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1. DNA TRANSFER TECHNIQUEs
Introduction
DNA transfer defined as the transfer of DNA segment from one cell to another cell via physical,
chemical & electrical ways. Gene is a segment of DNA that codes RNA for polypeptide molecule.
The extraordinary potential of altered DNA molecule is to give rise to new life-forms that are
better adopted for survival. Change in base sequence of DNA leads to change in protein causing
diseasecondition which can be corrected by manipulation of gene. Transgenicanimals and plants
can be obtained by introduction of exogeneous DNA into targeted animals and plants
accompanied by the stable expression. The techniques for the transfer of DNA into organisms
differ from organism to organisms.
DNA transfer mainly divided into four main category-
1.Physical method.
 Microinjection
 Partical bombardment
 Sonoporation
2.Chemical method
 DNA transfer by calcium phosphate
 Transfer of DNA by use polyethene glycol
 Use of DEAE-dextron for DNA transfer
 Liposome mediated transfer
3.Electrical method
 Electroporation
 Electrofusion
4.Biological method
 Agrobacterium mediated transfer
 Conjugation
 Viral transduction
Microinjection
DNA microinjection was first proposed by Dr. Marshall A. Barber in the early of nineteenth
century. This method is widely used for gene transfection in mammals. It involves delivery of
foreign DNA into a living cell (e.g. a cell, egg, oocyte, embryos of animals) through a fine glass

2. micropipette. The introduced DNA may lead to the over or under expression of certain genes. It
is used to identify the characteristic function of dominant genes.
Procedure
 The delivery of foreign DNA is done under a powerful microscope using a glass
micropipette tip of 0.5 mm diameter.
• Cells to be microinjected are placed in a container. A holding pipette is placed in the field of
view of the microscope that sucks and holds a target cell at the tip. The tip of micropipette is
injected through the membrane of the cell to deliver the contents of the needle into the
cytoplasm and then the empty needle is taken out.
Fig: Microinjection procedure for DNA transfer
Advantage
• No requirement of a marker gene.
• Introduction of the target gene directly into a single cell.
• Easy identification of transformed cells upon injection of dye along with the DNA.
• No requirement of selection of the transformed cells using antibiotic resistance or herbicide
resistance markers.
• It can be used for creating transgenic organisms, particularly mammals.

3. Partical bombardment
Prof Sanford and colleagues at Cornell University (USA) developed the original bombardment
concept in 1987 and coined the term “biolistics” (short for “biological ballistics”) for both the
process and the device. Also termed as particle bombardment, particle gun, micro projectile
bombardment and particle acceleration. It employs high-velocity micro projectiles to deliver
substances into cells and tissues.
Apparatus
The biolistic gun employs the principle of conservation of momentum and uses the passage of
helium gas through the cylinder with arrange of velocities required for optimal transformation of
various cell types. It consists of a bombardment chamber which is connected to an outlet for
vacuum creation. The bombardment chamber consists of a plastic rupture disk below which
macro carrier is loaded with micro carriers. These micro carriers consist of gold or tungsten micro
pellets coated with DNA for transformation. The apparatus is placed in Laminar flow while
working to maintain sterile conditions. The target cells is placed in the apparatus and a stopping
screen is placed between the target cells and micro carrier assembly. The passage of high
pressure helium ruptures the plastic rupture disk propelling the macro carrier and micro carriers.
Fig : Gene gun which is used in partical bombardment

4. Uses
• This method is commonly employed for genetic transformation of plants and many organisms.
• This method is applicable for the plants having less regeneration capacity and those which fail
to show sufficient response to Agrobacterium- mediated gene transfer in rice, corn, wheat,
chickpea, sorghum and pigeon-pea.
Sonoporation
Sonoporation involves the use of ultrasound for temporary permeabilization of the cell
membrane allowing the uptake of DNA, drugs or other therapeutic compounds from the
extracellular environment. This method leaves the compound trapped inside the cell after
ultrasound exposure. It employs the acoustic cavitation of micro bubbles for enhancing the
delivery of large molecules like DNA. The micro bubbles form complex with DNA followed by
injection and ultrasound treatment to deliver DNA into the target cells. Unlike other methods of
transfection, sonoporation combines the capability to enhance gene and drug transfer.
Fig: Schematic representation of sonoporation technique
Advantages
• Simple and highly efficient gene transfer method.
• No significant damage is cause to the target tissue.

5. Disadvantages
• Not suitable for tissues with open or cavitated structures.
• High exposure to low-frequency (<MHz) ultrasounds result in complete cellular death (rupture
of the cell). Thus cellular viability must be taken into consideration while employing this
technique.
Calcium phosphate mediated DNA transfer
The process of transfection involves the admixture of isolated DNA (10-100ug) with solution of
calcium chloride and potassium phosphate under condition which allow the precipitate of
calcium phosphate to be formed. Cells are then incubated with precipitated DNA either in
solution or in tissue culture dish. A fraction of cells will take up the calcium phosphate DNA
precipitate by endocytosis. Transfection efficiencies using calcium phosphate can be quite low,
in the range of 1-2 %. It can be increased if very high purity DNA is used and the precipitate
allowed to form slowly. Procedures have been developed where cell taking up exogenous DNA
could be upto 20%. This technique is used for introducing DNA into mammalian cells.
Fig: Ca3(PO4)2 mediated DNA transfer
DNA transfer by DAE-dextrone method
DNA transfer by DAE-Dextran method DNA can be transferred with the help of DAE Dextran also.
DAE-Dextran may be used in the transfection medium in which DNA is present. This is
polycationic, high molecular weight substance and convenient for transient assays in cos cells. It
does not appear to be efficient for the production of stable transfectants. If DEAE-Dextran
treatment is coupled with Dimethyl Sulphoxide (DMSO) shock, then upto 80% transformed cell
can express the transferred gene.

6. Liposome mediated transfer
• This can be done by packaging the DNA inside a fusogenic phospholipid vesicle which
interacts with the target cell membrane and facilitate DNA uptake.
• Briefly, bacterial cells will be transformed with suitable plasmid vector and treated with
chloramphenicol to amplify plasmid copy number.
• The cells will be treated with lysozyme to remove cell wall, resulting in protoplasts that
willbe centrifuged gently onto amonolayer of mammalian cells to promote fusion among
them using polyethylene glycol.
• Using lyposomes is more commonly used for this type of transfection. This method is
commonly known as lipofection. This method is far more efficient than chemical
transfection method. With this method up to 90% of cells in culture dish can be
transected.
Advantage
• Commercially available with reproducible results
• Applicable to a broad range of cell lines

7. • High efficiency and expression performance
• Fast and easy protocols which do not require media changes
Disadvantage
• Optimization may be necessary—some cell lines are sensitive to cationic lipids
• Some cell lines are not readily transfected with cationic lipids
Electroporation
Electroporation is a mechanical method used for the introduction of polar molecules into a host
cell through the cell membrane. This method was first demonstrated by Wong and Neumann in
1982 to study gene transfer in mouse cells. It is now a widely used method for the introduction
of transgene either stably or transiently into bacterial, fungal, plant and animal cells. It involves
use of a large electric pulse that temporarily disturbs the phospholipid bilayer, allowing the
passage of molecules such as DNA.
Procedure
The host cells and the DNA molecules to be transported into the cells are suspended in a solution
• When the first switch is closed, the capacitor charges up and stores a high voltage which gets
discharged on closing the second switch.
• Typically, 10,000-100,000 V/cm in a pulse lasting a few microseconds to a millisecond is
essential for electroporation which varies with the cell size.
• This electric pulse disrupts the phospholipid bilayer of the membrane causing the formation
of temporary aqueous pores.
• When the electric potential across the cell membrane is increased by about 0.5-1.0 V, the
charged molecules e.g. DNA migrate across the membrane through the pores in a similar manner
to electrophoresis.
• The initiation of electroporation generally occurs when the transmembrane voltage reaches at
0.5-1.5 V. The cell membrane discharges with the subsequent flow of the charged ions and
molecules and the pores of the membrane quickly close reassembling the phospholipid bilayer.

8. . The basic process inside an electroporation apparatus is represented in a schematic diagram
Fig: Schematic representation of Electroporation
Application
Electroporation is widely used in many areas of molecular biology and in medical field. Some
applications of electroporation include:
• DNA transfection or transformation
Electroporation is mainly used in DNA transfection/transformation which involves introduction
of foreign DNA into the host cell (animal, bacterial or plant cell).
• Direct transfer of plasmids between cells
It involves the incubation of bacterial cells containing a plasmid with another strain lacking
plasmids but containing some other desirable features. The voltage of electroporation creates
pores, allowing the transfer of plasmids from one cell to another. This type of transfer may also
be performed between species. As a result, a large number of plasmids may be grown in rapidly
dividing bacterial colonies and transferred to yeast cells by electroporation.
• Gene transfer to a wide range of tissues
Electroporation can be performed in vivo for more efficient gene transfer in a wide range of
tissues like skin, muscle, lung, kidney, liver, artery, brain, cornea etc. It avoids the vector-specific
immune-responses that are achieved with recombinant viral vectors and thus are promising in
clinical applications.

9. Agrobacterium mediated DNA transfer
Agrobacterium tumefaciens naturally transfers DNA into plant cells and is clearly one of the most
effectivemethodsof directedDNA transferpresentlyavailable.Twokindsof vectorsare commonlyused.
Cointegrative vectors have the foreign genes incorporated directly into the Ti plasmid. Binary vectors
carry two plasmids; the main Ti plasmid where most of the T-DNA has been removed, and a second
plasmM containing the foreign genes between the usual border sequences. The vir genes on the main
plasmidfunctiontomobilizethe foreigngenesintoaplantcell.Mostplanttransformationmethodsfollow
the procedure of cocultivating wounded tissue with vir-gene-induced bacteria.
Basic step of agrobacterium mediated DNA transfer
(1) bacterial colonization
(2) induction of bacterial virulence system,
(3) generation of T-DNA transfer complex
(4) T- DNA transfer and
(5) integration of T-DNA into plant genome
Fig:Agrobacterium mediated DNA transfer