecimiento del fago por lo que
EGM. TEMAS 1,2,3
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pueden ser substituidos durante procesos de clonaje. Los genes de la derecha son los de
recgulacin e inmunidad (cI, cro, N)
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La identificacin de una lisis plaque cuyos fagos lleven un gen de inters entre todas las plaques de lisis
crecidas se suelen realizar mediante mtodos de hibridacin.
Inserts: up to 25 Kb
Introduction of DNA in host cells: nude DNA and infection with in vitro reconstituted virus
High recuperation: up to 106-107 phage particles per plaque.
High sensitivity of detection with hybridization probes.
El fago lambda wild type no es adecuado para su utilizacin como vector ya q tiene
numerosas dianas de restriccin para cualquiern enzima y baja capacidad sin eliminar
parte del genoma propio para insertos de gran tamao. Por ello hay variantes aptas
para el clonaje, que han sido modificadas eliminando dianas o colocndolas en luhares
idneos (Izquierdo, p129):
-
Insertion vectors: Part or all the optional DNA has been removed an a unique
restriction site introduced at somo position within the trimmed down genome.
Made by the direct integration of the insert in a host target sequence.
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Permite cloner pequeos fragmentos de cDNA enla regin de cI. Por tanto los recombinantes
sern cI- . Los bacterifagos sin el DNA heterologo harn la lisogenesi i no formaran plaques.
Poseen dos regions de policlonage a ambos lados de una region de rlleno procedente de E.Coli
y que sera subtituida por un insert. Permite hasta unas 20kb, utilizando cualquiera de las
dianas de restriccin orientadas en sentido opuesto a ambos ados de las sequencias de
relleno.
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The lambda genome is linear but the two natural ends of the molecile have 12-nucleotidese
single stranded overhands (COS SITES)which have complementary sequences and so can base
pair to one another. A lambda cloning vector can therefore be obtained as a circular molecule
chich can be manipulated in the same way as a plasmid and re-introduced into E.coli by
transfection, the term used for uptake of naked phage DNA. Alternatively a more efficient
uptake system called in vitro packinh can be used. This procedure starts with the lineal version
of the cloning vector, the initial restriction cutting the molecule into two segments, the left
and the right arms, each with a cos site at one end. The ligation is carried out with quatities of
each arm and the DNA ti be cloned, the aim beig to produce concatamersn in which the
different fragments are linked together in the order left arm new DNA- right arm. The
concatamers are the added to an in vitro packing mix wich contains all the proteins needed to
make a lambda phage particle. The proteins form phage particles spontaneously and will place
inside tge particles any DNA fragment that is between 37-52kb out of concatamers and
EGM. TEMAS 1,2,3
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construct lambda phages around them. The phages are the mixed with E.coli cells and the
natural infection process transports the vector + new DNA into bacteria. (Genomes 2, pg 113)
ADN genmico y de alto peso molecular se trata con encimas para obtener un tamao medio
de DNA. Se perepara a su vez ADN del fago lambda para ser utilizado como vector de
sustitucin. El ADN del vector se trata con tambin con enzimas. Hhay q ser cuidadoso de crear
extremos cohesivos para que puedan unirse ambos extremos por complementariedad de las
secuencias. Se substituye un fragmento del vector por uno de inserto, lo que nos permite
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recuperar el tamao del genoma del fago original. El fago lambda solo empaqueta ADN de
cierto tamao, con lo que quedarn excluidos aquellos que unan brazos fgicos sin inserto.
La enzima ligasa unir covalentemente los extremos apareados por las dianas de restriccin y
los fagos se empaquetaran in vitro. El fago lambda normalmente se replica por el mtodo del
crculo rodante y empaqueta su ADN cortado en las regiones Cos. La regin Cos, cuando es
cortada da lugar a terminales cohesivos de 12 pb que constituirn los extremos del profago.
Para el paquetamiento in vitro de libreras genmicas se utiliza frecuentemente un mutante
del fago lambda deleccionado en las secuencias cos. Este mutante se mantiene en dorma
lisognica (como profago) en una cepa de E.Coli. Al introducir el ciclo ltico se origina una
acumulacin intracelular de todos los componentes del empaqietamiento, inclyendo las
precabezas vacias. El paso siguiente est bloqueado. Lisados de este mutante se pueden aadr
a ADN recombinante que posea secuencias Cos. De este modo, solo el ADN recombinado ser
empaquetado en el fago.
COSMIDS
Plsmidos a los que se les ha insertado la regin COS del fago lambda. Son partiularmente
tiles para clonar insertos de gran tamao. COS confiere al plasmido la facilidad para ser
empaquetado in vitro por l sistema del fago siempre que existan de 35-52kb entre COS y COS.
Lo que implica una seleccin efectiva para insertos de entre 40-45kb.
Una vez l fago haya inyectado el DNA en la bacteria este se comportara como n plsmido y
podr ser seleccionado por resistncia a ampicilina.
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The genome encodes 10 proteins and there is an intergenic region (IG) which contains the
origins of replication for both strands, + and -, the packaging signal for the + strand and a
transcription termination sequence.
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production of ssDNA for fragments cloned in plasmids which contain the ORI.
Required phage factors are provided by helper phages Helper phages have defects in their
replication origin that result in a poor growth, but they express all the transrequired elements
for production of phages
Bacterifago M13
Es un fago filamentoso, su DNA es circular de hebra sencilla y 6,4kb. Infecta bacterias
portadoras de un episoma F a travs de los filamentos sexuales F (pili). La replicacin del fago
no produce la lisi de la bacteria sino que esta continua creciendo lentamente excretabdi
particular al medio.
El DNA viral tiene una fase replicativa en la que se originan unas 100 copiasde DNA
nicartenario. Posteriormente se acumulan unas protenas de unin al DNA de banda simple
codificadas por el fago, que impiden la sintesi de l cadena complementaria, la maduracin
tiene lugar en la membrana, donde se sustituyen las protenas de unin a ADN de hebra
sencilla por las de la capside.
En los procesos de clonacin es mas fcil trabajar con ADN bicatenario. sta es la razn por la
que se utiliza preferentemente la forma replicativa de M13, esta forma se puede aislar de
clulas infectadas, manipular como cualquier ADN de plsmido y reintroducir en clulas por
transformar. El ADN de doble banda se incorporar al ciclo de replicacin y generar aprtculas
fagicas que contendrn solo una de las dos hebras del ADN + i la otra nunca se empaquetar.
Tanto el ADN de la forma replicativa como el de hebra sencilla del fago pueden transfectar
clulas competentes de E.Coli y proseguir con el ciclo vital.
No hay grandes restricciones respecto al tamao del inserto puesto que el tamao final de la
particula viene determinado por la longitud del ADN, sin embargo los los insertos grandes son
muy inestables y acaban por ser eliminados o acortados.
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Estos viriones sern de dos tipos: recombinantes y auxiliares. En la mayora de los casos no
ser necesario separar los unos de los otros ya q se emplearan primers especficos que solo
estn presentes en los recombinantes.
Helper phage
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CLONING IN YEAST
Production of proteins with a commercial value:
Post translational.protein modification
No toxins
Expression with genomic DNA
Large mutant collection available: facilitates detection and isolation of recombinants
Small genome (1.4 X10E7 versus 3X10E9)
Rapid growth (<2 hours to divide). They can grow in suspension or form colonies on agar
Division by sexual or asexual reproduction
Can be grown as haploids or diploids facilitating the isolation of recessive mutations
Yeast have a polysaccharide wall around the cell membrane which hampers DNA uptake.
Las levaduras mantienen las ventajas de los organismos en cuanto a que son unicelulares, de
facil manipulacin y crecimiento rpido. Su organismo celular es eucariota lo que permite la
realizacin de procesos de expresin y maduracin caracteristicos de las clulas animales i
vegetales.
Son capaces llevar a cabo prcesos que no realizan las bacterias y que pueden ser importantes
para la expresin de un gen eucaritico en un sistema heterlogo. Realizan acetilaciones Nterminales, N-glicosilaciones, fosforilaciones, eliminacin de la metionina inicial de la protena
madura, secrecin proteica, procesamientos proteoltico, permiten unin y ensamblaje de
subunidades en macromolculas.
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Spheroplast transformation.
To transform cells, spheroplasts are produced by removing the wall enzymatically or by
treatment with lithium acetate
Spheroplasts behave as competent bacteria and can take DNA from the environment in the
presence of CaCl2. The cell wall can be synthesized again in the appropriate growth medium.
Large DNA fragments can be cloned in YACS
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and no conclusions can be made about the presence or absence of growth on selective media. This is
particularly useful if there are questions about the age or viability of the cells on the original plate.
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Los vectores ARS/CEN son vectores ARS a los que se les ha aadido secuencias centromricas
para augmentar su estabilidad. La regin centromrica es responsable de la secrecin de las
clulas hijas al crear una estructura cromatnica especial. No pa
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YACs
Los vectores YAC (yeast astificial chromosome) son vectores ARS/CEN a los que se les ha
aadido telmeros cromosmicos. Permiten clonar los fragmentos ms grandes de DNA
siendo especialmente tiles cuando interesa aislar genes con muchos intrones que ocupan una
gran extensin del genoma.
Incluyen un plsmido procaritico para su propagacin y preparacin en E.Coli. Se puede
cortar con EcoRI en dos fragmentos que constituyen los brazos cromosmicos. El brazo
izquierdo tiene un telmero, un centrmero, una secuncia ARS i un gen de resistncia a
ampicilina para la seleccin. El brazo derecho solo tiene otro telmero y otro gen de seleccin
URA3.
Se emplea principalmente para elaborar libreras de genomas muy grandes .
El cromosoma artificial de levaduras o YAC (Yeast artificial chromosome) forma parte de los vectores de
clonacin de alta capacidad siendo, de hecho, el de mayor capacidad (200 kb - 3.000 kb). Fueron
descritos por primera vez en 1983 por Murray y Szostack.
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Homologous recombination
Per determiner la function de un gen, a veces se puede producer una mutacin y ver el efecto
que ello genera en la clula o organismo. Permite producir mutacione sin vivo a nivel del gen.
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Mutantes letales tambien pueden ser rescatados en levaduras iploides donde al inducir la
esporulacin en un mediopobre, habr igual proporcin de esporas haploides letales como
normales
.
EGM. TEMAS 1,2,3
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