A ] ENZYMES
DNA primarily acquired from prokaryotes
i) Lytic Enzymes
• Various enzymes are used for obtaining naked
protoplast. After which the protoplast is further
fractionated to obtain the DNA content.
• The lytic enzyme are used to isolate the DNA
from the cell.
ii) DNA ligase
• It is used to join the sticky ends
iii) Synthesising enzymes
• DNA pol is used to amplify a particular DNA
fragment
• Rev.Transcriptase is used to synthesise DNA
over RNA template.
iv) Alk. Phosphatase
• It separates the phosphate group attached to
the terminal end of DNA strand, thus avoiding
recircularisation after being cleaved by RE
v) Cleaving enzymes
• These enzymes cut the DNA strands.
• There are 3 types of DNAase
a) Exonuclease
• It removes nucleotides from terminal ends
b) Endonuclease
• It cleaves only 1 strand of the DNA molecule
c) RE/ Mol.Scissor
• to be discussed in class
• Types of RE: to be discussed in class
• Why are RE synthesised & who synthesises
them? : to be discussed in class
• What is the action of RE enzyme? To be
discussed in class
• How is a RE named? To be discussed in class
B ] VECTOR
• A vector is a vehicle DNA that forms r-DNA
after:
• Step 1: being cleaved by a certain RE with non-
methylated sticky ends
• Step 2: it is integrated with a fragment of alien
DNA having a GOI which was separated from an
organism by applying the same RE.
Types of vectors
i) Plasmids
• These are natural but artificially manipulated
circ.
ii) Disarmed phage DNA or Plasmid
• It may no longer be considered as infectious ,
rather it can be used as a good vector.
iii) Cosmids & Phagemids
• These are artificially constructed by joining a
piece of Phage DNA & Plasmids
• They are essential for handling very large length
of alien DNA
iv) Artificial chromosome
• They are constructed by taking centromeric &
telomeric DNA of certain chromosome & can be
used to handle eukaryotic alien DNA
• Most common are YAC,MAC,HAC
Characteristics of a good Vector
• A commercially purposeful vector must have
the following parts
i) Ori : At least 1 ori must be present
ii) ROP & MCS genes
• ROP is responsible for Replication of Plasmid
frequently.
• Sometimes a site called MCS also solves the
purpose
• MCS is responsible for independent Multiple
Cloning
iii) Selectable Marker
• to be discussed in class
SEARCH FOR NEW TYPES OF VECTORS WITH NEW
ADVANTAGES IS CONTINUOUSLY TAKING PLACE.
ONE OF THE BEST EXAMPLE IS HERE .
• Natural Genetic Engineer: Agrobacterium
tumifaciens
• An organism which follows procedure of RDT &
transfers certain genes into specific host inside
which their productivity also occurs & is
expressed can be called as Natural genetic
engineer
• A classical example is A.tumifaciens
• This bacterium has a plasmid called TI-plasmid
which has a gene called T-DNA responsible for
tumours in Dicots called Crown-Gall-Disease
• This bacteria transfers this plasmid easily to
mature cells of the Dicots in which the T-DNA
gene integrates with the host genome & causes
Crown-Gall
• Biotechnologists have taken up this tendency of
TI plasmid as an advantage.
• The TI plasmid can be disarmed for use as a
Vector for many dicots
• SOME IMPORTANT DEFINATIONS
• Amplification: Production of numerous copies
of a required fragment of DNA
• Disarm: Removal of certain segments of DNA
which is responsible for infection
• Cleave: cutting of DNA at a specific sequence
usually a palindrome
• Clone: Integrating b/w a vector & an alien DNA
& then allowing multiplication of these
integrated form (r-DNA)
• Recombinant protein: The product produced
from the GOI inside the CH by rDNA
• C ] COMPETENT HOST
• After synthesis of rDNA it is required to insert it
into a host cell
• The host cell should be competent enough i.e
capable of synthesising the RP
• Insertion of the rDNA (or only GOI w/o vector)
may be done directly or indirectly
• 1) DIRECT METHODS OR ASSURED INSERTION
• a) Heat shock method:
• The treatment with Cal. Chl. & giving heat shock
at 42’C for 1-2 mins
• This process is successful if the CH is a bacteria
• Divalent ions like Cal.ions help in attachment of
rDNA to the CW of a bacterium.
• Heat shock forces the plasmid to enter the
cytoplasm
• b) Microinjection or Microinjection pipeting
• This is done in case of animal cell as a competent
host
• DNA is injected directly into the nucleus of the
CH by means of this gadget.
• c) Gene gun method / Biolistic Method
• In this technique microparticles of Au or W
coated with the rDNA are fired from the gun into
the CH(usually a plant cell)
• 1) INDIRECT METHODS OR ABSORPTION
INSERTION
• Methods in which the CH absorbs the rDNA
easily into its cytoplasm
• a) Transformation in PK cell
• b) Transjection in EK cells
• e.g by Disarmed TI plasmid for Dicots
by Disarmed Retroviral genome for humans
PCR
• It is a technique for making several copies of a piece
of DNA
• Every cycle of PCR produces 2 copies of DNA from a
single copy.
• Note that the replication of DNA is carried out in-vitro
i.e o/s the protoplasm in a test tube.
REQUIREMENTS IN PCR TECHNOLOGY
• 1) DNA sample : Usually 1 type of DNA fragment is
used taken from Gel- Electrophoresis block.
• 2) Some ions like Mg.
• 3) Primers : They are short oligonucleotide stretches
that can behave as initiating primers for
polynucleotide synthesis.
• 4) Enzyme : A highly Thermostable / Thermoresistant
DNA pol is used. As it should tolerate about 92’C
without losing its property.
• This is achieved by taking Taq-DNA-Pol
• Note: Taq DNA pol is extracted from the bacteria
Thermus aquaticus living in hot springs
• 5) A high precision oven with quick temperature
regulating system called as Thermal Cycler
MECHANISM
• A test tube with the sample of DNA required to be
copied & the other raw materials are taken into a
Thermal Cycler
• Step 1: Denaturation
• The test tube is heated upto 92’C-94’C
• At this temperature , the dsDNA denatures to ssDNA
• Step 2: Annealing
• The test tube is cooled to a temp. b/w 40-60’C
• At this temperature , the primers get attached to a
particular part of the DNA strand complementarily.
• Step 3: Polymerisation/ Extension
• After maintaining the annealing process for 1 min,
addition of DNA nucleotides starts on the 3' end of
primer.
• Polmerisation is best affected at 72’C
Gel Electrophoresis
• This is a process for separating DNA segments
required for our Expts. Selectively under electric field
• Other molecules with a residual charge on it will
also be separated like RNA , acidic protein & basic
proteins etc. , but too much branched molecule can’t
be separated.
• DNA before being Electrophorised is first treated
with a particular RE.
• For this purpose we use a Agarose Gel block
• What is agar? To be discussed in class
• Procedure
• A rectangular box of agar culture is taken.
• Note that the block of agar should be devoid of any
other substance i.e it should be totally sterile & not
polluted.
• Four to five wells are dug on one end of the agar
block
• Samples of DNA after fragmentation are placed in
each cell.
• Potential difference is applied across the block such
that cathode is applied to the well side & anode to
the opposite edge.
• Smaller DNA fragments travels at higher speed
towards anode & larger fragments trails behind due
to sieving effect .
• After sometime DNA with different masses are
arranged as bands across the block.
• Note: There is 99.9% Probability the DNA fragments of
same molecular wt must be having same length of
nucleotide as well as same sequence of nucleotides
Treatment with Ethidium Bromide Solution & exposed to UV
ray.
• DNA remains invisible in the gel block
• The block is wetted with Ethidium Bromide soln
slowly.
• Then the block is viewed in UV light in a dark room &
the bands of DNA appear bright Orange in colour
Elution
• The orange bands are cut from the gel & the DNA is
extracted from every band separately.