Download Recombinant DNA technology

Recombinant DNA technology
Recombinant DNA technology
• Methods used to join together
(recombine) different DNA segments that
are not found together in nature.
• This technique is used in genetic analysis
to serve several applications:
1. In Vitro Mutagenesis It is much easier to make
mutation in isolated gene than when it is part of a
complex organism structure
2. Study of the gene properties (genotype)
3. Study the expression of gene product (phenotype)
4. Produce large quantity of medically or
agriculturally or industrially important gene product
e.g Insulin production in large quantity by bacteria
Hydrolysis of DNA
by restriction
endonucleases
Fig. 13-4, p.333
Production of
recombinant DNA
Fig. 13-5, p.334
Restriction Endonucleases
(r.e)
1. Enzymes produced by bacteria that hydrolyze
the phosphodiester backbone of DNA at specific
sequences
2. The sequences targeted by r.e are palindromes,
meaning their sequence reads the same on
both strands going in the same direction
3. Most r.e cut DNA in a way that leaves sticky ends
that are very useful for recombining DNA from
different sources.
Table 13-1, p.335
p.335
Dolly
1996-2003
In July, 2016, four identical
clones of Dolly (Daisy, Debbie,
Dianna and Denise) were alive
and healthy at nine years old.
p.331
The cloning process that produced Dolly
The cloning of a virus
Fig. 13-6, p.336
The cloning of cells
Fig. 13-7, p.336
The cloning of
human DNA
fragments with a
viral vector
Fig. 13-8, p.336
DNA Plasmid
Extra chromosomal self-replicating genetic elements of a bacterial cell
& can be transferred from one strain of a bacterial species to another
by cell-to-cell contact.
p.338
For A Successful Experiment
When bacteria take up a plasmid, we say they have
been transformed
Bacteria are encouraged to take up foreign DNA by:
1.heat-shock the bacteria at 42 C. followed by placing
them on ice.
2. Place them in an electric field “electroporation”
Then selection through selectable markers on the
plasmid.
A vector cloning site containing multiple restriction sites
Fig. 13-11, p.339
Cloning with pU plasmids
Fig. 13-12, p.340
Cloning
1. It refers to creating a genetically identical population
2. DNA can be combined by using r.e that create sticky ends in
the DNA. This rDNA has a target DNA sequence of interest
3. The target DNA sequence is carried in some type of vector,
usually a bacterial plasmid or a virus
4. The target DNA sequence is inserted into a host organism &
the natural doubling time of the organism is used to create
many copies of the target DNA sequence
5. Organisms that are carrying the target DNA are identified
through a process called selection, which often involves
antibiotic resistance.
Synthesis of insulin in
humans
Fig. 13-15, p.345
Production of recombinant human insulin
Fig. 13-16, p.346
Electroporation. Foreign DNA can be introduced into plant
cells by electroporation, the application of intense electric
fields to make their plasma membranes transiently
permeable.
Genetic Engineering (g.e)
1. It is the process of inserting genes of interest into
specific organisms for either a medical or scientific
benefit
2. Gene therapy is the process of inserting a missing
gene into an organism
3. Bacteria are often used as the factories to produce a
protein from a cloned gene. This has led to the
production of human insulin & erythropoietin..etc.
4. The gene must be cloned into an expression vector,
usually a plasmid with special features that allows it
to be transcribed & translated in a host cell.
Genetic Engineering in Agriculture
1.
2.
3.
4.
Disease resistance e.g. corn & cotton
Nitrogen fixation
Frost-free plants e.g strawberries & potatoes
Tomatoes with a long shef life deactivating the
gene in tomato which produce ethylene.
5. Increased milk production giving cows bovine
somatotropin (BST) “growth hormone”….
6. Good predator attraction straw berry gene on
mustard plants produces a chemical attractant for
predator mites that eat the herbivorous spider
mites.
Transgenic tomato plant : Recombinant DNA methods have produced plants
that resist defoliation by caterpillars, with longer shelf life.
Fig. 13-18, p.349
Steps of making recombinant DNA
• Isolation of DNA  Cutting DNA in to small
pieces with restriction enzymes Ligate the
pieces into cloning vector Transform
recombinant DNA molecule into host cell The
transformed cell divides many times to form a
colony of millions of cells, each carries the
recombinant DNA molecule (DNA clone).
1. Isolation of nucleic acids
Is the separation of DNA free from other major
molecules such as RNA and proteins, lipids and
polysaccharides.
The isolation procedure mainly involve:
DNA isolation from bacteria involves lysis of cell wall
by a lysozyme enzyme then alkali denaturation
treatment followed by solvents extraction
(phenol/chloroform/isoamyl alcohol mixture ) which
separates chromosomal DNA from plasmid DNA
(remains circular shape and not affected by alkali
treatment).
• DNA extraction from human uses blood
sample as a source of DNA. In particular, the
DNA of white blood cells is isolated in a
procedure almost similar to the bacterial DNA
without the need for the use of lysozyme
enzyme.
2. Cutting DNA
• DNA can be cut into large fragments by
Restriction enzymes (r.e).
• They are group of endonucleases found as
protective enzymes in bacteria to destroy foreign
DNA in a process called restriction. The host
bacterial DNA itself is methylated by a
modification enzyme (a methylase) to be
protected from the restriction enzyme’s activity.
Inverted repeat palindromes are more common and
have greater biological importance than mirror-like
palindromes.
The product of restriction enzymes cutting to the
DNA is either a sticky (complementary) or blunt(noncomplementary) two ends.
In sticky ends: the terminal part of DNA are unequal
cohesive single strands which can easily combined
together due to complementary property between
them.
sticky ends
However , because sticky ends are usually
more needed in recombinant DNA
technology than blunt end , an enzyme called
Terminal deoxynucleotidyl transferase
can be used to add nucleotides to the bluntends of DNA chains to convert them into
sticky ends.
3. Joining of hybrid DNA
By cutting a donor DNA and receiver DNA with
the same restriction enzyme, the ends of the
two DNA will have the same sticky ends. These
two DNAs are mixed in a tube to rejoin together
due to the matching of their sticky ends. A small
gap will be left that can be sealed by ligase.
4. Amplification of recombinant DNA
I. Cloning of recombinant DNA
It is the method of replicating recombinant
DNA inside living cell to generate large
population of cells containing identical copies
of this type of DNA. The objective of cloning is
to replicate recombinant DNA in large
amounts, so that it can be used for genetic
analysis.
• Since the chemical structure of DNA
fundamentally the same in all living
organisms, any segment of foreign DNA
from an organism is inserted into host
DNA of living organism capable of
replication, then the foreign DNA will be
replicated along with the host cell's DNA
during cell division.
Molecular cloning is based on two
basic principles
1.DNA fragments are inserted into plasmid
vectors to produce recombinant DNA
2. The insert-vector recombinant molecules are
transported into living cells, usually E.coli,
which is grown up in colonies to make copies
of the recombinant DNA.
Selecting for recombinant DNA in a bacterial plasmid
Fig. 13-9, p.338
Specific steps of cloning
A. The selection of a vector
B. Generation of foreign DNA fragments (containing
particular gene) and ligate the foreign DNA into vector to
make insert-vector recombinant.
C. The selection of appropriate (competent) E. coli strain
D. Transforming competent E. coli with recombinant vector
E. Grow E. coli colonies to replicate recombinant vectors and
select colonies containing the recombinants DNA.
F. Characterizing the properties of DNA inserts by genetic
analysis.
A. Cloning Vectors
Cloning vector is a plasmid that can be modified
to carry new genes, which must have:
• An origin of replication.
• A selectable marker (antibiotic resistance
gene, such as ampicillin resistance( ampr) or
tetracycline resistance( tetr).
• Multiple cloning site (MCS) a site where
insertion of foreign DNA will not disrupt
replication or inactivate essential markers.
Plasmid pBR322
Fig. 13-10, p.339
B. Generation and Ligation of Inserts into
Vector
The standard procedure for creating the
recombinant molecule involves cleaving the
DNA of interest [the insert] and the vector with
the same restriction enzyme, followed by
incubation with DNA ligase to ligate the insert
into the vector.
DNA Library
1. It is a collection of clones of an entire genome
2. The genome is digested with r.e & the pieces are
cloned into vectors & transformed into cell lines
3. Specific radioactive probes to a sequence of
interest are reacted to filters that have copies of
the bacterial colonies in the library. The probe
binds to the sequence of interest, and the colony’s
location can be seen by autoradiography
4. A cDNA library is constructed by using reverse
transcriptase to make DNA from mRNA in a cell.
This cDNA is then used to construct a library
similar to a genomic DNA library.
Formation of
cDNA
Fig. 13-21, p.352
Steps involved in the
construction of a
DNA library
Fig. 13-19, p.350
C. Choice of an E. coli host
• Usually an E. coli mutant called lacZΔM15 is used as a
host for the recombinant vector. This mutant is
characterized by having inactive β-galactosidase
activity due to deletion in the N-terminal part of the
enzyme protein coded by defective lacZ gene.
• In the mean time , the defective part of this lacZ gene
has been inserted into the vector(plasmid DNA).So,
when the plasmid vector is transferred the bacterial
host the combined parts complement to each others
to give the active enzyme. A test for the formation of
active enzyme can be detected by converting a blue
colored X-gal dye into white color.
D. Transformation of E. coli with
Recombinant Vector
• Transformation is the process of making the
bacteria to take up the recombinant vector
molecule. Nucleic acids do not enter
bacteria under their own power but usually
they require certain procedure involving
treating the bacteria with ice-cold solutions
of CaCl2 followed by short heating to 42
Clone selection via
Inclusion of ampicillin in the agar allows only
blue/white screening bacteria with a plasmid to grow, because these
plasmids provide antibiotic resistance
Fig. 13-13, p.341
Three types of colonies are produced:
1. Transformed bacteria containing recombinant
plasmid
2. Transformed bacteria containing nonrecombinant plasmid
3. Non-transformed bacteria
Identification of cells containing plasmids
Inclusion of X-gal allows for blue-white colony
screening. Recombinant clones will have white
colonies while non-recombinant will have blue
colonies.