Download RESTRICTION ENZYMES AND VECTORS


Genetic engineering is the manipulation of
genetic materials which can be introduced
in the host organisms and thus change the
phenotype of the host organism.
o
o
o
o
o
o
o
o
o
o
RESTRICTION ENZYMES
DNA LIGASE
S1 NUCLEASE
REVERSE TRANSCRIPTASE
T4 POLYNUCLEATIDE KINASE
TAQ POLYMERASE
KLENOW FRAGMENT
ALKALINE PHOSPHATASE
RNase
TERMINAL DEOXYNUCLEOTIDE
TRANSFERASE

FUNCTION
The restriction enzymes called ‘’
Molecular scissor’’ and responsible for cutting
of DNA
Restriction Enzymes belong to a class of
enzymes called Nucleases and are of two
types:
Exonucleases
it cleave the DNA molecules at their
ends.
Endonucleases
it cleave the DNA molecules internally
at specific recognition sequences.




Type 1 Restriction endonucleases are complex
endonucleases and have recognition sequences of
about 15 bp; they cleave the DNA about 1000bp
away 5’-end of the recognition site, e.g. Ecok,
EcoB,etc.
Type 2 Restriction endonucleases are remarkably
stable and induce cleavage either in within or
immediately outside their recognition sequences
.they require Mg ion for cleavage.
Type 3 Restriction endonucleases are intermediate
b/w the Type1&Type2 enzymes ; they cleave
DNA in the immediate vicinity of their recognition
sites, e.g, Ecop1 , Ecop15, Hinf3 etc.

FUNCTION
It used for ligation of foreign DNA and
vectors during gene cloning. It ligate stick
ends of DNAs.

FUNCTION
It removes single- strand protruded
from its ends ;both 3’- and 5’-extension

FUNCTION
It used to produce cDNA (
complementary DNA) copies of , usually ,
mRNA that are used for creation of cDNA
rnase enzymes
o
FUNCTION
Rnase A- it digest RNA but not DNA .
RNase H- it also digest RNA and hybrid of
RNA-DNA.

FUNCTION
It is used for addition of phosphate group
to an end having a free 5’- OH .

It is used for addition of single stranded
sequences to 3’-ends of blunt ended
fragments.



It is used for
removing 5’-phosphate
group from DNA ends
.
This enzyme is
dimeric glycoprotein
with a molecular
weight 14,000.
It is made up of two
identical or similar
subunit each of
molecular weight
69,000.
FUNCTION
It is used in polymerase of DNA (in PCR)
activity at pH of 9 and temperature around
75C


FUNCTION
Klenow fragment of E.coli DNA
polymerase to make protruding ends doublestranded by extending the strands.
o
A Vector is a DNA molecule that has the
ability to replicate autonomously in an
appropriate host cell and into which the
DNA fragment to be cloned is integrated
for cloning .
vector must have an origin of DNA
replication (ori) that function in the host
cell . Any extra-chromosomal small
genome .e.g.,plasmid, phage or virus .
o
o
o
o
o
o
It should be able to replicate autonomously .
A Vector should be ideally less 10 kb in size .
The vector should be easy to isolate and
purify.
It should be easily introduced into the host .
The should have a suitable marker genes.
A vector should contain unique target site for
restriction enzymes.
plasmid
vector
They
have high copy
no. ( 1 to more than 50
per cell)
Plasmid are circular ,
double-stranded DNA
molecules.
Plasmid have origin of
replication (ori).
Plasmid have also
antibiotic resistance
genes as markers and
unique restriction site
Cosmid
vector
Cosmids
are
essentially a
combination of
plasmid & lambda
phage .
They have origin of
replication ,unique
restriction site,
selectable markers
from plasmid.
They can be used to
cloned DNA inserts
of upto 40kb.





It contain an origin of
replication.
It accommodate larger
DNA insert.
It ensure the recombinant
phage to be always lytic
It contain more than one
restriction sites.
Some lambda vector
contain promoter,operator
and lacZ gene of E.coli
lac operon. Insertion of
DNA fragment in such
vectors inactive and act as
selectable marker
A typical Fish vector is a
plasmid.e.g.,pRSV.
o
Selector Marker,
e.g.,ampicillin resistance
(amp)
o
Ori from E. coli plasmid
pBR322.
o
An enhancer/promoter
sequence (SV40)
o
A multiple cloning site
for insertion of the DNA
insert
o
A terminal site including
polyadenylation site.




Drosophila P elements
developed as valuable
vectors for invaluable
genetic material.
P elements are
transposons of 2.9kb; they
contain 3 gene flanked by
short (31bp) inverted
repeat sequence at their
ends.
P element vectors are
shuttle vector ; like YAC,
DNA insert of upto 40kb
into P element vector
o
o
o
o
It is spherical virus
with circular, doublestranded 5,243bp
chromosome.
It encodes 5 protein
,viz, small-T , LargeT,VP1,VP2and VP3.
Large –T is essential for
viral replication, while
VP1,VP2,VP3 form viral
capsid.
ORI (About 80 bp)






It contains pBR322 ori and a
selectable marker for cloning in
E.coli.
Retroviral 5’LTR and 3’LTR are
needed for efficient transcription of
proviral DNAand for generating 3’end of length transcript.
LTRs are essential for intregration
of the proviral DNA into host
genome.
It also has R, U5, U3, P and Pu
encoding sequence (involved in
reverse transcription).
S sequence(needed for splicing to
produce functional mRNA for
envelope protein synthesis.
Psi sequence (necessary for
packaging into virions).

These Vectors have been designed to replicate
in cells of two different species; therefore , they
contain two origins of replication , one specific for
each host species, as well as those genes
necessary for their replication and not provided
by host cells . These vector are created by
Recombinant DNA techniques . Some of them can
be grown in two different prokaryotic species ,
while others can propagate in a prokaryotic
species ,e.g yeast, animal, plants . These vectors
can be grown in one host and then moved into
another without extra manipulation.




OriS, origin of
replication from E.coli
plasmid.
repE , encodes a Rep
protein that
specifically binds to
oriS to initiate
replication.
DNA insert of upto
300kb
Low copy no.






These are linear vectors that
behave like an yeast
chromosome hence they are
called (YACs).
An ARS sequence for
replication
CEN4 sequence for
centromeric function
Telomeric sequence at the
ends for protection from
exonuclease action.
One or two selectable marker
gene
SUP4, a selctable marker
into which DNA insert is
intregrated