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Gene Cloning
Recombinant DNA:
—DNA formed by
combining
DNA segments from two or
more
different sources or
different
regions of a
genome is
termed as recombinant DNA
(rDNA).
—rDNA is any artificially created DNA
molecule
which brings together DNA
sequences
that are not usually found
together
in
nature.
—Crossing-over produces rDNA under
natural condition.
Recombinant DNA
Technology:
—A technology which
involves
joining
together
of
DNA molecules
from two or
more
different sources that are inserted
into a host organism to produce new
genetic combinations
that are of value to science, medicine, agriculture
and
industry
—This technology was
invented in
the early 1970s.
—It
enables us
to enhance the ability of
an
organism to:
—produce
a particular
chemical product (ex-penicillin
from fungus)
—prevent
synthesis of a chemical
product (ex- β-ODAP in Lathyrus)
—enable
an organism to produce an entirely
different product (ex- insulin in microorganism)
Essential Components for cloning a gene:
—Enzyme of cutting DNA fragments: Restriction endonuclease (RE)
—Enzyme
of
joining DNA fragments: DNA ligase
—Vehicles for introducing the recombinant molecule into the host cell: Vector
—DNA fragments: Gene
libraries
—Selection: Selection of the transformed cells for the presence
of the rDNA
Steps of cloning:
—Identification of the gene of interest (GOI).
—PCR
amplification of the GOI
with
gene-specific primer with specific
RE sites (Restriction endonuclease sites).
—Cutting
the PCR product (insert DNA) using the specific Restriction Endonuclease (RE).
—Selecting a cloning
vector
(a small molecule capable of
self-replicating
inside
host cells), and
cutting
the
cloning
vector with the
same RE.
—Incubating the vector and insert
DNA together to anneal and then joining
them using DNA ligase. The resultant DNA is
called recombinant DNA.
—Transferring
the recombinant
DNA to
an appropriate host such as bacteria,
virus or yeast which will provide necessary
bio-machinery for DNA replication.
—Identifying the host cells that contain the recombinant DNA.
Restriction Endonuclease (RE)
—Also
called
restriction
enzymes
—1962:
“Molecular scissors”
discovered
in
bacteria
—E.
coli
bacteria
have an enzymatic
immune
system that recognizes and destroys foreign
DNA
—3,000 enzymes have been identified,
around 200 have unique properties, many are purified and
available commercially
—RE: Molecular
scissors that
cut double-stranded DNA molecules at specific points.
—An
important
tool
for manipulating DNA
Werner
Arbor,
Hamilton
Smith and Daniel Nathans shared the 1978 Nobel prize for Medicine and Physiology for their
discovery of Restriction
Enzymes.
Nomenclature:
Named for
bacterial
genus, species,
strain,
and
type:
Example:
HinDII (1st RE isolated)
Genus:
Haemophilus
Species: influenzae
Strain: D
Order discovered:
II
Example:
EcoRI
Genus: Escherichia Species: coli
Strain:
R
Order discovered: I
Biological Role:
—Most bacteria use
RE
as a defense against bacteriophages.
—REs
prevent the replication of the phage by cleaving its DNA
at specific
sites.
—Restriction Modification System:
REs
are paired with methylases.
—Methylases
are enzymes that add methyl groups to specific
nucleotides
within
the
recognition sequence. The methylation prevents recognition by the RE. Therefore, the RE within
a cell doesn’t destroy its own DNA.
¢RE are bacterial enzymes
that
recognize specific 4-8
bp sequences called restriction sites and cleaves
both the DNA
strands at this
site
(site
specific).
¢They
cleave
the DNA within
the molecule, hence, endonucleases.
¢RE
has 3 functions:
—Recognition
—Cleavage
—Modification
Type IIs: Separate endonuclease and methylase;
recognition site is asymmetrical;
cleavage occurs on one side of
the
recognition sequence up to 20
bp
away
Type II Restriction endonucleases:
—Simple
enzymes having separate endonuclease and methylase activities
—Recognize
a specific nucleotide sequence and cut a DNA
molecule at this site and
nowhere
else
—Mostly
recognize a hexanucleotide sequence
—Very
stable and
only require Mg2+ as a cofactor
—Many
REs make a simple double-stranded cut in the middle of
the
sequence
and result in ‘blunt ends’
—Other
REs do not cut both strands of the DNA at the same position and result in ‘staggered
end’
or ‘cohesive
ends’
or ‘sticky
ends’.
Base
pairing
between
these
ends can stick the DNA fragments back together again.
—This
type
is
used for gene
cloning.
- Enzymes recognize specific 4-8 bp sequences
- Some enzymes cut in a staggered fashion - “sticky ends”
- Some
enzymes
cut
in a direct
fashion
– “blunt ends”
Uses for Restriction Enzymes:
¢RFLP
analysis (Restriction Fragment Length Polymorphism)
¢DNA sequencing
¢DNA storage – libraries
¢Gene
cloning &
Transformation
¢Large
scale analysis –
gene
chips
Digestion Conditions
¢XbaI
—Buffer
2: (10 mM Tris-HCl, 10 mM MgCl2, 50 mM NaCl, 1
mM
DTT, pH 7.9).
—100
μg/ml
BSA
(optional)
—1
Unit digest
1 μg DNA
—Incubate
at 37°C for
1
hour
—Heat inactivate 65°
for 20 min
20 μl reaction:
10
μl
DNA (~1 μg
total)
7
μl
water
2 μl 10X reaction buffer
1 μl RE 10 units/μl
Incubate
1
hour at
appropriate
temperature
Note:
1.10 fold
excess enzyme ensures complete digestion.
2. Enzyme should never
exceed 1/10th of
reaction volume.
3.BSA is
often recommended
because it stabilizes the enzyme.
Double Digestion for directional cloning
Isoschizomers and Neoschizomers
¢Restriction
enzymes that
have the same recognition sequence
as well
as
the same cleavage
site are Isoschizomers.
Eg:
SphI (CGTAC/G) and BbuI (CGTAC/G) are
¢Restriction
enzymes that
have the same
recognition sequence
but cleave
the
DNA at a
different site within that
sequence
are Neoschizomers.
Eg: SmaI
and XmaI
Star activity
Some
restriction enzymes may cleave sequences other than their defined recognition sequence under sub-optimal reaction conditions. In
general, these conditions include high salt concentration, presence of
impurities,
or excessive enzyme compared to substrate DNA. This altered specificity is called star activity.
DNA Ligase
¢During replication DNA ligase
catalyzes
the
formation of 3’ –
5’ phosphodiester bonds between the short fragments
of the lagging
strand of DNA in the replication fork.
¢In
rDNA technology, purified DNA ligase is
used
to covalently
join
the
ends
of the
restriction
fragments in vitro.
¢This enzyme
catalyzes the formation of 3’ → 5’ phosphodiester bond between
the 3’OH– end of one
restriction fragment and the 5’ phosphate end of another
restriction fragment.
¢The process is
called
ligation.
DNA Ligase – enzyme catalyzing the formation of phosphodiester
bond between 3’-OH group of one end of the DNA molecule and 5’-phosphate group
of the second end
of
DNA.
Ligase cofactors:
1. ATP
•DNA
ligases
of bacteriophages (phage
T4,
T7)
•DNA
ligases
of mammals
2. NAD+
•DNA
ligases
of bacteria (Escherichia coli,
Bacillus
subtilis, Salmonella typhimurium)
•T4
DNA ligase
can ligate sticky as well
as
blunt
ends
•E.
coli
DNA ligase can
ligate
only blunt ends
T4 DNA ligase
•T4
DNA Ligase catalyzes the joining of two strands of
DNA between
the 5´- phosphate and the 3´- hydroxyl
groups of
adjacent
nucleotides in either a cohesive-ended or
blunt
ended configuration.
•The enzyme has
also
been shown to catalyze the joining of
RNA
to either a DNA or RNA strand in a
duplex molecule but will
not join single-stranded nucleic acids.
Inactivation of T4 ligase:
Heat
to
70°C
for 10 minutes.
Features:
—Requires
ATP as a co-factor
—Optimal
pH
(7.2-7.8)
— Requires bivalent ions
(Mg2+, Mn2+)
and
reducing
factors
(β mercaptoethanol or ditiotreitol)
— Inhibitors: polyamines (spermin, spermidine), high concentration of
ions (Na+,
K+,
Li+,
NH4+)
— Can connect
both
cohesive and blunt ends (but for blunt ends reaction is slower and requires
higher concentrations
of enzyme)
Typical ligation reaction:
COMPONENT
|
20 μl
REACTION
|
T4
DNA Ligase Buffer (10X)*
|
2 μl
|
Digested
Vector DNA (4 kb)
|
50 ng (0.020 pmol)
|
Digested
Insert DNA (1 kb)
|
37.5
ng (0.060
pmol)
|
Nuclease-free water
|
to
20 μl
|
T4
DNA Ligase
|
1 μl
|
Incubate at 16°C overnight
or room temperature
for 2 hours
What should be optimized for a successful ligation:
1. The ratio of the molar concentration of vector to insert.
-
Optimum
ratios may vary from 8:1 to as high as 1:16
vector: insert, though generally fall in
the range
of 3:1
to
1:3.
2. Amount of DNA.
- Usually 10-200 ng
of plasmid
is
used
for
reaction.
3. The volume of reaction.
- Usually a
minimal
volume
is
recommended (e.g.
10 µl).
4. Amount of ligase.
- Each ligation reaction
generally
requires 1-10 units of high-quality ligase.
5. Incubation time and temperature.
The
ligation
incubation time and temperature may also need
to
be optimized. In general:
-
blunt-ended
ligations are performed at 4°C overnight;
-- sticky-end ligations are performed for 1-3 hours (at 22ºC or 16ºC)
or
overnight
at 4°C.
-In general, ligation reactions performed at lower temperatures require longer incubation times.
DNA Modifying Enzymes
¢DNA modifying enzymes include enzymes
that are involved in degradation, synthesis, and alteration of DNA.
Nucleases:
¢Enzymes
that degrade nucleic acids by breaking the phosphodiester bond
¢Endonucleases: Breaks
a phosphodiester bond at an intercalary position
¢Exonuclease: Breaks
a phosphodiester bond from the ends
¢Major
nucleases used in cloning:
¢Restriction
endonucleases (Endonuclease)
¢DNaseI
(Endonuclease)
¢S1-nuclease (Endonuclease)
¢Bal31
(Exonuclease)
¢Exonuclease
III (Exonuclease)
¢Ribonuclease
(Cleaves RNA)
Polymerases:
¢
Synthesize
copies
of nucleic acid molecules
¢‘DNA-dependent’
or ‘RNA-dependent’
¢
Synthesis
proceeds
in a 5’ →
3’ direction
¢Major
polymerases used in gene cloning are:
¢DNA polymerase I (nick
translation procedure for radiolabelling DNA)
¢Klenow fragment (radiolabelling
by primed synthesis and DNA sequencing by the dideoxy
method)
¢Reverse transcriptase (preparation
of complementary
DNA or cDNA)
Choosing Klenow enzyme or Exonuclease III (T4 DNA Polymerase)?
End modifying enzymes:
¢ Alkaline phosphatase:
¢removes
phosphate groups from the 5’ ends
of DNA, leaving a 5’-OH
group
¢used
to prevent unwanted ligation of DNA molecules
¢Terminal transferase:
¢repeatedly
adds nucleotides to any available 3’
terminus
¢used
to add homopolymer
tails to DNA molecules prior to the construction of recombinants
¢Polynucleotide
kinase:
¢involved
in the removal or addition of phosphate groups
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