Download Selective propagation of the clones

Survey
yes no Was this document useful for you?
   Thank you for your participation!

* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project

Document related concepts

Mutation wikipedia , lookup

Chromosome wikipedia , lookup

Mutagen wikipedia , lookup

Genome (book) wikipedia , lookup

Metagenomics wikipedia , lookup

DNA repair wikipedia , lookup

Comparative genomic hybridization wikipedia , lookup

DNA profiling wikipedia , lookup

Mitochondrial DNA wikipedia , lookup

Human genome wikipedia , lookup

SNP genotyping wikipedia , lookup

Zinc finger nuclease wikipedia , lookup

Genome evolution wikipedia , lookup

Nutriepigenomics wikipedia , lookup

DNA polymerase wikipedia , lookup

Cloning wikipedia , lookup

Cancer epigenetics wikipedia , lookup

Nucleosome wikipedia , lookup

Bisulfite sequencing wikipedia , lookup

Replisome wikipedia , lookup

Genetic engineering wikipedia , lookup

Genealogical DNA test wikipedia , lookup

Gene wikipedia , lookup

United Kingdom National DNA Database wikipedia , lookup

DNA damage theory of aging wikipedia , lookup

Primary transcript wikipedia , lookup

Point mutation wikipedia , lookup

Genomics wikipedia , lookup

Gel electrophoresis of nucleic acids wikipedia , lookup

Cell-free fetal DNA wikipedia , lookup

Plasmid wikipedia , lookup

Epigenomics wikipedia , lookup

Nucleic acid double helix wikipedia , lookup

Genome editing wikipedia , lookup

Designer baby wikipedia , lookup

Non-coding DNA wikipedia , lookup

Microevolution wikipedia , lookup

Nucleic acid analogue wikipedia , lookup

DNA vaccination wikipedia , lookup

Therapeutic gene modulation wikipedia , lookup

DNA supercoil wikipedia , lookup

Site-specific recombinase technology wikipedia , lookup

Extrachromosomal DNA wikipedia , lookup

Cre-Lox recombination wikipedia , lookup

Deoxyribozyme wikipedia , lookup

No-SCAR (Scarless Cas9 Assisted Recombineering) Genome Editing wikipedia , lookup

Vectors in gene therapy wikipedia , lookup

Molecular cloning wikipedia , lookup

Helitron (biology) wikipedia , lookup

Artificial gene synthesis wikipedia , lookup

History of genetic engineering wikipedia , lookup

Genomic library wikipedia , lookup

Transcript
VECTOR BIOLOGY AND GENE
MANIPULATION
INTRODUCTION TO CLONING
OVERVIEW OF CLONING:
 Discovery of DNA Modifying and Restriction
Enzymes.
 Cutting and joining DNA molecules by
restriction enzymes and ligases, respectively.
 Selective amplification of genes of interest using
PCR (Polymerase Chain Reaction).
 Attachment of fragments of DNA to suitable
replicons called cloning vectors.
 Small plasmids and bacteriophages are most
suitable.
 Maintenance does not require integration into the
host genome.
 Genetic transformation of Escherichia coli.
 Recombinant DNA can be isolated in intact form.
Pre requisites to gene manipulation:
 The vector DNA must be purified and cut open.
 The passenger DNA must be inserted into the
vector molecule to create the recombinant DNA.
 Cutting and joining reactions are monitored by
gel electrophoresis.
 The recombinant must be introduced into the
host - E.coli.
Separation of nucleic acids:
 Agarose and polyacrylamide gels are used to
separate nucleic acids (DNA, RNA).
 Northern blot: separation of RNA molecules on
agarose gel, blotting and hybridizing with specific
labeled probe or gene
Southern blot: separation of DNA molecules on
agarose or polyacrylamide ges, blotting to nylon or
nitrocellulose membranes and hybridizing with
specific labeled probe or gene
 Gel retardation or band shift assay
(EMSA).
 Reduction in electrophoretic mobility upon
binding of proteins to DNA.
 Western – Proteins from acryl amide gels
to membranes.
DNA CLONING VECTORS
Characterization of the DNA of any organism:
 DNA cloning: A desired DNA fragment is identified
and selectively amplified so that its structure can be
studied using a variety of different techniques such
as Restriction enzyme analysis, DNA sequencing,
in vitro expression studies.
 Molecular Hybridization: The DNA fragment is not
amplified but rather studied as it is found in a
complex mixture of DNA fragments. The restriction
analysis can be performed as well as the
chromosomal location. RNA expression can also be
studied in this way.
How to introduce DNA into cells: Vectors
Vectors used to move DNA between species, or
from the lab bench into a living cell, must meet
three requirements:
 They must be autonomously replicating
DNA molecules in the host cell.
-The most common vectors are designed for
replicating in bacteria or yeast, but there are
vectors for plants, animals and other species.
 They must contain a selectable marker so
cells containing the recombinant DNA can be
distinguished from those that do not. An
example is drug resistance in bacteria.
 They must have an insertion site to
accommodate foreign DNA.
-Usually a unique restriction cleavage site in a
nonessential region of the vector DNA.
-Later generation vectors have a set of about
15 or more unique restriction cleavage sites.
How to clone DNA:
1.Cell based DNA cloning.
2. Cell free DNA cloning.
Cell based Cloning of DNA:
 Construction of a recombinant DNA library
Ligation of the target DNA into a suitable vector.
 Transformation of the library into a host
E.coli or yeast to amplify cloned DNA fragments.
 Selective propagation of the clones
Plating of clones on selection media to allow
screening.
 Isolation of the recombinant clones
Identification and expansion of clones of interest.
PLASMID CLONING VECTORS
 Plasmids are autonomously replicating circular DNA
molecules found in bacteria.
 They have their own origin of replication, and they
replicate independently of the origins on the "host"
chromosome.
 A plasmid that was widely used in many recombinant
DNA projects is pBR322. It replicates from an origin
derived from a colicin-resistance plasmid (ColE1).
 This origin allows a fairly high copy number,
about 100 copies of the plasmid per cell.
 Plasmid pBR322 carries two antibiotic
resistance genes, each derived from different
transposons (jumping genes) that were initially
found in R-factors, which are larger plasmids
that confer antibiotic resistance.
Plasmid pBR 322
(beta lactamase)
Features of plasmid pBR322:
The gene conferring resistance to ampicillin (ApR) can
be interrupted by insertion of a DNA fragment into the
PstI site, and the gene conferring resistance to
tetracycline (TcR) can be interrupted by insertion of a
DNA fragment into the BamHI site.
Use of the TcR and ApR genes allows for easy
screening for recombinants carrying inserts of foreign
DNA.
For instance, insertion of a restriction fragment in the
BamHI site of the TcR gene inactivates that gene. One
can still select for ApR colonies, and then screen to see
which ones have lost TcR .
Plasmid pUC19 with lacZ gene and Ampr gene and an
E. coli plasmid replicon.
(IPTG: lacZ inducer
X-gal: substrate for beta galactosidase)
Yeast vectors:
The yeast 2µm plasmid: REP1 and REP2 are involved
in replication of the plasmid, and FLP codes for a
protein that can convert the A form of the plasmid
(shown here) to the B form, in which the gene order has
been rearranged by intramolecular recombination. The
function of D is not exactly known.
 Development of cloning vectors for yeast has been
stimulated greatly by the discovery of the 2 µm plasmid
that is present in most strains of S. cerevisiae.
 The 2 µm plasmid is an excellent basis for a cloning
vector. It is 6 kb in size which is ideal for a vector, and
exists in the yeast cell at a copy number of between 70
and 200.
 Replication: makes use of a plasmid origin, several
enzymes provided by the host cell, and the proteins
coded by the REP1 and REP2 genes carried by the
plasmid.
 Some yeast cloning vectors carry genes conferring
resistance to inhibitors such as methotrexate and copper
for selection.
 Most of the popular yeast vectors make use of a
radically different type of selection system.
 In practice a normal yeast gene is used, generally one
that codes for an enzyme involved in amino acid
biosynthesis.
 In order to use LEU2 as a selectable marker, a special
kind of host organism is needed. The host must be an
auxotrophic mutant that has a nonfunctional LEU2 gene.
 Such a leu2- yeast is unable to synthesize leucine and
can survive only if this amino acid is supplied as a
nutrient in the growth medium.
 Selection is possible because transformants contain a
plasmid-borne copy of the LEU2 gene, and so are able
to grow in the absence of the amino acid.
 In a cloning experiment, cells are plated out onto
minimal medium, which contains no added amino
acids.
 Only transformed cells are able to survive and form
colonies.
CLONING STRATEGY USING THE YEAST
2 µm PLASMID
LAMBDA AND COSMID VECTORS
 Bacteriophages (lambda and M13) can be used as
cloning vectors.
 Lamda phage has a genome of about 50 kb of linear
DNA.
 Its life cycle is conducive to the use as a cloning
vector.
 The lytic cycle can be supported by only a portion of
the genes found in the lambda genome.
 This type of lambda vector is also called as the
lambda replacement vector (because the lambda DNA
is removed and replaced with foreign DNA).
Lambda life cycle:
 The lytic life
cycle
produces
phage
particles
immediately.
 The
lysogenic
life cycle requires
genes in the middle
of the genome,
which
can
be
replaced.
Cos sites:
 at either end of the lamda DNA molecules
(double stranded), a short 12 nucleotide stretch,
in which DNA is single stranded.
 two single strands are complementary and
base pair with one another to form a circular,
completely double stranded molecule.
act as recognition sequences for an
endonuclease cleaves the catenane at the cos sites
to produce individual genomes.
LAMBDA INSERTION AND REPLACEMENT
VECTORS
 Only 37 to 52 kb DNA fragments can be packaged
into the lambda head.
 This can be done in vitro.
The middle portion of the lambda genome can be
replaced (that region is non essential) and DNA can be
cloned (up to 20 kb).
 These vectors are used for construction of genomic
DNA libraries.
LAMBDA GENOME
Lamda DNA transfection efficiency low; when packed more efficient.
the bacterial chromosome integrated with lamda DNA-lysogen
M13 AND PHAGEMID VECTORS
 These vectors are used to produce DNA for sequencing.
 M13 is a virus with a genome of single stranded DNA.
It has a nonessential region into which foreign genes can
be inserted.
 It has been modified to carry a gene for β-galactosidase
(lacZ) as a way to screen for recombinants.
 The replicative form is duplex, allowing one to cleave
with restriction enzymes and insert foreign DNA.
 Some vectors are hybrids
between plasmids and
single-strand phage;
these are called phagemids.
 One example is pBluescript.
Phagemids are plasmids (with the modified, highcopy number ColE1 origin) that also have an origin
of replication of the phage f1 which is related to
phage M 13.
 The vector has a multiple
cloning site inserted into
the lacZ gene and
which is flanked by
two different phage
RNA polymerase promoters.
One can easily obtain either double- or
single-stranded forms of these plasmids.

 The "blue" comes from the blue-white screening
for recombinants that can be done with the multiple
cloning sites which are in the β-galactosidase gene.
 The "script" refers to
the ability to make RNA copies
of either strand in vitro
with phage RNA polymerases.
 Infection of transformed bacteria (containing the
phagemid) with a helper virus (e.g. derived from
M13) will cause the M13 origin to be activated, and
progeny viruses carrying single-stranded copies of
the phagemid can be obtained.
COSMID VECTORS

A cosmid is a hybrid between a lambda vector and
a plasmid.
 The COS sites are the only thing that is necessary
for lambda DNA packaging.
 Therefore if one can ligate COS sites about 50 kb
apart then the ligation products can be in vitro
packaged.
BACTERIOPHAGE P1
 These vectors are like lambda and can hold up
to 100 kb of DNA.
 This DNA can then be packaged by the P1
phage protein coat.
 The use of T4 in vitro packaging systems can
enable the recovery of 100 kb inserts.
YEAST ARTIFICIAL CHROMOSOMES
(YACs)
 YACs are yeast vectors with centromeres and
telomeres.
 They can carry about 200 kb or larger fragments (in
principle up to 1000 kb = 1 Mb).
 Thus very large fragments of DNA can be cloned in
yeast.
 In practice, chimeric clones with fragments from
different regions of the genome are obtained fairly
often, and some of the inserts are unstable.
Chromosome Structure
 Centromere is required for the chromosome to be
distributed correctly to daughter cells during cell
division.
 Two telomeres, the structures at the ends of a
chromosome, which are needed in order for the ends
to be replicated correctly and which also prevent the
chromosome from being nibbled away by
exonucleases.
 The origins of replication, which are the positions
along the chromosome at which DNA replication
initiates, similar to the origin of replication of a
plasmid.
Chromosome Structure
The structure and use of a YAC vector
 Several YAC vectors have been
developed but each one
is constructed along
the same lines, with pYAC3 being a typical example.
 pYAC3 is essentially a pBR322 plasmid into which
a number of yeast genes have been inserted.
 The genes, URA3 from YIp5 and TRP1-ori from
YRp7 have been included in pYAC3.
 The DNA fragment
containing TRP1 is extended
even further to include the sequence
called CEN4, which is the DNA
from the centromere region
of chromosome 4.
The TRP1–origin–CEN4 fragment therefore contains
two of the three components of the artificial
chromosome.
 The third component, the telomeres, is provided by
the two sequences called TEL. These are not themselves
complete telomere sequences, but once inside the yeast
nucleus they act as seeding sequences onto which
telomeres will be built.
 SUP4, is the selectable marker into which new
DNA is inserted during the cloning experiment.
The cloning strategy with pYAC3 is as follows :
 The vector is first digested with a combination of
BamHI and SnaBI restriction enzymes, cutting the
molecule into three fragments.
 The fragment flanked by
BamHI sites is discarded,
leaving two arms, each bounded by one
TEL sequence and one SnaBI site.
 The DNA to be cloned, which must have blunt ends
(SnaBI is a blunt end cutter, recognizing the sequence
TACGTA), is ligated between the two arms, producing
the artificial chromosome.
 Protoplast transformation is then used to introduce
the artificial chromosome into S. cerevisiae.
 The yeast strain that is used is a double auxotrophic
mutant, trp1- ura3-, which is converted to trp1+ ura3+
by the two markers on the artificial
chromosome.
Any cell transformed with an incorrect artificial
chromosome, containing two left or two right arms
rather than one of each, is not able to grow on
minimal medium as one of the markers is absent.
 The presence of the insert DNA in the vector can
be checked by testing for insertional inactivation of
SUP4, which is carried out by a simple colour test:
red colonies are recombinants, white colonies are
not.
A YAC vector and
the way it is used to
clone large pieces of
DNA.