rDNA Technology-Biochemden by SATISH

Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology
Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology (Biotechnology) Recombinant DNA Technology Recombinant DNA Technology I.Satish Kumar Recombinant DNA Technology Recombinant DNA Technology Lecturer in Biochemistry Recombinant DNA Technology Recombinant DNA Technology Visit: http://biochemden.in http://biochemistryden.blogspot.com Recombinant DNA Technology Recombinant DNA Technology http://bioscienceden.blogspot.com Email: satishkumar.i1980@gmail.com Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology
Recombinant DNA technology
Life sciences Study materials Molecular Biology ----------------------------------------------------------------------------------------
Recombinant DNA technology

Biotechnology:
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 the capabilities of microorganisms, cultured tissue cells and part of them. The controlled use of biological agents, such as microorganisms (or) cellular components, for beneficial use The last definition is brief and comprehensive and may be used by students, if they have 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, when microorganisms were already used for processes like Fermentation. First Discovery: In 1920, Clostridium acetobutylicum was used by Chaim Weizmann for converting starch into butanol and Acetone. The latter was an essential component of explosives during World War-I. Second Discovery: During Second World War (1940s), the production of Penicillin (as an antibiotic discovered by Alexander Fleming in 1929) on a large scale from cultures of Pencillium notatum Third Discovery: The third discovery of Biotechnology is its recent reincarnation in the form of Recombinant DNA technology, which led to the development of a variety of gene technologies and is thus considered to be the greatest scientific revolution of this century.
Old Vs New Biotechnology:

Fermentation by some microorganisms, formation of yoghurt (curd), cheese from milk, Vinegar from molasses, production of antibiotics like Penicillin from certain fungi, Process of baking and brewing are often included in describing what is called Old Biotechnology PCR (Polymerase Chain Reaction), rDNA technology, Cell culture and fusion and bio-processing, which became possible only through the researches in molecular biology have been described as New Biotechnology
---------------------------------------------------------------------------------------------------------------------------Prepared by I.Satish Kumar, Lecturer in Biochemistry,
http://biochemistryden.blogspot.com
http://biochemden.in
Recombinant DNA Technology

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 a phenotypic bacterium such as Salmonella. The explanation of this phenomenon led to the discovery of the plasmid, where by the gene conferring antibiotic resistance residues in a form of extra-chromosomal DNA. The resistance is due to an enzyme that causes 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) 2) 3) 4) 5) 6) 7) Restriction endonucleases DNA ligases Alkaline Phosphatase Polynucleotide Kinase Terminal Deoxynucleotidyl Transferase RNA dependent DNA polymerase (Reverse Transcriptase) RNase H
1) Restriction endonulceases: The Restriction endonucleases recognizes a specific base sequence of four to eight 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 II enzymes are frequently used in the rDNA technology. Type I and type III are not use because these type enzymes cleaves the DNA far from the recognition sites. The Restriction endonuclease recognizes particular specific sequences are 4 to 6 nucleotides recognize by the type II enzymes because these enzymes only cleave at this site. The palindrome sequence is Two-fold symmetry as shown below. The type II restriction enzymes are discovered and characterized by Hamilton Smith and Daniel Nathans 5- GATATC 3 in 1960. Approximately 200 Restriction 3- CTATAG 5 endonucleases are isolated from bacterial species like- E.coli, Bacillus, Two fold symmetry axis Haemophillus, Streptococcus and (Cleaved by EcoRV) thermus aquaticus. Restriction endonucleases is named by the first letter of the genus of the bacterium that produced it and the first two letters of its species, followed by its serotype (or) strain designation, if any,
and a roman numerical if the bacterium contains more than one type of restriction enzymes. 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 stuartil 2) DNA ligases: The complimentary ends of the DNAs specifically associate under annealing conditions and are covalently joined through the action of an enzyme named 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 an enzyme-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 of the 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 of ATP to a dephosphorylated end of DNA or RNA. The enzyme is encoded by a gene of phage T4 and is extracted from E.coli cells infected with the phage. This enzyme used after the Alkaline Phosphatase activity and introduces 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 using a template. Any nucleotide that is provided in the reaction mixture is utilized to elongate the DNA strand.
If only one kind of nucleotide is provided a mononucleotide polymer will be produced.
5. Alkaline Phosphatase The enzyme alkaline phosphatase removes the phosphate moiety at the 5-end of DNA strand, whether it is part of blunt end single extension 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 and Calf 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:
RNase H is an endonuclease that is useful for degrading the RNA strand from a DNA:RNA hybrid molecule. It cut up the RNA into short fragments.
VECTORS
Introduction:
The DNA fragment (or) the gene of interest can be linked to a carrier molecule, which can transport the gene of interest into the host cell. This carrier molecule is referred to as a Cloning vector (or) Cloning vehicle; the cloning vehicle is the central component of a gene cloning experiment and it constitutes the 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 plating test. It should contain an assortment of unique Restriction endonucleases cleavage sites. It should occur in large number of copies.
Type of vector: There are three main types of vectors in use. They are 1) Plasmids 2) Cosmids 3) Phagemids
1) Plasmids:
Plasmids are extra chromosomal, autonomously replicating , small circular molecules of DNA found in many prokaryotes and in a few eukaryotes 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, toxin production, for antibody production, for degradation of a large number of
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unusual substrates such as herbicides (or) industrial effluents and genes for nitrogen fixation. These confer the phenotypic traits of plasmids. They rely on the DNA replication enzymes of the host cells for their replication; however, the initiation of replication is controlled by plasmid genes. 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 about 5 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 range in size from 1X106 Daltons to greater than 200X106.
Number of plasmids:
The number of copies of plasmid in a cell is referred to as Copy number. When there are one (or) two copies, the copy number is called low copy number. When there are twenty (or) more copies per cell, the copy number is called high copy number
Plasmid classification:
The naturally occurring plasmids are classified based on the main characteristics coded by the plasmid genes. It is grouped into FIVE main types: a) F-plasmids b) R-Plasmids c) Col plsamids d) Degradative plasmids e) Virulence plasmids a) F-Plasmids (Fertility plasmids): These plasmids carry only tra genes (transfer gene) and no characteristic beyond the ability to promote conjugate transfer of plasmids. The presence of tra genes promotes bacterial conjugation. These plasmids may 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.
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 RP4 found 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 pathogenic bacteria. The plasmid genes after encode for enzyme that chemically inactivate the 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 that prevent 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 that are liberated by strains of E.coli are called Colicins. The colicins are simple proteins. Several different types of colicins have been isolated which kill sensitive cells by different mechanisms. The plasmids containing genes for such toxic substance, colicin is called Col plasmid. Depending upon the nature of colicins, 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 the bacterium to metabolize unusual substrates such as Toluene, xylene and salicylic acid. These plasmids are also called Dissimilation plasmids. This plasmid type (Ptol) is responsible for the ability of certain Pseudomonas species to break down different to degrade industrial solvents such as toluene and xylene. A combination of several plasmids, when transferred to pseudomonas bacteria, allows the bacteria to break down complex hydrocarbons and other compounds present in crude oil. The bacteria, containing these plasmids have a potential use for treatment of environments contaminated with oil spills. e) Virulence plasmids: The plasmids have genes that confer pathogenicity on the host bacterium. For example, Ti-plasmids found in Agro bacterium tumefacience. They include crown gall disease on dicotyledonous plant.

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. It is 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 the enzyme DNA ligase, which link together the two DNA fragments end to end. The resulting 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, the complementary single strand region is abbreviated as Cos site. The cos site consists of two complementary single strands held together by complementary base pairing both these two strands. At the cos site, 3-end of each of the DNA strands does not establish 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 of generations. The cosmid DNA does not code for the synthesis of viral proteins. The cosmid does not participate in the multiplication of phage particles. 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 the bacterial cell. The cosmid pickup even long sized genes. Hence it is used in the genome of the organisms. The cosmids are also used in the study of some non-sense sequences found in the genome of the organisms.
Disadvantages: Each cosmid requires two cos sites for the successful packing of recombinant cosmid within the protein coat of bacteriophage. In the 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, which is commonly used in molecular biology laboratories, is Blue script II KS, which is derived from PUC19, and is 2961 bp long. The KS designation indicates the Orientation of poly linkers, such that the transcription of lac Z gene precedes from the restriction site for Kpn I to that for Sac I. The detailed 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 to be read in opposite directions on the two strands. An inducible Lac promoter (Lac I), upstream of Lac Z region, which complements with E.coli (lac Z) and provides the facilities for selection of chimeric vector DNA (recombinant DNA) using the criterion of white colonies (as against blue colonies obtained if no foreign DNA is inserted). f(+) and f(-) origins of replication derived from a filamentous phage for 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 used in the absence of helper phage. A gene for amphicillin resistance for antibiotic selection of chimeric vector.

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1) Cohesive end ligation:
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 the nature of the cutting of DNA by the restriction enzyme. The different types of insertion of foreign DNA fragment into the plasmid DNA are explained below: The EcoRI cuts the DNA and produces double stranded DNA with cohessive tails. This complementary single stranded tails are often called Sticky mortise and- tanon termini. This property of the enzyme was used to join both ends of a foreign gene with the ends of plasmid DNA. The Restriction endonucleases like EcoRI, Bam HI, Sau 3A etc cut the DAN around the axis (or) at the line of symmetry of the restriction site. As a result of the cutting, linear double-stranded DNA fragments are formed; each DNA fragment has a single stranded tail at each of both strands. The single stranded ends are ready to form base pairing with each other; hence such DNA fragments are called sticky ended molecules. The same restriction enzyme is also used, to cut the foreign DNA. The complementary bases found at the single-stranded tails of foreign DNA and that those of the vector DNA undergo hydrogen bonding. But hydrogen bonding cannot seal the nick present in between the two DNA fragments. Then an enzyme DNA ligase seals the nick. As a result, the chimeric plasmid DNA is formed. The chimeric plasmid is then inserted into the bacterial cells for bacterial transformation.
2) Blunt end ligation:
Hargobind khorana (1970) first discovered the use of T4-DNA ligase in gene cloning. The blunt end ligation is practiced when Restriction endonucleases cuts the two strands of DNA along the line of symmetry of their restriction sites. Such enzymes produce blunt ended DNA fragments when the DNAs contained in the solution is treated with the restriction enzyme. The DNA fragments do not have sticky ends for ligation. In such cases, both types of DNAs are separately treated with the restriction enzyme, which produces blunt-ended DNA fragments. Then the two types of DNA fragments are mixed together to induce ligation. The ligation reaction is catalyzed by a special group of enzyme known as T4-DNA ligase, which joins the blunt-ended molecules.
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The enzyme links the ends of the foreign DNA fragments. Phosphodiester bond is established between the 3-hydroxyl group of one fragment and the 5phosphate group of another DNA fragment. Disadvantages: 1) A large number of plasmids are re-circularized by the action of T4DNA ligase enzyme; the enzyme even links the two ends of the same DNA fragment. 2) The percentage of formation of recombinant plasmids containing the foreign DNA is less than that of homopolymer tailing technique of insertion of foreign gene.
3. Homopolymer tailing:
P.Lobban and Kasier introduced this method. In this method also the foreign DNA 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 transferase is used to add nucleotides to the 3-hydroxyl group of the DNA fragments. It is a special kind of polymerase enzyme, which does not require template strand to add nucleotides to 3Hydroxyl group end of the DNA fragments. But the exact sequence of the nucleotides added to the 3,-hydroxyl group depends upon the kind of nucleotides available in the pool. In this method, both plasmid DNA and foreign DNA are separately treated with a restriction enzyme to produce linear DNA fragments. Then the plasmid DNAs are treated with Terminal nucleotide transferase enzyme in presence of ATP molecules. The enzyme adds Adenine nucleotides to the DNA results in the formation of polyadenine tail [Poly (A) tail] at 3-hydroxyl group end of plasmid DNA fragment. On the other hand, the foreign DNA fragments are treated with terminal nucleotide transferase in the presence of TMP nucleotides; the enzyme adds thymine nucleotides to 3Hydroxyl group of foreign DNA fragments, forms poly thymine tail (poly T) at 3hydroxyl 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 carries this out. The DNA ligase is used to seal the nick found in between the two fragments.

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4. Joining with Linkers:

Linkers are short pieces of double stranded DNA containing a restriction site. It is used to join blunt ended DNA fragments. In this process the linkers are attached to the blunt ends of desired DNA with a restriction enzyme, which cuts the linker to produce sticky ends. The same enzyme is used 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 blunt end 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 attached to the blunt ended foreign DNA fragment with the help of DNA ligase. As a result the blunt ended foreign DNA becomes sticky ended. It can be joined with the plasmid sticky ended DNA fragments in the normal method.
6. Alkaline phosphatase method:

By treating the liberalized plasmid vector DNA with alkaline phosphatase to remove 5-terminal phosphate groups, both recircularization and plasmid dimmer formation are prevented. In this case, circularization of the vector can occur only by insertion of nonphosphatase treated foreign DNA which provides one 5-terminal phosphate at each join. One nick at each join remains unligated but after transformation of host bacteria, cellular repair mechanism reconstitutes the intact duplex.
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 DNA into genome. In organisms like bacteria and other microbes, (or) even in higher plants, the uptake of genes by cells is often described by the term Transformation. However in animals this term has been replaced by the term transfection, because the term Transformation in animal cell culture is used to describe phenotypic alteration of cells.
Transformation methods

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Properties of a Good Host
A 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. coli K12 sub strain HB 101, (5) does not have methylases since these enzymes would methylate the replicated recombinant DNA which, as a result, would become resistant to useful restriction enzymes, and (6) is deficient in normal recombination function so that the DNA insert is not altered by recombination events.
Transformation methods in animals

Several approaches have been used for the introduction of DNA into animal cells/ embryos, which are listed as follows: 1) Calcium phosphate Precipitation method 2) DEAE-Dextran mediated transfection 3) Lipofection 4) Electroporation 5) Microinjection method 6) Retroviral infection method 1) Calcium Phosphate method: The DNA preparation to be used for transfection is first dissolved in a phosphate buffer, calcium chloride solution is then added to the DNA solution; this leads to the formation of insoluble calcium phosphate which co-precipitates with the DNA. The calcium-phosphate DNA precipitate is added to the cells to be transfected. The cells take in the precipitate particles by Phagocytosis. Initially, 1-2% of the cells were transfected by this approach. In a small proportion of the transfected cells, the DNA becomes integrated into the cell genome producing stable (or) permanent transfection.

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2) DEAE-Dextran mediated transfection method: DEAE-dextran (Diethyl amino ethyl dextran) is water soluble and polycationic i.e., has a multiple positive charge. It is added to the transfection solution containing the DNA. DEAE-dextran brings about DNA uptake by the cells through Endocytosis. Posing its interaction with the negatively charged DNA molecules and with the components of the cell surface plays an important role. 3) Lipofection: The delivery of DNA into cells using liposomes are Lipofection. Liposomes are small vesicles prepared from a suitable lipid. Initially, nonionic lipids were usd for preparing liposomes so that DNA had to be introduced within the vesicles following specific encapsidation procedures. Usually liposomes are prepared by dispersion of a Phospholipid like Phosphotidyl choline (PC) in water by mechanical methods like Sonication, which tend to destroy DNA. DNA of up to 1 kb has been incorporated into sonicated liposomes. Use of choline cationic liposomes to which DNA binds on the outside by electrostatic attraction. These liposomes cause perturbations in plasma membrane due to which they fuse and the DNA enter into the cytoplasm. Cationinc liposomes are available commercially (marketed as LIPOFECTIN by Gibco-BRL). The positively charged liposomes not only complex with DNA, but also bind to cultured animal cells and are efficient in transforming them, by fusion with the 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 brief period (few milliseconds) to a very high voltage gradient (eg: 4000 to 8000v/cm). This includes transient pores in the cell membranes through which DNA seems to enter the cells. Linearized DNA is far more efficient in transfection than circular supercoiled DNA.
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5. Microinjection: DNA solution is injected directly into the nucleus of a cell (or) into the male pronucleus of a fertilized one-to two-cell ovum. The general procedure for microinjection is as follows. Donors females are induced to superovulated using appropriate hormone treatments. Female mice are subjected to a regime of pregnant mare serum gonadotrophin (PMSG), which stimulate growth, and development of follicles, which contain the developing oocytes. The ovulation induced by the subsequent treatment with human chronic gonadotropin (HCG). The superovulated females re then mated with fertile males, and collect fertilized eggs surgically. The transgene construct is prepared in a buffer solution and is injected into the male pronuclei of fertilized eggs using a microinjection assembly. The linearized transgene constructs is injected into the cytoplasm of a single ovum. The microinjection embryos are cultured invitro up to the morula (or) blastocyst stage. The surviving embryos are then transferred into the uterus of surrogate mothers. These embryos develop to full term and give rise to normal mice. The microinjection method is frequently using in transgenic biology for producing transgenic animals and Transgenic plants. 5. Retroviral infection: Recombinant retroviruses produce virions, which are used to infect animal cells and mice embryos. Generally, early 4 to 16 celled embryos are used for this method. The recombinant retrovirus RNA genome is copied by Reverse transcriptase to yield a DNA copy (reverse transcription) which becomes integrated into the cells genome. The reverse transcriptase is encoded by the retrovirus. And is produced immediately after infection. The recombinant retrovirus integrates into the cellular genome at random sites & usually is not accompanied with dilations (or) rearrangements.

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Transformation methods in Plants:

In plants some of the methods are using present days. Some are discusses below they are, 1) Agrobacteriaum mediated gene transfer method 2) Agroinfection 3) DNA mediated gene transfer a) b) c) d) e) f) Chemical method-gene transfer Electroporation Microprojectile gun method Micro and macro injection Lipofection Pollen transformation method
1) Agrobacterium mediated gene transfer method: The most commonly used vectors for gene transfers in higher plants are based in tumor inducing mechanism of the soil bacterium Agrobacterium tumefaciens, which is the causal organism for Crown gall disease. A closely related species Agrobacterium rizogenes causes Hairy root disease.
The DNA segment, which is transfected is called T-DNA and is part of a large Ti-plasmid(Tumor inducing), found in virulent strains of Agrobacterium tumafaciens. Similarly Ri (Root inducing) megaplasmids are found in the virulent strains of Agrobacterium rhizogenes. The Ti- and Ri plasmids inducing 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

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i)
Region- A: It contains T-DNA, which is responsible for tumor induction so that mutations in this region lead to the production of tumors with altered morphology. This region transferred to plant nuclear genome, so that the region is described as T-DNA (Transferred DNA).
ii)
Region-B: Responsible for Replication
iii)
Region-C: Responsible for Conjugation
iv)
Region-D: Responsible for Virulence, so that mutation in this region abolishes virulence. This region is therefore called Virulence (vir) region and plays 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. tms1 Representing Shooty locus tms2
tmr
Representing Rooty locus
These genes are responsible for the biosynthesis of two phytohormones, namely, Auxins and Cytokinins. These phytohormones in their turn alter the developmental program, leading to the formation of crown gall.
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b) nos region: This region responsible for the synthesis of unusual amino acid (or) sugar derivatives, which are collectively given the name Opines. 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 the opines, which are utilized as a source of carbon and nitrogen. The T-DNA regions on all Ti and Ri-plasmids are flanked by almost perfect 25 bp direct repeat sequences, which are essential for TDNA transfer.
Virulence region (vir): The vir region (~35kbp) is organized into SIX operons, namely Vir-A Vir-B Vir-D Vir-G
Vir-C Vir-E Tumor formation
Polycistronic
The vir-A and vir-G gene products regulate the expression of other vir loci. T-DNA transfer process: The T-DNA transfer process will start by the formation of nick at the third and fourth base of the bottom strand of each 25 base pair sequence repeat. This initiates DNA synthesis from the nick in the right hand 25bp repeat sequence in 53 direction. This newly formed DNA will forms complex with protection vir-E and gets transporated to the plant neucleus. The vir-D operon encodes an endonuclease that produces the nicks in the border sequences. The vir-B operon coded protein identified in the bacterial envelope, alocation , which suggests that may play a role in directing T-DNA transfer extra cellularly. Role is not at clear.
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The functions of several other vir gene products are largely unknown.
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 this recombinant plasmid for co-culture with the plant cells is called Agroinfrction. This may also lead to the integration of viral DNA so that transgenic plant containing integrated viral DNA can be produced. 3) DNA mediated gene transfer: The DNA mediated genes transfer (DMGT) mechanisms are conducting in different methods by molecular biologists. i) Chemical method DNA uptake by protoplast: Direct DNA uptake by protoplast can be stimulated by chemicals like Polyethylene Glycol (PEG). The technique is so efficient that virtually every protoplast system has proven transformable. PEG is also used to stimulate the uptake of liposomes and to improve the efficiency of electoporation. PEG at high concentration (15 to 25%) will precipitate ionic macromolecules such as DNA and stimulate their uptake by endocytosis without any gross damage to protoplasts. ii) Electroporation: This method is based on the use of electric impulses of high field strength. These impulses increase the permeability of DNA by forming pores on protoplast membrane. The DNA will contact with membrane it enter into the cells. It is important to test whether electoporation could transfer genes into walled cells. iii) Microprojectile gun method: Klein and coworkers for transient assay in onion epidermis introduced the method. The main component for conducting the method is Helium pressure device. The device contains gas acceleration tube, rupture disc, stopping screen, macro carrier carrying particles coated with DNA and target cells. These components are enclosed by a chamber, create partial vacuum and reduces damage to plant cells and also provides partial acceleration. In this method, 1 to 2 m tungsten (or) gold particles coated with the DNA to be used for transformation are accelerated to velocities which enable their entry into plant cells / nuclei. DNA
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coating is to mix 1.25 to 18mg micro particles with 0.5 to 70 m of plasmid DNA in a calcium chloride (0.25 to 2.5 M) and spermidine solution (0.1M). The helium gas release into gas acceleration tube to break the rupture disc. This generates helium shock waves, which accelerated the macro projectile to which DNA coated micro projectiles are attached. A stopping screen stops the macro projectile and the micro projectiles pass through this screen and become embedded in the cells. iv) Micro and macroinjection: The DNA solution is directly injected inside the cell using capillary glass micropippette with the help micromanipulators of a microinjection assembly. This microinjection method is demanding and time-consuming method, maximum of 40 to 50 protoplasts can be microinjected in one hour. The injection of plasmid DNA into the lumen of developing inflorescence using a hypodermic syrunge is called Microinjection. It is the DNA is taken up by microspores during some specific stage of their development. v) Lipofection: Introduction of DNA into cells via liposomes is known as lipofection. It is the method of choice for DNA delivery into animal and plant cells also. The plasmid DNAs of 9kb and larger have been integrated intact. vi) Pollen tub transformation method: The DNA can be taken up by the germinating pollen and can either integrate into sperm nuclei (or) reach the zygote through the 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 vectors b) 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.

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The marker gene (or) reporter produces a phenotype, which is used for the identification 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 the recombinant cells. It do not allow selective multiplication of the cells having them; 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 and r amp (for Tetracycline resistance and Amphicillin resistance respectively)]. The nontransformed bacterial cells are eliminated by plating them on a medium containing BR322 the selection agent (eg: Tetracyclin (or) Amphicillin in the case of P ). Identification of clones having recombinant vectors: In this method the recombinant vectors will separate from the various unaltered vectors. In case, the vector has at least two selectable markers, eg: PBR322, the DNA insert may be placed within one of these markers say, ampr gene. The other marker tetr is used for elimination of the non-transformed cells. The transformed clones are then replica plated on amphicillin containing medium. The recombinant will be sensitive to amphicillin due to inactivation of the gene ampr by insertion of the DNA fragment. Another identification is PUC vector containing gene Lac Z, when the desired gene integrate into the Lac Z gene, which forms White colonies (or) plaques on a medium containing X-gal. The LacZ genes express -galctosidase enzyme. 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 for the identification for isolating highly precise clone cells. 1) Colony hybridization method 2) Immuno chemical method 3) Hybridization method 1) Colony hybridization method: This technique is used to identify those bacterial colonies in a plate which contain a specific 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 onto the nitrocellulose filter, the bacterial cells sticking on to the velvet are transferred onto the filter. A reference point is marked both on the master plate and the on replica plate to facilitate later comparisons. The nitrocellulose filter is removed from the agar plate and treated with Alkali to lyse the bacterial cells. This also denatures the DNA released from these cells. ---------------------------------------------------------------------------------------------------------------------------22 Prepared by I.Satish Kumar, Lecturer in Biochemistry,
The filter is treated with Proteinase.K to digest and remove the proteins; the denatures 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 the sequence of DNA segment used for transformation). The unhybridized probe is removed by repeated washing. The colonies, whose DNA hybridizes with the probe are detected by Auto radiography, only these colonies show up in the autoradiography. The positions of colonies showing up in the autoradiography are compared with the master 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 the antigen molecule. Antibody molecules absorb very strongly to plastics such as polyvinyl, from which they are 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 are then lysed by exposure to chloroform vapor by the result releases the antigen from positive colonies. The plate places on the unlabelled antibody coated polyvinyl sheet, the antigen complex with the bound IgG. The sheet is removed and exposed to 125I-labelled IgG. The 125 I-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 image detect positively reacting colonies. The required clones can then be recovered from the replica 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 of double stranded nucleic acids to undergo denaturation (or) melting (separation into single strands) and for complemetary single strands to spontaneously anneal (form a duplex). Hybridization can occur between DNA & DNA; DNA & RNA; RNA & RNA and may be intramolecular (or) intermolecular. The hybridization can occur between nucleic acids in solution (or) where one is in solution and the other immobilized (either on a solid support). Hybridization methods are mainly FOUR types, namely: a) b) c) d) Southern hybridization Nothern hybridization Western hybridization Polymerase chain reaction
a) Southern hybridization: The name of this technique is derived fro the following: ---------------------------------------------------------------------------------------------------------------------------Prepared by I.Satish Kumar, Lecturer in Biochemistry,
23
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 gel to the nitrocellulose filter resembles blotting. DNA sample is first digested with a restriction enzyme and digested sample is gel electrophoresed. The DNA bands in the gel are denatures into single strands with the help of an alkali solution.subsequently, the gel is laid on top of a buffer saturated filter paper, placed on a solid support, with its two edges of the filter paper immersed in the buffer. Asheet of niteocellulose membrane is placed on top of the gel and a stack of many papers on the top of this membrane. 500grams weight placed on the top of paper towels. The buffer solution moves, due to capillary action, from the bottom filter paper through the gel carrying with it the denatured DNA present in the gel; the DNA becomes trapped in the nitrocellulose membrane as the buffer phases through it. This process is known as blotting and takes several hours to complete. While passing through the gel, the buffer carries with it single stranded DNA, which binds on to the nitrocellulose membrane, when the byffer passes through it to the paper towels. After leaving this arrangement for a few hours (or) overnight, paper towels are removed and discarded. The nitrocellulose membrane with single stranded DNA bands blotted on to it., is baked at 800C 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 be used for hybridization with radioactively labelled DNA (or) RNA probe. The membrane may then be washed to remove any unbound DNA and X-ray film is exposed to the hybridized membrane 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 of Amino benzyl oxy methyl paper. The procedure of this method is being related to southern blotting was called Nothern blotting. The two techniques, show the following differences: In southern hybridization DNA is seperated by gel electrophoresis while in nothern blotting RNAs are seperated.
In southern hybridization DNA has to be denatured before blotting, while this step is not needed in nothern hybridization. ---------------------------------------------------------------------------------------------------------------------------24 Prepared by I.Satish Kumar, Lecturer in Biochemistry,
In southern hybridization Nitrocellulose filter is using; In nothern hybridization Amino benzyl oxymethyl paper will use.
Hybridization with probe interactions are In southern hybridization DNA & DNA hybrid molecule In southern hybridization RNA & DNA hybrid molecule Application: Nothern hybridization is useful in the identification and seperation of the RNA that is complementary to a specific DNA probe; this is a sensitive test for the detection of transcription of a DNA sequence that is used as probe.
c) Western hybridization:
In this method, proteins are seperated by using the method resemble to Sandwich 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 the protein bands. These second molecules may be labelled with radioactive, enzyme (or) fluorescent tags; these labelled molecules binds on antigen & detect by various probes.
d) Polymerase Chain Reaction(PCR):

The polymerase chain reaction (PCR) technique was first developed by Kary Mullis in 1985,. 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 are Thermocyclers. 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 triphosphates E.g.: TTP, dCTP, dATP, dGTP Heat stable DNA polymerase E.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 DNA polymerase. Step 1:(Denatureation step): The reaction mixture is heated to a temperature (usually 90 to 980C) that assures DNA denaturation. ---------------------------------------------------------------------------------------------------------------------------Prepared by I.Satish Kumar, Lecturer in Biochemistry,
25
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. These sequence locate at 3 ends of two strands of the desired segments Step3: The temparature adjusts for the primer extending process through the 3hydroxyl group of primers (only annealed primer molecules). The primers are extended towards 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 completes the 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 the DNA 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 of the 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 are expected. The cycle may be repeated upto 60 times, but usually 20 to 30 cycles are adequate. After PCR cycles, the amplified DNA segment is purified by gel electrophoresis and can be used for the desired purpose.

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Applications of PCR:
1) PCR can be used to amplify a specific gene in different individuals of a species. These copies can be used for cloning. 2) PCR can be used to study & identify the multiplicaton changes in the amplified (or) clones gene. 3) PCR can be used to study DNA polymorphism in the genome using known 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 no 32 need to radioactive atoms(P ) for transgene identification. This PCR detection takes in one day for completing this identification 6) 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. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. Principles of gene manipulations, Old & Primrose. Fundamentals of Biochemistry, Voet & Voet. Principles of Biochemistry, Lehninger, Nelson & Cox, CBS publications. Molecular biology & Genetic engineering, SARAS publications. Molecular biology, S.Chand Publications. Molecualr Biology, David freifelder. Practical Biochemistry, Wilson & Walker, 4/e, Cambridge low price edition. Elementals of Biotechnology, P.K.Guptha, Rastogi publications. Advanced Molecular Biology, Richard M. Twyman, 2/e, VIVA BOOK PRIVAT LIMITED. Biotechnology, B.D.Singh, Kalyani publications. Genetics, P.K.Guptha, Rastogi publications.

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Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology

Recombinant DNA technology

Life sciences Study materials Molecular Biology ----------------------------------------------------------------------------------------

Recombinant DNA technology

Old Vs New Biotechnology:

Recombinant DNA Technology

Life sciences Study materials Molecular Biology ----------------------------------------------------------------------------------------

Enzymes involved in rDNA technology:

Life sciences Study materials Molecular Biology ----------------------------------------------------------------------------------------

Life sciences Study materials Molecular Biology ----------------------------------------------------------------------------------------

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

Life sciences Study materials Molecular Biology ----------------------------------------------------------------------------------------

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Life sciences Study materials Molecular Biology ----------------------------------------------------------------------------------------

---------------------------------------------------------------------------------------------------------------------------Prepared by I.Satish Kumar, Lecturer in Biochemistry,

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Life sciences Study materials Molecular Biology ----------------------------------------------------------------------------------------

---------------------------------------------------------------------------------------------------------------------------Prepared by I.Satish Kumar, Lecturer in Biochemistry,

Life sciences Study materials Molecular Biology ----------------------------------------------------------------------------------------

1) Cohesive end ligation:

Methods of insertion of foreign gene into the vector

2) Blunt end ligation:

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Life sciences Study materials Molecular Biology ----------------------------------------------------------------------------------------

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Life sciences Study materials Molecular Biology ----------------------------------------------------------------------------------------

4. Joining with Linkers:

5. Joining with Adapters:

6. Alkaline phosphatase method:

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Life sciences Study materials Molecular Biology ----------------------------------------------------------------------------------------

Properties of a Good Host

Transformation methods in animals

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Life sciences Study materials Molecular Biology ----------------------------------------------------------------------------------------

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Life sciences Study materials Molecular Biology ----------------------------------------------------------------------------------------

Transformation methods in Plants:

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Life sciences Study materials Molecular Biology ----------------------------------------------------------------------------------------

Region-B: Responsible for Replication

Region-C: Responsible for Conjugation

Representing Rooty locus

Life sciences Study materials Molecular Biology ----------------------------------------------------------------------------------------

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Life sciences Study materials Molecular Biology ----------------------------------------------------------------------------------------

Selection of transformed cells:

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Life sciences Study materials Molecular Biology ----------------------------------------------------------------------------------------

Life sciences Study materials Molecular Biology ----------------------------------------------------------------------------------------

Life sciences Study materials Molecular Biology ----------------------------------------------------------------------------------------

Northen hybridization methods:

Life sciences Study materials Molecular Biology ----------------------------------------------------------------------------------------

d) Polymerase Chain Reaction(PCR):

Life sciences Study materials Molecular Biology ----------------------------------------------------------------------------------------

---------------------------------------------------------------------------------------------------------------------------Prepared by I.Satish Kumar, Lecturer in Biochemistry,

Life sciences Study materials Molecular Biology ----------------------------------------------------------------------------------------

---------------------------------------------------------------------------------------------------------------------------Prepared by I.Satish Kumar, Lecturer in Biochemistry,

Life sciences Study materials Molecular Biology ----------------------------------------------------------------------------------------

---------------------------------------------------------------------------------------------------------------------------Prepared by I.Satish Kumar, Lecturer in Biochemistry,