rDNA Technology-Biochemden by SATISH

8/3/2019 rDNA Technology-Biochemden by SATISH

1/28

ecombinant DNA Technology Recombinant DNA Technolog





ecombinant DNA Technology Recombinant DNA Technologecombinant DNA Technology Recombinant DNA Technolog





















Recombinant

DNA technology(Biotechnology)I .Satish K umar

Lecturer in Biochemistry

Visit:http://biochemden.inhttp://biochemistryden.blogspot.com

http://bioscienceden.blogspot.com

Email: [email protected]


2/28

Life sciences Study materials Molecular Biology

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

----------------------------------------------------------------------------------------------------------------------------

Prepared by I.Satish Kumar, Lecturer in Biochemistry,

http://biochemistryden.blogspot.com http://biochemden.in

2

Recom binant DNA techno logyBiotechnology:

Some of the available definitions of Biotechnology,

It is the application of biological organisms, system (or) process to manufacturing and

service industries

The integrated use of Biochemistry, Microbiology and engineering sciences of thecapabilities of microorganisms, cultured tissue cells and part of them.

The controlled use of biological agents, such as microorganisms (or) cellularcomponents, for beneficial use

The last definition is brief and comprehensive and may be used by students, if theyhave to learn only one definition.

Historical Resume:

The term Biotechnology was coined by the scientists Carl neuberg in 1919.The origin of biotechnology can be traced back to prehistoric times, whenmicroorganisms were already used for processes like Fermentation.

First Discovery: In 1920, Clostridium acetobutylicum was used by ChaimWeizmann for converting starch into butanol and Acetone. The latter was anessential component of explosives during World War-I.

Second Discovery: During Second World War (1940s), the production ofPenicillin (as an antibiotic discovered by Alexander Fleming in 1929) on a largescale from cultures of Pencillium notatum

Third Discovery: The third discovery of Biotechnology is its recent reincarnationin the form of Recombinant DNA technology, which led to the development of avariety of gene technologies and is thus considered to be the greatest scientificrevolution of this century.

Old Vs New Biotechnology:

Fermentation by some microorganisms, formation of yoghurt (curd),cheese from milk, Vinegar from molasses, production of antibiotics likePenicillin from certain fungi, Process of baking and brewing are often includedin describing what is called Old Biotechnology

PCR (Polymerase Chain Reaction), rDNA technology, Cell culture andfusion and bio-processing, which became possible only through the researchesin molecular biology have been described as New Biotechnology


3/28


- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

----------------------------------------------------------------------------------------------------------------------------



3

Introduction:An early demonstration of the transfer of DNA from one cell type to another

concerned drug resistance of certain strains of E.coli could be transformed to aphenotypic bacterium such as Salmonella. The explanation of this phenomenon led to

the discovery of the plasmid, where by the gene conferring antibiotic resistanceresidues in a form of extra-chromosomal DNA. The resistance is due to an enzyme thatcauses the degradation of the antibiotic.

Enzymes involved in rDNA technology:Genetic engineering means creation of new DNA, for the cloning purpose. In

these rDNA technology two types of enzymes are using:

1) Restriction endonucleases2) DNA ligases

3) Alkaline Phosphatase4) Polynucleotide Kinase5) Terminal Deoxynucleotidyl Transferase6) RNA dependent DNA polymerase (Reverse Transcriptase)7) RNase H

1) Restriction endonulceases:

The Restriction endonucleases recognizes a specific base sequence of four toeight bases in double-stranded DNA and cleaves both strands of the duplex.

There are known type of restriction endonucleases : type I,II and III. Type IIenzymes are frequently used in the rDNA technology. Type I and type III are notuse because these type enzymes cleaves the DNA far from the recognition sites.The Restriction endonuclease recognizes particular specific sequences are 4 to 6nucleotides recognize by the type II enzymes because these enzymes only cleaveat this site. The palindrome sequence is Two-fold symmetry as shown below.

The type II restriction enzymes arediscovered and characterized byHamilton Smith and Daniel Nathansin 1960. Approximately 200 Restriction

endonucleases are isolated frombacterial species like- E.coli, Bacillus,Haemophillus, Streptococcus andthermus aquaticus.

Restriction endonucleases isnamed by the first letter of the genus of the bacterium that produced it and the firsttwo letters of its species, followed by its serotype (or) strain designation, if any,

5- GATATC 3

3- CTATAG 5

Two fold symmetry axis

(Cleaved by EcoRV)

Recom binant DNA Technology


4/28


- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

----------------------------------------------------------------------------------------------------------------------------



4

and a roman numerical if the bacterium contains more than one type of restrictionenzymes.

E.g.: EcoRI is produced by E.coli strain RY13

Bam HI isolated from Bacillus amyloliquefaciens

Bgl II isolated from Bacillus globigis

Hae III isolated from Haemophilus aegyptius

Pst I isolated from Providencia stuartil2) DNA ligases:

The complimentary ends of the DNAs specifically associate underannealing conditions and are covalently joined through the action of an enzymenamed DNA ligase. The enzyme produced by bacteriophage T4.

Mechanism of DNA ligase activity:

The E.Coli and T4 ligases share the property of sealing nicks that have 3'-OH and 5- P termini. Both enzymes under take a two step reaction, involving anenzyme-AMP complex.

The E.Coli and T4 enzyme use different cofactors.

The E.Coli enzymes uses NAD as a cofactor, T4 enzyme uses ATP.

The AMP of the enzyme complex becomes attached to the 5-Phosphate ofthe nick; and then a phosphodiester bond is formed with the 3-OH terminus of

the nick, releasing the enzyme and the AMP.

3. Polynucleotide Kinase:

It is a phosphorylating enzyme that transfers the gamma phosphate ofATP to a dephosphorylated end of DNA or RNA.

The enzyme is encoded by a gene of phage T4 and is extracted fromE.coli cells infected with the phage.

This enzyme used after the Alkaline Phosphatase activity andintroduces 32P label (by using ATP).of DNA and RNA strands.

Mg+2 and dithiothreitol are used in the reaction.

4. Terminal Deoxynucleotidyl Transferase

The enzyme is a DNA polymerase that extend a strand without usinga template.

Any nucleotide that is provided in the reaction mixture is utilized toelongate the DNA strand.


5/28


- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

----------------------------------------------------------------------------------------------------------------------------



5

If only one kind of nucleotide is provided a mononucleotide polymerwill be produced.

5. Alkaline Phosphatase

The enzyme alkaline phosphatase removes the phosphate moiety atthe 5-end of DNA strand, whether it is part of blunt end singleextension or a recessed end of a double-stranded DNA.

The phosphate of RNA terminal is also removed by this enzyme.

The commercial source of this enzymes is two sources: Bacterial andCalf intestinal phosphatases.

6. RNA dependent DNA polymerase:

RNA dependent DNA polymerase is Reverse Transcriptase (RT).

This enzyme synthesizes a single strand of DNA along an RNA

template. It can also synthesize a second strand along the first one to make a ds

complementary or cDNA. RT is usually utilized to copy mRNAs into ss or ds cDNA, and to make

short labeled probes.7. RNase H:


6/28


- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

----------------------------------------------------------------------------------------------------------------------------



6

RNase H is an endonuclease that is useful for degrading the RNAstrand from a DNA:RNA hybrid molecule. It cut up the RNA into shortfragments.

VECTORS

Introduction:

The DNA fragment (or) the gene of interest can be linked to a carriermolecule, which can transport the gene of interest into the host cell. This carriermolecule is referred to as a Cloning vector (or) Cloning vehicle; the cloningvehicle is the central component of a gene cloning experiment and it constitutesthe gene transfer system.

Important features of vectors:

It must be able to replicate. There must be some way to introduce vector DNA into a cell.

There must be some means of detecting its presence, preferably by a platingtest.

It should contain an assortment of unique Restriction endonucleasescleavage sites.

It should occur in large number of copies.

Type of vector:There are three main types of vectors in use. They are1) Plasmids2) Cosmids3) Phagemids

1)Plasmids:

Plasmids are extra chromosomal, autonomously replicating , smallcircular molecules of DNA found in many prokaryotes and in a feweukaryotes such as the yeast Saccharomyces cerevisiae.

Properties of Plasmids:

They replicate independently (or) autonomously Most of them are circular duplex of DNA molecules.

They have an origin of replication naturally in them

They are passed on to the daughter cells during cell division.

They may carry very important genes for antibiotic resistance, toxinproduction, for antibody production, for degradation of a large number of


7/28


- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

----------------------------------------------------------------------------------------------------------------------------



7

unusual substrates such as herbicides (or) industrial effluents and genesfor nitrogen fixation. These confer the phenotypic traits of plasmids.

They rely on the DNA replication enzymes of the host cells for theirreplication; however, the initiation of replication is controlled by plasmidgenes.

Certain plasmids do not show any phenotypic traits such as plasmids are

called Cryptic plasmids. They have high transformation efficiency.

They have convenient selectable markers such as antibiotic resistance,toxin production etc, for transformants and recombinants.

They have the ability to clone reasonably large pieces of DNA say about5 kilo base pairs.

They are of low molecular weight.

They are easily isolated and purified.

Size of plasmids:

Plasmids are duplex, supercoiled DNA molecules and they rangein size from 1X106 Daltons to greater than 200X106.

Number of plasmids:

The number of copies of plasmid in a cell is referred to as Copynumber. When there are one (or) two copies, the copy number is calledlow copy number. When there are twenty (or) more copies per cell, thecopy number is called high copy number

Plasmid classification:

The naturally occurring plasmids are classified based on the maincharacteristics coded by the plasmid genes. It is grouped into FIVE maintypes:

a) F-plasmidsb) R-Plasmidsc) Col plsamidsd) Degradative plasmidse) Virulence plasmids

a) F-Plasmids (Fertility plasmids):

These plasmids carry only tra genes (transfer gene) and nocharacteristic beyond the ability to promote conjugate transfer of plasmids.The presence of tra genes promotes bacterial conjugation. These plasmidsmay be denoted as F+ and F-, which means those having the fertility (F)factor and those without it. These plasmids are not used in gene cloning.Most of the tra genes are involved in pili synthesis (sex pili) on donor.


8/28


- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

----------------------------------------------------------------------------------------------------------------------------



8

b) R-Plasmids (Drug resistance):

These carry genes conferring on the possessor resistance to one (or)more antibacterial agents such as Chloramphenicals, Amphicillin,Tetracycline and any metal. The R strands for Resistance. Plasmid RP4found in pseudomonas is an example of R-plasmid. This R-factor was

discovered in Japan in 1955.

The R-factor is wide spread in contain strain of almost all pathogenicbacteria. The plasmid genes after encode for enzyme that chemically inactivatethe drug (or) by active export eliminate it from the cell.

c) Colicinogenic (or) Col plasmid:

Col plasmids are E.coli plasmid able to produce colicins, proteins thatprevent growth of susceptible bacterial strains that do not contain a col plasmid.The bacterial toxins are generally called Bacteriocin these bacteriocin are

active only against closely related strains of bacteria toxins of this. Types thatare liberated by strains of E.coli are called Colicins. The colicins are simpleproteins. Several different types of colicins have been isolated which killsensitive cells by different mechanisms. The plasmids containing genes for suchtoxic substance, colicin is called Col plasmid. Depending upon the nature ofcolicins, there are different types of col plasmids. They are col B, colE1, col E2,col I and col V. the toxin from Pseudomonos is called Pyrocins.

d) Degradative plasmids:

These are plasmids, which have genes for enzymes that enable thebacterium to metabolize unusual substrates such as Toluene, xylene andsalicylic acid. These plasmids are also called Dissimilation plasmids. Thisplasmid type (Ptol) is responsible for the ability of certain Pseudomonas speciesto break down different to degrade industrial solvents such as toluene andxylene. A combination of several plasmids, when transferred to pseudomonasbacteria, allows the bacteria to break down complex hydrocarbons and othercompounds present in crude oil. The bacteria, containing these plasmids have apotential use for treatment of environments contaminated with oil spills.

e) Virulence plasmids:

The plasmids have genes that confer pathogenicity on the hostbacterium. For example, Ti-plasmids found in Agro bacterium tumefacience.They include crown gall disease on dicotyledonous plant.


9/28


- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

----------------------------------------------------------------------------------------------------------------------------



9

2)Cosmids:Cosmids is a hybrid DNA formed by the joining of a plasmid and

(lambda) phage DNA carrying a cos site in brief cosmid is a plasmid carrying

the cos site of a phage DNA. The cosmid is not naturally found in living cells. Itis a constructed vector.

Example: Col E1 cosmid is a typical cosmid used in genetic engineering.

Construction of Cosmids:col E1 cosmid is constructed from col E1 plasmid and -phage DNA. The

plasmid is cut with a restriction endonuclease enzyme, which removes a portion

of DNA from the plasmid. The same restriction enzyme is used to cut the -phage DNA to get a DNA fragment containing Cos site.

These two DNA fragments are mixed together in the presence of theenzyme DNA ligase, which link together the two DNA fragments end to end. Theresulting recombinant plasmid is called Col E1 cosmid.

Characteristic features:

The cosmid is a plasmid containing cos site.

It is a circular double stranded DNA.

H contains complementary single strand regions, thecomplementary single strand region is abbreviated as Cos site.

The cos site consists of two complementary single strands heldtogether by complementary base pairing both these two strands.

At the cos site, 3-end of each of the DNA strands does notestablish covalent bond with 5-end of the same chain that is a

definite nick is present in each of the two strands. The nicks are restrained in the cosmid for a number ofgenerations.

The cosmid DNA does not code for the synthesis of viral proteins.

The cosmid does not participate in the multiplication of phageparticles.

The cosmid DNA packed with in the protein coat of bacteriophage.Thus the transformed virus particle is formed.

Advantages:

The cosmids transfers a somewhat larger foreign gene into thebacterial cell.

The cosmid pickup even long sized genes. Hence it is used in thegenome of the organisms.

The cosmids are also used in the study of some non-sensesequences found in the genome of the organisms.


10/28


- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

----------------------------------------------------------------------------------------------------------------------------



10

Disadvantages:

Each cosmid requires two cos sites for the successful packing ofrecombinant cosmid within the protein coat of bacteriophage. Inthe recombinant cosmids the packaging enzyme fails to pack the

DNA into the protein coat of bacteriophage. The transfer of gene from the transformed cells is difficult the

cosmids need additional work for this gene transfer. The package fails when the distance of separation exceeds 54,000

bps (or) when it is less than 38,000 bps. Main use is gene cloning for successful result.

3) Phagemid:Phagemids are prepared artificially by combining features of

phages with plasmids as the name suggests. One such phagemid, whichis commonly used in molecular biology laboratories, is Blue script II KS,which is derived from PUC19, and is 2961 bp long. The KS designationindicates the Orientation of poly linkers, such that the transcription of lacZ gene precedes from the restriction site for Kpn I to that for Sac I. Thedetailed structure of Phagemid blue script II KS (+/-) is given in the figure.

It may be noted that it has the following features:

A multiple cloning site (MCS) flanked by T3 and T7 promoters tobe read in opposite directions on the two strands.

An inducible Lac promoter (Lac I), upstream of Lac Z region, whichcomplements with E.coli (lac Z) and provides the facilities forselection of chimeric vector DNA (recombinant DNA) using thecriterion of white colonies (as against blue colonies obtained if noforeign DNA is inserted).

f(+) and f(-) origins of replication derived from a filamentous phagefor recovery of sense (+) and anti sense (-) strands of lac Z gene,when host is coinfected with a helper phage.

An origin of replication (Col EI ori) derived from plasmid, and usedin the absence of helper phage.

A gene for amphicillin resistance for antibiotic selection of chimeric

vector.


11/28


- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

----------------------------------------------------------------------------------------------------------------------------



11

Ligation methods

Methods of insertion of foreign gene into the vector

There are two types of ligation of foreign gene with plasmid DNA. They

are cohesive end ligation and blunt end ligation. The ligation depends upon thenature of the cutting of DNA by the restriction enzyme. The different types ofinsertion of foreign DNA fragment into the plasmid DNA are explained below:

1) Cohesive end ligation:

The EcoRI cuts the DNA and produces double stranded DNA withcohessive tails.

This complementary single stranded tails are often called Stickymortise and- tanon termini.

This property of the enzyme was used to join both ends of a foreigngene with the ends of plasmid DNA.

The Restriction endonucleases like EcoRI, Bam HI, Sau 3A etc cut theDAN around the axis (or) at the line of symmetry of the restriction site.As a result of the cutting, linear double-stranded DNA fragments areformed; each DNA fragment has a single stranded tail at each of bothstrands.

The single stranded ends are ready to form base pairing with eachother; hence such DNA fragments are called sticky ended molecules.

The same restriction enzyme is also used, to cut the foreign DNA. Thecomplementary bases found at the single-stranded tails of foreignDNA and that those of the vector DNA undergo hydrogen bonding. Buthydrogen bonding cannot seal the nick present in between the two

DNA fragments. Then an enzyme DNA ligase seals the nick. As a result, the chimericplasmid DNA is formed. The chimeric plasmid is then inserted into thebacterial cells for bacterial transformation.

2) Blunt end ligation:

Hargobind khorana (1970) first discovered the use of T4-DNA ligase in genecloning.

The blunt end ligation is practiced when Restriction endonucleases cuts the twostrands of DNA along the line of symmetry of their restriction sites.

Such enzymes produce blunt ended DNA fragments when the DNAs containedin the solution is treated with the restriction enzyme.

The DNA fragments do not have sticky ends for ligation. In such cases, bothtypes of DNAs are separately treated with the restriction enzyme, whichproduces blunt-ended DNA fragments.

Then the two types of DNA fragments are mixed together to induce ligation. Theligation reaction is catalyzed by a special group of enzyme known as T4-DNAligase, which joins the blunt-ended molecules.


12/28


- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

----------------------------------------------------------------------------------------------------------------------------



12

The enzyme links the ends of the foreign DNA fragments. Phosphodiester bondis established between the 3-hydroxyl group of one fragment and the 5-phosphate group of another DNA fragment.

Disadvantages:1) A large number of plasmids are re-circularized by the action of T4-

DNA ligase enzyme; the enzyme even links the two ends of the sameDNA fragment.2) The percentage of formation of recombinant plasmids containing the

foreign DNA is less than that of homopolymer tailing technique ofinsertion of foreign gene.

3. Homopolymer tailing:

P.Lobban and Kasier introduced this method. In this method also the foreignDNA and the plasmid are separately treated with a restriction enzyme to cut

DNA into fragments. But this method need not require to produces cohesive ended (or) blunt ended

molecules are used to cut the at their restriction site. The enzyme Terminal nucleotide transferaseis used to add nucleotides to the

3-hydroxyl group of the DNA fragments. It is a special kind of polymeraseenzyme, which does not require template strand to add nucleotides to 3-Hydroxyl group end of the DNA fragments.

But the exact sequence of the nucleotides added to the 3,-hydroxyl groupdepends upon the kind of nucleotides available in the pool.

In this method, both plasmid DNA and foreign DNA are separately treated with arestriction enzyme to produce linear DNA fragments.

Then the plasmid DNAs are treated with Terminal nucleotide transferaseenzyme in presence of ATP molecules.

The enzyme adds Adenine nucleotides to the DNA results in the formation ofpolyadenine tail [Poly (A) tail] at 3-hydroxyl group end of plasmid DNA fragment.

On the other hand, the foreign DNA fragments are treated with terminalnucleotide transferase

in the presence of TMP nucleotides; the enzyme adds thymine nucleotides to 3-Hydroxyl group of foreign DNA fragments, forms poly thymine tail (poly T) at 3-hydroxyl end of DNA fragment.

The two kinds of DNA fragments are then mixed together in a solution to establish

the insertion of foreign DNA into the plasmid DNA. Complementary base pairing carriesthis out. The DNA ligase is used to seal the nick found in between the two fragments.


13/28


- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

----------------------------------------------------------------------------------------------------------------------------



13

4. Joining with Linkers:

Linkers are short pieces of double stranded DNA containing a restrictionsite. It is used to join blunt ended DNA fragments.

In this process the linkers are attached to the blunt ends of desired DNA with arestriction enzyme, which cuts the linker to produce sticky ends. The same enzyme isused to cut the plasmid. Then the desired DNA and plasmid DNA fragments are mixed.Recombinant DNA and the linker of desired DNA.

5. Joining with Adapters:

Adaptor is a short DNA fragment containing one sticky end and another bluntend

The adaptor is very similar to linkers, but it differs in having one sticky end. It is

used to join blunt ended foreign DNA with plasmid DNA. The adaptors can be attachedto the blunt ended foreign DNA fragment with the help of DNA ligase. As a result theblunt ended foreign DNA becomes sticky ended. It can be joined with the plasmid stickyended DNA fragments in the normal method.

6. Alkaline phosphatase method:

By treating the liberalized plasmid vector DNA with alkaline phosphatase to remove5-terminal phosphate groups, both recircularization and plasmid dimmer formation areprevented. In this case, circularization of the vector can occur only by insertion of non-

phosphatase treated foreign DNA which provides one 5-terminal phosphate at eachjoin. One nick at each join remains unligated but after transformation of host bacteria,cellular repair mechanism reconstitutes the intact duplex.

Transformation methods

Change that a normal cell undergoes as it becomes malignant; also, permanent,heritable alteration in a cell resulting from the uptake and incorporation of foreign DNAinto genome.

In organisms like bacteria and other microbes, (or) even in higher plants, theuptake of genes by cells is often described by the term Transformation. However inanimals this term has been replaced by the term transfection, because the termTransformation in animal cell culture is used to describe phenotypic alteration of cells.


14/28


- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

----------------------------------------------------------------------------------------------------------------------------



14

Properties of a Good HostA good host should have the following features:(1) is easy to transform,(2) Supports the replication of recombinant DNA,(3) is free from elements that interfere with replication of recombinant DNA,(4) Lacks active restriction enzymes, e.g., E. coliK12 sub strain HB 101,

(5) does not have methylases since these enzymes would methylate thereplicated recombinant DNA which, as a result, would become resistant touseful restriction enzymes, and

(6) is deficient in normal recombination function so that the DNA insert is notaltered by recombination events.

Transformation methods in animals

Several approaches have been used for the introduction of DNA intoanimal cells/ embryos, which are listed as follows:

1) Calcium phosphate Precipitation method

2) DEAE-Dextran mediated transfection3) Lipofection4) Electroporation5) Microinjection method6) Retroviral infection method

1) Calcium Phosphate method:

The DNA preparation to beused for transfection is firstdissolved in a phosphate buffer,

calcium chloride solution is thenadded to the DNA solution; thisleads to the formation ofinsoluble calcium phosphatewhich co-precipitates with theDNA. The calcium-phosphateDNA precipitate is added to thecells to be transfected. The cellstake in the precipitate particlesby Phagocytosis. Initially, 1-2%of the cells were transfected by

this approach. In a smallproportion of the transfectedcells, the DNA becomesintegrated into the cell genomeproducing stable (or) permanenttransfection.


15/28


- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

----------------------------------------------------------------------------------------------------------------------------



15

2) DEAE-Dextran mediated transfection method:

DEAE-dextran (Diethyl amino ethyl dextran) is water soluble andpolycationic i.e., has a multiple positive charge. It is added to thetransfection solution containing the DNA. DEAE-dextran brings about DNA

uptake by the cells through Endocytosis. Posing its interaction with thenegatively charged DNA molecules and with the components of the cellsurface plays an important role.

3) Lipofection:

The delivery of DNA into cellsusing liposomes are Lipofection.Liposomes are small vesiclesprepared from a suitable lipid.Initially, nonionic lipids were usd for

preparing liposomes so that DNAhad to be introduced within thevesicles following specificencapsidation procedures. Usually liposomes are prepared

by dispersion of a Phospholipidlike Phosphotidyl choline (PC)in water by mechanical methodslike Sonication, which tend todestroy DNA. DNA of up to 1 kbhas been incorporated into sonicated liposomes.

Use of choline cationic liposomes to which DNA binds on the outside byelectrostatic attraction. These liposomes cause perturbations in plasmamembrane due to which they fuse and the DNA enter into the cytoplasm.Cationinc liposomes are available commercially (marketed asLIPOFECTIN by Gibco-BRL).

The positively charged liposomes not only complex with DNA, but also bindto cultured animal cells and are efficient in transforming them, by fusion withthe plasma membrane. The use of liposomes as a transformation (or)transfection system is called Lipofection.

4) Electoporation:

Transfection mixture containing cells and DNA is exposed to very briefperiod (few milliseconds) to a very high voltage gradient (eg: 4000 to8000v/cm). This includes transient pores in the cell membranes throughwhich DNA seems to enter the cells. Linearized DNA is far more efficient intransfection than circular supercoiled DNA.


16/28


- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

----------------------------------------------------------------------------------------------------------------------------



16

5. Microinjection:

DNA solution is injected directlyinto the nucleus of a cell (or) into themale pronucleus of a fertilized one-to

two-cell ovum. The general procedurefor microinjection is as follows. Donorsfemales are induced to superovulatedusing appropriate hormone treatments.Female mice are subjected to a regimeof pregnant mare serum gonadotrophin(PMSG), which stimulate growth, anddevelopment of follicles, which containthe developing oocytes. The ovulationinduced by the subsequent treatmentwith human chronic gonadotropin

(HCG). The superovulated females rethen mated with fertile males, andcollect fertilized eggs surgically.

The transgene construct isprepared in a buffer solution and isinjected into the male pronuclei offertilized eggs using a microinjectionassembly. The linearized transgeneconstructs is injected into the cytoplasm of a single ovum. The microinjection embryosare cultured invitro up to the morula (or) blastocyst stage. The surviving embryos arethen transferred into the uterus of surrogate mothers. These embryos develop to fullterm and give rise to normal mice. The microinjection method is frequently using intransgenic biology for producing transgenic animals and Transgenic plants.

5. Retroviral infection:

Recombinant retroviruses produce virions, which are used to infect animal cells andmice embryos. Generally, early 4 to 16 celled embryos are used for this method. Therecombinant retrovirus RNA genome is copied by Reverse transcriptase to yield aDNA copy (reverse transcription) which becomes integrated into the cells genome. Thereverse transcriptase is encoded by the retrovirus. And is produced immediately afterinfection. The recombinant retrovirus integrates into the cellular genome at randomsites & usually is not accompanied with dilations (or) rearrangements.


17/28


- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

----------------------------------------------------------------------------------------------------------------------------



17

Transformation methods in Plants:

In plants some of the methods are using present days. Some are discussesbelow they are,

1) Agrobacteriaum mediated gene transfer method

2) Agroinfection

3) DNA mediated gene transfer

a) Chemical method-gene transferb) Electroporationc) Microprojectile gun methodd) Micro and macro injectione) Lipofectionf) Pollen transformation method

1) Agrobacterium mediated gene transfer method:

The most commonly used vectors forgene transfers in higher plants are basedin tumor inducing mechanism of the soilbacterium Agrobacterium tumefaciens,which is the causal organism for Crowngall disease. A closely related speciesAgrobacterium rizogenes causes Hairyroot disease.

The DNA segment, which is transfected is called T-DNA and is part of alarge Ti-plasmid(Tumor inducing), found in virulent strains of Agrobacteriumtumafaciens. Similarly Ri (Root inducing) megaplasmids are found in thevirulent strains of Agrobacterium rhizogenes. The Ti- and Ri plasmidsinducing Crown gall disease and Hairy root disease.

Structure of Ti-Plasmid:

Most Ti-plasmids have FOUR regions, which are given.

a) Region A b) Region B c) Region C d) Region - D


18/28


- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

----------------------------------------------------------------------------------------------------------------------------



18

i) Region- A:

It contains T-DNA, whichis responsible for tumorinduction so that mutationsin this region lead to the

production of tumors withaltered morphology.

This region transferred toplant nuclear genome, sothat the region is describedas T-DNA (TransferredDNA).

ii) Region-B:

Responsible for Replication

iii) Region-C:

Responsible for Conjugation

iv) Region-D:

Responsible for Virulence, so that mutation in this region abolishesvirulence. This region is therefore called Virulence (vir) region andplays crucial role in this transfer of T-DNA into the plant nuclear

genome.

T-DNA:

It contains two regions:a) onc region:

It contains three genes, they are tms1, tms2, tmr.

tms1Representing Shooty locus

tms2

tmr Representing Rooty locus

These genes are responsible for the biosynthesis of twophytohormones, namely, Auxins and Cytokinins. Thesephytohormones in their turn alter the developmental program,leading to the formation of crown gall.


19/28


- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

----------------------------------------------------------------------------------------------------------------------------



19

b) nos region:

This region responsible for the synthesis of unusual amino acid(or) sugar derivatives, which are collectively given the nameOpines.

Opines are derived from a variety of compounds (eg: Arginine +Pyruvate), that are found in plant cells.

Two most common opines are Octopine and Nopaline. These two opines synthesis responsible enzymes coding are

present in T-DNA.

Outside the T-DNA, Ti-plasmid carries genes that catabolize theopines, which are utilized as a source of carbon and nitrogen.

The T-DNA regions on all Ti and Ri-plasmids are flanked by almostperfect 25 bp direct repeat sequences, which are essential for T-DNA transfer.

Virulence region (vir):The vir region (~35kbp) is organized into SIX operons, namely

Vir-AVir-B

Vir-D PolycistronicVir-G

The vir-A and vir-G geneproducts regulate the expression ofother vir loci.

T-DNA transfer process:

The T-DNA transferprocess will start by theformation of nick at thethird and fourth base of the bottom strand of each 25 base pairsequence repeat. This initiates DNA synthesis from the nick inthe right hand 25bp repeat sequence in 53 direction.

This newly formed DNA will forms complex with protection vir-Eand gets transporated to the plant neucleus.

The vir-D operon encodes an endonuclease that produces thenicks in the border sequences.

The vir-B operon coded protein identified in the bacterialenvelope, alocation , which suggests that may play a role indirecting T-DNA transfer extra cellularly. Role is not at clear.

Vir-C

Vir-E Tumor formation


20/28


- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

----------------------------------------------------------------------------------------------------------------------------



20

The functions of several other vir gene products are largelyunknown.

2) Agro infection method:

Introduction of a viral genome into plant cells by placing it within

the T-DNA of a Ti-plasmid, and using the Agrobacterium containing thisrecombinant plasmid for co-culture with the plant cells is calledAgroinfrction. This may also lead to the integration of viral DNA so thattransgenic plant containing integrated viral DNA can be produced.

3) DNA mediated gene transfer:

The DNA mediated genes transfer (DMGT) mechanisms areconducting in different methods by molecular biologists.

i) Chemical method DNA uptake by protoplast:

Direct DNA uptake by protoplast can be stimulated bychemicals like Polyethylene Glycol (PEG). The technique is soefficient that virtually every protoplast system has proventransformable. PEG is also used to stimulate the uptake ofliposomes and to improve the efficiency of electoporation. PEG athigh concentration (15 to 25%) will precipitate ionicmacromolecules such as DNA and stimulate their uptake byendocytosis without any gross damage to protoplasts.

ii) Electroporation:

This method is based on the use of electric impulses of highfield strength. These impulses increase the permeability of DNA byforming pores on protoplast membrane. The DNA will contact withmembrane it enter into the cells. It is important to test whetherelectoporation could transfer genes into walled cells.

iii) Microprojectile gun method:

Klein and coworkers for transient assay in onion epidermisintroduced the method. The main component for conducting themethod is Helium pressure device. The device contains gasacceleration tube, rupture disc, stopping screen, macro carriercarrying particles coated with DNA and target cells. Thesecomponents are enclosed by a chamber, create partial vacuumand reduces damage to plant cells and also provides partial

acceleration. In this method, 1 to 2 m tungsten (or) gold particlescoated with the DNA to be used for transformation are acceleratedto velocities which enable their entry into plant cells / nuclei. DNA


21/28


- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

----------------------------------------------------------------------------------------------------------------------------



21

coating is to mix 1.25 to 18mg micro particles with 0.5 to 70 m ofplasmid DNA in a calcium chloride (0.25 to 2.5 M) and spermidinesolution (0.1M).

The helium gas release into gas acceleration tube to breakthe rupture disc. This generates helium shock waves, which

accelerated the macro projectile to which DNA coated microprojectiles are attached. A stopping screen stops the macroprojectile and the micro projectiles pass through this screen andbecome embedded in the cells.

iv) Micro and macroinjection:

The DNA solution is directly injected inside the cell usingcapillary glass micropippette with the help micromanipulators of amicroinjection assembly. This microinjection method is demandingand time-consuming method, maximum of 40 to 50 protoplasts can

be microinjected in one hour.

The injection of plasmid DNA into the lumen of developinginflorescence using a hypodermic syrunge is calledMicroinjection. It is the DNA is taken up by microspores duringsome specific stage of their development.

v) Lipofection:

Introduction of DNA into cells via liposomes is known aslipofection. It is the method of choice for DNA delivery into animal

and plant cells also. The plasmid DNAs of 9kb and larger havebeen integrated intact.

vi) Pollen tub transformation method:

The DNA can be taken up by the germinating pollen andcan either integrate into sperm nuclei (or) reach the zygote throughthe pollen tube pathway.

Selection of transformed cells:

When recombinant vector condensation completed, the E.coli cells are identified

the following methods, namely,

a) Selection of clones containing recombinant vectorsb) Selection of clones containing a specific DNA insert

a) Selection of clones containing recombinant vectors:

This is generally achieved by inserting a selectable marker gene into the vector.


22/28


- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

----------------------------------------------------------------------------------------------------------------------------



22

The marker gene (or) reporter produces a phenotype, which is used for theidentification of the recombinants. The markers are either selectable (or)Scorable.

The selectable marker genes conferring resistance to antimetabolites.Eg: Kanamycin is good selectable markers.

Scorable markers produce distinct phenotypes, for easy identification of therecombinant cells. It do not allow selective multiplication of the cells havingthem; they only enable their easy identification.

Elimination of non-transformed cells:

A good vector has at least two marker genes. [PBR322

has the genes tetr

andamp

r(for Tetracycline resistance and Amphicillin resistance respectively)]. The non-

transformed bacterial cells are eliminated by plating them on a medium containingthe selection agent (eg: Tetracyclin (or) Amphicillin in the case of P

BR322).

Identification of clones having recombinant vectors:

In this method the recombinant vectors will separate from the various unalteredvectors. In case, the vector has at least two selectable markers, eg: P

BR322, the DNA

insert may be placed within one of these markers say, amp r gene. The othermarker tet

ris used for elimination of the non-transformed cells. The transformed

clones are then replica plated on amphicillin containing medium. Therecombinant will be sensitive to amphicillin due to inactivation of the gene amp

r

by insertion of the DNA fragment.

Another identification is PUCvector containing gene Lac Z, when the desiredgene integrate into the Lac Z gene, which forms White colonies (or) plaques

on a medium containing X-gal. The LacZ genes express -galctosidaseenzyme. The unchanged vectors form Blue colonies on same medium.

b) Selection of the clone containing a specific DNA insert:

The techniques are used for identification of the single clone from among the

thousands obtained from a cloning experiment. The various strategies are used forthe identification for isolating highly precise clone cells.

1) Colony hybridization method2) Immuno chemical method3) Hybridization method

1) Colony hybridization method:

This technique is used to identify those bacterial colonies in a plate which contain aspecific DNA sequence.

The recombinant bacterial cells are plated onto a suitable agar plate; this is the

Master plate. The sterilized vector cloth containing block cork are lowered into the master plate till

the velvet touches all the colonies; the block is withdrawn and gently lowered ontothe nitrocellulose filter, the bacterial cells sticking on to the velvet are transferredonto the filter.

A reference point is marked both on the master plate and the on replica plate tofacilitate later comparisons.

The nitrocellulose filter is removed from the agar plate and treated with Alkali tolyse the bacterial cells. This also denatures the DNA released from these cells.


23/28


- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

----------------------------------------------------------------------------------------------------------------------------



23

The filter is treated with Proteinase.K to digest and remove the proteins; thedenatures DNA remains bound to the filter.

The filter is now backed at 800C to fix the DNA; in this the DNA-print in bacterial

colonies in the same relative positions in the master plate.

The filter is now hybridized with the radioactive probe (the probe represents thesequence of DNA segment used for transformation). The unhybridized probe isremoved by repeated washing.

The colonies, whose DNA hybridizes with the probe are detected by Autoradiography, only these colonies show up in the autoradiography.

The positions of colonies showing up in the autoradiography are compared with themaster plate to identify these colonies; these colonies contain the DNA segment.The colonies are then picked up for further studies.

2) Immuno chemical method:Immuno chemical methods depends upon three points:

An immune serum contains several IgG types that bind to different determinants on theantigen molecule.

Antibody molecules absorb very strongly to plastics such as polyvinyl, from which theyare not removed by washing. IgG antibody can be readily radio labeled with

125I by

iodination in vitro.

The transformed cells are plated onto a suitable agar plate. The bacterial colonies arethen lysed by exposure to chloroform vapor by the result releases the antigen from positivecolonies. The plate places on the unlabelled antibody coated polyvinyl sheet, the antigencomplex with the bound IgG. The sheet is removed and exposed to

125I-labelled IgG. The

125I-IgG can react with antigen, this bind another side of the antigen (opposite side of

unlabelled antibody binding). Washing the sheet and making an auto radiographic imagedetect positively reacting colonies. The required clones can then be recovered from thereplica plate.

This mthod is mainly used in identifing the Phage expression vector gt 11. This

vector carries the Ecoli lac Z gene, encoded the protein -galactosidase.

3) Hybridization methods:

Nuckeic acid hybridization describes a range of techniques which exploit the ability ofdouble stranded nucleic acids to undergo denaturation (or) melting (separation into singlestrands) and for complemetary single strands to spontaneously anneal (form a duplex).Hybridization can occur between DNA & DNA; DNA & RNA; RNA & RNA and may beintramolecular (or) intermolecular.

The hybridization can occur between nucleic acids in solution (or) where one is insolution and the other immobilized (either on a solid support).

Hybridization methods are mainly FOUR types, namely:

a) Southern hybridizationb) Nothern hybridizationc) Western hybridizationd) Polymerase chain reaction

a) Southern hybridization:

The name of this technique is derived fro the following:


24/28


- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

----------------------------------------------------------------------------------------------------------------------------



24

The name of its inventor, E.M.Southern The DNA-DNA hybridization that forms its basis.

It is also called Southern blotting, since the procedure for transfer of DNA from the gelto the nitrocellulose filter resembles blotting.

DNA sample is first digested with a restriction enzyme and digested sample is gelelectrophoresed. The DNA bands in the gel are denatures into single strands with the help ofan alkali solution.subsequently, the gel is laid on top of a buffer saturated filter paper, placedon a solid support, with its two edges of the filter paper immersed in the buffer. Asheet ofniteocellulose membrane is placed on top of the gel and a stack of many papers on the topof this membrane. 500grams weight placed on the top of paper towels.

The buffer solution moves, due to capillary action, from the bottom filter paper throughthe gel carrying with it the denatured DNA present in the gel; the DNA becomes trapped inthe nitrocellulose membrane as the buffer phases through it. This process is known asblotting and takes several hours to complete.

While passing through the gel, the buffer carries with it single stranded DNA, whichbinds on to the nitrocellulose membrane, when the byffer passes through it to the papertowels. After leaving this arrangement for a few hours (or) overnight, paper towels are

removed and discarded. The nitrocellulose membrane with single stranded DNA bandsblotted on to it., is baked at 80

0C for two to three hours to fix the DNA permanently on the

membrane. This membrane now has a replica of DNA bands from agarose gel, and can beused for hybridization with radioactively labelled DNA (or) RNA probe. The membrane maythen be washed to remove any unbound DNA and X-ray film is exposed to the hybridizedmembrane to get autoradiograph.

b) Northen hybridization methods:

This hybridization method is using for blot transfer of RNA. Instead mRNA bands from

the gel were blot transferred into a chemically reactive paper, prepared by diazotization ofAmino benzyl oxy methyl paper. The procedure of this method is being related to southernblotting was called Nothern blotting. The two techniques, show the following differences:

In southern hybridization DNA is seperated by gel electrophoresis while in nothernblotting RNAs are seperated.

In southern hybridization DNA has to be denatured before blotting, while this step isnot needed in nothern hybridization.


25/28


- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

----------------------------------------------------------------------------------------------------------------------------



25

In southern hybridization Nitrocellulose filter is using; In nothern hybridization Aminobenzyl oxymethyl paper will use.

Hybridization with probe interactions areIn southern hybridization DNA & DNA hybrid moleculeIn southern hybridization RNA & DNA hybrid molecule

Application:

Nothern hybridization is useful in the identification and seperation of the RNAthat is complementary to a specific DNA probe; this is a sensitive test for the detection oftranscription of a DNA sequence that is used as probe.

c) Western hybridization:

In this method, proteins are seperated by using the method resemble toSandwich reaction by adding IgG and radiolabelled immunoglobulin molecules.

The protein bands seperated by PAGE. The protein bands are transferred onto a nitrocellulose (or) nylon membrane. Initially

this was achieved by a capillary movement of buffer done by electrophoresis. The specific protein bands are identified in a variety of ways.Antibodies are the most commonly used as probes for detecting antibodies.Lectins are used as probes for glycoprotein identification.

The species-specific second antibody is used to bind to the antibodies bound to theprotein bands. These second molecules may be labelled with radioactive, enzyme(or) fluorescent tags; these labelled molecules binds on antigen & detect by variousprobes.

d) Polymerase Chain Reaction(PCR):

The polymerase chain reaction (PCR) technique was first developed by Kary Mullisin1985,. By using the PCR technique. We can multiply (or) increases the DNA from microgram

(g) level to the higher amount level. The PCR process doing by the machines, they areThermocyclers.

The PCR is carried out in vitro ( in test tube). It utilizes:

Desired DNA fragment

Two nucleotide primers (about 20 bases long)

The four deoxynucleoside triphosphatesE.g.: TTP, dCTP, dATP, dGTP

Heat stable DNA polymeraseE.g.: Taq polymerase (isolated from bacterium the Thermus aquaticus)

Pfu polymerase (isolated from Pyrococcus furiosus)

Procedure:

Before starting PCR, reaction mixture will be prepare. It contains amplifable DNA,excess of the two primer molecules, four deoxyribonucleoside triphosphates and DNApolymerase.

Step 1:(Denatureation step): The reaction mixture is heated to a temperature (usually90 to 98

0C) that assures DNA denaturation.


26/28


- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

----------------------------------------------------------------------------------------------------------------------------



26

Step 2:(Annealing step): The mixture is now cooled at a temparature (40 to 600C) that

permits annealing of the primer to the complementary sequences in the DNA. Thesesequence locate at 3 ends of two strands of the desired segments

Step3: The temparature adjusts for the primer extending process through the 3-hydroxyl group of primers (only annealed primer molecules). The primers are extendedtowards each other so that the DNA segment lying between the two primers is copied .In this polymerization Taq polymerase enzyme plays an important role in the

polymerization process in the replication in vitro. The completion of the step3 completesthe first cycle of amplification each cyle may take place one to three minutes.

Step 4: The next cycle of amplification is initiated by denaturation, which seperates theDNA strand from newly synthesized complementary DNA strand.

Step 5: Annealing allows the primers to base pairs with both the new and old strands,the totla number of strands being twice their oiginal number.

Step 6: Synthesis of new strands takes place, which doubles the number of copies ofthe desired DNA segment present at the end of step1. This completes the second cycle.

Thus at each cycle, both new and old strands anneal to the primers and serve as

emplates for DNA synthesis. The end of n cycles 2n

copies of the segment areexpected. The cycle may be repeated upto 60 times, but usually 20 to 30 cycles areadequate.

After PCR cycles, the amplified DNA segment is purified by gel electrophoresis andcan be used for the desired purpose.


27/28


- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

----------------------------------------------------------------------------------------------------------------------------



27


28/28


- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

---------------------------------------------------------------------------------------------------------------------------- 28

Applications of PCR:

1) PCR can be used to amplify a specific gene in different individuals of aspecies. These copies can be used for cloning.

2) PCR can be used to study & identify the multiplicaton changes in theamplified (or) clones gene.

3) PCR can be used to study DNA polymorphism in the genome usingknown sequences as primers.4) PCR can be used to identify the transgenic animals among the normal

animals and detect the presence of a gene trasferred into an organism(transgene).

5) In another hybridization methods (Southern, Nothern & Western)radioactive atoms will use for identify transgene. But by using PCR noneed to radioactive atoms(P

32) for transgene identification. This PCR

detection takes in one day for completing this identification6) Use to estimate the DNA frequencies in the species.7) PCR use to determine the physical locaton of genes in chromosomes.8) By using PCR, to determine the sex of embryos.

Reference books:

1. Principles of gene manipulations, Old & Primrose.2. Fundamentals of Biochemistry, Voet & Voet.3. Principles of Biochemistry, Lehninger, Nelson & Cox, CBS publications.4. Molecular biology & Genetic engineering, SARAS publications.5. Molecular biology, S.Chand Publications.6. Molecualr Biology, David freifelder.7. Practical Biochemistry, Wilson & Walker, 4/e, Cambridge low price edition.8. Elementals of Biotechnology, P.K.Guptha, Rastogi publications.9. Advanced Molecular Biology, Richard M. Twyman, 2/e, VIVA BOOK PRIVAT LIMITED.10. Biotechnology, B.D.Singh, Kalyani publications.11. Genetics, P.K.Guptha, Rastogi publications.

rDNA Technology-Biochemden by SATISH

Documents