BIOLOGY 3 Marks Question

1. Small volumes of culture can be taken out from the reactor for testing.
2. It has a foam breaker for regulating the foam.
3. It has a control system that regulates the temperature and pH.

1. Palindromic nucleotide sequence is the recognition (specific) sequence present both on the vector and on a desired/alien DNA for the action of the same(specific) restriction endonuclease to act upon.
2. Same restriction endonuclease binds to both the vector and the foreign DNA, cut each of the two strands of the double helix at specific points in their sugar phosphate backbone of recognition sequence for restriction endonucleases/palindromic sequence of vector and foreign DNA, to cut strand a little away from the centre of the palindrome sites, creates overhanging stretches/sticky ends.

1. ampR/ampicillin resistance genes, tetR/tetracycline resistance gene.

They help in identifying and eliminating non-transformants/non-recombinants and selectively permitting the growth of the transformants/recombinants.

2. Simpler process/less cumbersome, in the presence of chromogenic substrate recombinants are colourless, and non-recombinants are blue in colour.

1. Escherichia coli/E.coli.
2. ori.
3. amp^R is the marker gene that helps in identification and elimination of the non transformant growing in ampicillin medium/selectively permitting the growth of the transformant resistant to ampicillin // tet^R is the marker gene that helps in identification and elimination of the non transformant growing in tetracycline medium/selectively permitting the growth of the transformant resistant to tetracyline.

DNA fragments on the agarose gel are negatively charged molecules, and they move towards the anode (The fragments separate according to their size) The separated DNA fragments can be visualised after staining with ethidium bromide.

Followed by exposure to UV radiation Separated fragments are extracted from the gel by elution.

DNA from every tissue from an individual, shows the same degree of polymorphism and is heritable, hence very useful in DNA finger printing.

1. ori - origin of replication.
2. amp^R - ampicillin antibiotic resistant gene.
3. rop - gene to produce the proteins involved in the replication of the plasmid.

Separation/denaturation of two strands of two dsDNA, using two sets of primers/small chemically synthesised oligonucleotides complementary to regions of DNA and (thermostable) DNA polymerase/Taq polymerase, extension of the primers, by enzyme using nucleotides replicates the DNA and if the process of replication is repeated many times multiple copies of DNA are produced.

The following diagram can be considered in lieu of the explanation.

Application = Produces larger biomass leading to higher yields of desired protein/recombinant protein/processing large volume of culture/conversion of raw materials into specific product biologically.

1. Cuts at specific position within the DNA/cuts DNA at specific nucleotide/cuts at palindromic nucleotide sequence.
2. Separation of DNA fragments. (under the influence of electric field)
3. Helps in Identifying and eliminating non-transformants from transformants/selection of transformants.

1. Pst I/Pvu I/EcoR I/Cla I/Hind III/BamH I/Sal I/ Pvu II.
2. ori, rop.
3. amp^R/tet^R.

1. -AATTC / Sticky end.
2. -Ori / Origin of Replication.

2. Pallindromic sequence, because the sequence of base pairs reads same on the two strands when orientation of readigreadingpt the same.
3. PCR - Polymerase Chain Reaction, Importance - amplification of gene of interest (in vitro).

1. a = ampicillin, b = EcoR-I, c = Hind-III, d = tetracycline.
2. Insertion of alien gene into coding sequence of a-galactosidase, results into inactivation of the enzyme// (insertion inactivation), these colonies do not produce any colour (in the presence of chromogenic substrate) hence are identified as recombinant colonies.

Which is isolated from bacterium Thermus aquaticus, and is used to amplify DNA in vitro (by PCR),

It remains active even under high temperature at which DNA denatures.

1. Restriction enzymes EcoRI/Hind II,
2. They recognise and cut at a specific sit on DNA.

1. Restriction enzymes:

1. Restriction enzymes belongs to class of enzymes nucleases which breaks nucleic acids by cleaving their phosphodiester bonds.
2. Since Restriction endonucleases cuts DNA at specific recognition site, they are used to cut the donor DNA to isolate the desired gene.
3. The desired gene has sticky ends which can be easily ligated to cloning vector cut by same restriction enzymes having complementary sticky ends to form recombinant DNA.
4. An example is EcoR1 which is obtained from E.coli bacteria “R” strain which cuts DNA at specific palindromic Recognition site.

5‘ GAATTC 3‘

3‘ CTTAAG 5‘

2. Plasmids:

1. Plasmids are autonomous, extra chromosomal circular double stranded DNA of bacteria.
2. Since they are small and self replicating, they are used as cloning vectors in genetic engineering.
3. Some plasmids have antibiotic resistance genes which can be used as marker genes to identify recombinant plasmids from non recombinant ones.
4. The plasmids are cut and ligated with desired genes and transformed into host cell for amplification to obtain the desired products.
5. An example of artificial modified plasmids are pBR322 ( constructed by bolivar and rodriguez) or pUC (constructed at university at california).

1. Small volume cultures cannot yield appreciable quantities of products. To produce in large quantities, the development of bioreactors, where large volumes (100 - 1000 litres) of culture can be processed, was required. Thus, bioreactors can be thought of as vessels in which raw materials are biologically converted into specific products, individual enzymes, etc., using microbial plant, animal or human cells.
2. The most commonly used bioreactors are of stirring type. A stirred - tank reactors is usually cylindrical or with a curved base to facilitate the mixing of the reactor contents. The stirrer facilitates even mixing and oxygen availability throughout the bioreactor. The bioreactor has an agitator system, an oxygen delivery system and a foam control system, a temperature control system. pH control system and sampling ports so that small volumes of the culture can be withdrawn periodically.

1. DNA fragments are negatively charged molecules. Using this property of DNA it is forced to move towards an anode under an electric field through an agarose medium. The DNA fragment separates according to their size through sieving effect provided by agarose gel. Hence the smaller the fragment in size, farther it moves.
2. The DNA fragments after separation are treated with ethidium bromide followed by UV radiation exposure. This enables us to visualize the DNA fragments.

1. Ori-gene: The sequence from where replication start/ any piece of DNA when linked to this sequence can be made to replicate within the host cell, this sequence control the copy number of linked DNA.

2. Antibiotic resistance genes: Help in identifying and eliminating non transformant from transformant/ acts as selectable marker/ helps in ligation of alien DNA at recognition site (present in one of the two antibiotic resistance gene).

3. Rop: Codes for proteins, involved in the replication of plasmids.

Two features that the engineered vectors made to posses are:

1. ORI.
2. Selectable marker.

1. Denaturation: Double-helical DNA is denatured by providing high temperature (95-degree Celsius). DNA polymerase does not get degraded in such high temperatures. The DNA polymerase used in this reaction is thermostable and is isolated from the thermophilic bacteria, Thermus aquaticus (Taq).

2. Annealing: It is the step in which primers are annealed to single-stranded DNA templates. Two sets of primers are used. The temperature of the reaction mixture is lowered to 50-65°C for some seconds to allow annealing of primers. DNA polymerase extends the primer in 5' to 3' direction.

3. Extension: Replication of DNA occurs in vitro.

4. This cycle is repeated several times to generate up to 1 billion identical copies of the DNA.

• If a foreign DNA is ligated at the BamH I site of tetracyclin-resistance gene in the pBR 322, the recombinant plasmid will lose the tetracycline-resistance.
• The transformant can be selected out from the nontransformants by culturing on a medium containing ampicillin.
• The transformants are transferred to a culture medium containing tetracyclin; there they fail to grow but non-transformants grow in tetracyclincontaining medium as well as ampicillin-containing medium.
• Here, one antibiotic gene facilitates selection of transformants, while the other antibiotic gene gets inactivated by the insertion of alien DNA and facilitates selecting the recombinants.

• When an alien DNA or recombinant DNA is ligated within the coding sequence of an enzyme, the enzyme becomes inactivated; this phenomenon is called insertional inactivation.
• A recombinant loses the ability to produce a (blue) colour with a chromogenic substrate; hence, it can be distinguished from the non-recombinants, which produce blue-coloured colonies with the chromogenic substrate.
• This method is preferred to the antibiotic-resistance method as the latter is a cumbersome procedure that requires simultaneous plating on two plates having two different antibiotics.

1. Isolation of DNA.
2. Fragmentation of DNA by restriction endonucleases.
3. Isolation of desired DNA fragment.
4. Ligation of DNA fragment into vector.
5. Transferring recombinant DNA into host.
6. Culturing host cells at large scale.
7. Extraction of the desired product.

1. A - Denaturation process
2. B - Primers
3. C - Taq DNA polymerase. Tag polymerase is a thermostable enzyme, which remains active during the high temperature and induces denaturation of DNA,

1. The protein coded by a recombinant gene in the transgenic or genetically modified organism, is called a recombinant protein.
2. This can be harvested on a large scale by culturing the transgenic cells in large volumes in bioreactors.
3. The precautions include:

• The optimum temperature must be maintained.
• There should be foam control.
• Suitable pH must be provided.

1. A- Vector DNA,/ Plasmid DNA; B- Foreign DNA.
2. 5'- GAATTC - 3'; 3'-CTTAAG - 5'
3. DNA ligase.

• In gel electrophoresis, the molecules resolve according to their molecular size by the sieving effect provided by the gel.
• DNA being negatively charged, moves towards the anode in the electric field.
• Use of DNA isolated:

1. The DNA fragments may be used to construct recombinant DNA by joining them with cloning vectors.
2. The desired DNA fragments may be amplified, i.e. making multiple copies, by polymerase chain reaction (PCR).

• The restriction enzyme, ECORI recognises the palindromic sequence, shown below:

• It cuts the DNA strands a little away from the centre of the palindrome site, but between the same two bases on both the strands (indicated by the arrows)
• This leaves single-stranded stretches, called sticky ends, overhanging at the end of the strands.

• DNA ligase can catalyse hydrogen bond formation between the cut complementary parts of the vector DNA and foreign DNA.

• This enzyme is used to prevent unwanted self-ligation of vector DNA molecule.
• Sources: from bacteria (BAP), from calf intestine (CAP)

• Restriction endonucleases are used to cut the DNA strands of vector and the source/ foreign DNA at specific locations, containing the genes of our interest).
• When cut by the same restriction enzyme, the resultant DNA fragments have the same kind of sticky ends, which are easily joined by a DNA ligase.

• 'Ori' is the sequence of nucleotides in a DNA, where replication starts.
• When a piece of alien DNA is ligated to it, it replicates and forms multiple copies (cloning) within the host.

• Gel electrophoresis is used to separate the DNA fragments formed by the restriction digestion.
• The separated DNA fragments are isolated and used in biotechnological experiments.

• Taq polymerase.
• It is isolated from Thermus aquaticus bacterium.

• By treating bacteria with cold calcium chloride or lysozyme.
• Escherichia coli, Bacillus subtilis.

1. Microinjection.
2. Biolistics/ gene gun.
3. Heat-shock method.
4. Using disarmed pathogen vectors.

1. He would have loaded the samples at the end A. In the wells near the end A.
2.  

• The DNA fragments resolve or separate according to their size, through the sieving effect, provided by the agarose gel matrix.
• The smaller the DNA fragment, the farther it moves and vice-versa.

• He must have stained the DNA with ethidium bromide followed by exposure to UV-radiation.
• he DNA is visible as orange coloured bands.

• Vector is a DNA molecule that can carry a foreign/ desired DNA segment and replicates inside the host cell.
• pBR 322 vector.

• Genes encoding antibiotic resistance are useful as selectable markers because the normal E.coli cells do not carry resistance against any of these antibiotics.
• The ligation of alien DNA is carried out at a restriction site present in one of the antibioticresistance genes, say a foreign DNA is ligated at the BamHI site of tetracycline resistance gene in the vector pBR 322.
• The recombinant plasmids will lose tetracycline resistance due to insertional inactivation.
• The recombinants could be selected out from the non-recombinants by plating the transformants on ampicillin-containing medium.
• The transformants growing on ampicillin-containing medium are transferred to tetracycline-containing medium.
• The recombinants cannot grow on this medium, but non-recombinants will grow on this medium too.

• The DNA formed by combining DNA molecules from different sources/ genomes, is called a recombinant DNA.
• It shows features of those organisms from where the DNAs are isolated.
• The same restriction endonuclease is used to cut both vector DNA and the source DNA at specific locations: hence, the same kind of sticky ends are formed.
• DNA ligase joins together (end-to-end) the complementary cut counterparts by forming hydrogen bonds.

• Bioreactors are the large vessels in which the raw materials are biologically converted into specific products in large quantities, i.e. on commercial scale.
• The bioreactors provide optimum conditions of (i) pH, (ii) substrate concentration, (ii) mineral salts, (iv) vitamins, (v) temperature and (vi) oxygen.

1. Recombinant DNA Technology/ Genetic Engineering.
2. Three steps are:

• Isolation of human DNA with desirable gene.
• DNA segment is incorporated into bacterial plasmid to form recombinant DNA.
• Recombinant DNA is introduced in bacterial cell, which makes pistein directed by human DNA.

1. Any protein encoded gene when expressed in a heterologous host, it is called as recombinant protein.
2. For the large scale production of industrial products.
3. He is curious, inquisitive and having interest in science.

1. The DNA fragments resolve according to their size through the sieving effect of the agarose gel; hence the smaller fragments (in D) have moved farther than those (in C) which are comparatively larger.
2. B is the anode.
3. The matrix provides the sieving effect for the separation of DNA fragments according to their sizes.
4. The separated DNA fragments are stained by ethidium bromide followed by exposure to ultra violet radiation.

• Agrobacterium tumefaciens is a pathogen of several dicot plants.
• It is able to deliver a piece of DNA, called T-DNA into the plant cells and transform them into tumour cells; it dictates the host cells to synthesise its nutrients.
• The Tumour inducing (Ti) plasmid of this bacterium is modified and made non-pathogenic.
• Though it is non-pathogenic, it still has the capacity to deliver its Ti plasmid to the plants and hence, the genes of interest ligated to it; thus it acts as a suitable vector in biotechnology experiments.
• It has been used to transfer nematode-specific genes into tobacco plants, to make them resistant to the nematode, Meloidegyne incognitia, that attacks the roots of tobacco plant.

• Three types- Type I, II and III.
• Because they cut the DNA molecules at a specific site and generate fragments.

1. DNA sample that was loaded on the gel may have got contaminated with nuclease (exo- or endo- or both) and completely degraded.
2. Electrodes were put in opposite orientation in the gel assembly, i.e., anode towards the wells (where DNA sample is loaded).

Since DNA molecules are negatively charged, they move towards anode and hence move out of the gel instead of moving into the matrix of gel.

3. Ethidium bromide was not added at all or was not added in sufficient concentration and so DNA was not visible.
4. After staining with Ethidium bromide it was not observed under UV.

• The first letter of the name comes from the genus of the bacterium.
• The second and third letters come from the name of the species of the prokaryote cell from where it is isolated.
• The next letter comes from the strain of the prokaryote.
• The Roman Numbers following these four letters indicate the order in which the enzymes were isolated from that strain of the bacterium, e.g. EcoRI is isolated from E.coli, RY 13.

1. Positive terminal- ‘B’

Negative terminal- ‘A’

1. DNA is negatively charged. Because of its negative charge, DNA moves towards the positive electrode (anode).
2. The separated DNA fragments are separated by elution. The separated bands of DNA are cut out from the agarose gel and extracted from the gel piece.

1. A bioreactor is used for obtaining a desired gene product in large quantities by processing large volumes of cultures of microbial, plant or animal cells.
2. The stirring type of bioreactors (Simple stirred-tank bioreactor or sparged stirred-tank bioreactor) are commonly used.
3. The components of simple stirred-tank bioreactor include:

• A stirrer.
• Flat-bladed impeller.
• An oxygen delivery system.
• A foam control system.
• pH control system.
• Sampling port.

1. He would have loaded the samples near end A; in the wells.
2. The DNA fragments separate (resolve) according to their size through sieving effect provided by the agarose gel. Hence, the smaller the fragment size, the farther it moves.
3. After staining the DNA with ethidium bromide followed by exposure to UV radiations the DNA bands appear coloured.

1. Biotechnology deals with the techniques of using live organisms or enzymes from organisms to produce products and processes useful to human.
2. These are formed by integration of a segment of DNA of the bacterium Agrobacterium tumifaciens into the host DNA.
3. Rahul's uncle has a positive desire to know about latest discoveries and developments in the field of science.

1. EcoRl and BamHI as restriction enzymes.
2. ori and rop genes.
3. amp^R (an antibiotic resistant gene).

1. Selectable marker in the cloning vector helps to identify and eliminate the non-recombinants and to selectively permit the growth of recombinants.
2. Ori (origin of replication) is a sequence of DNA from where replication starts; any piece of alien DNA ligated to this can be made to replicate within the host cut and it determines the copy number too.
3. Rop codes for the proteins involved in the replication of the plasmid.

• The DNA fragments are separated by electrophoresis using agarose as the matrix.
• Since, DNA fragments are negatively charged, they move towards the anode under the electric field through the medium and separate/ resolve according to their size due to the sieving effect of agarose gel.
• The separated fragments can be viewed by staining the DNA with ethidium bromide followed by exposure to UV radiation.
• Elution is the process in which the separated bands of DNA are cut out from the gel and extracted.

• Polymerase Chain Reaction.
• Denaturation, Annealing and Extension.

1. It helps in identifying or selecting transformants and eliminating non-transformants by Selectively permitting the growth of the trarsformants.
2. This is a sequence from where replication starts and any piece of DNA, when linked to this sequence can be made to replicate within the host cells. This sequence is also responsible for controlling the copy number of the linked DNA.
3. Rop codes for the protein involved in the replication of the plasmid.

1. Microinjection: In this method, the recombinant DNA is directly injected into the nucleus of an animal cell.

2. Gene gun/ Biolistics: In this method, used for plant cells, the cells are bombarded with high velocity microparticles of gold or tungsten coated with DNA.

3. Heat shock method: In this method, the rDNA is forced into the competent cell by incubating the cell with rDNA on ice followed by placing them briefly at 42°C (heat shock) and then putting them back on Ice.

1. Isolation of DNA.
2. Fragmentation of DNA by restriction endonucleases.
3. Isolation of a desired DNA fragment.
4. Amplification of the desired DNA fragment.
5. Ligation of the DNA fragment into a vector.
6. Transferring the recombinant DNA into the host.
7. Culturing the host cells and extraction of the desired gene product on a large scale.
8. Downstream processing, i.e. separation and purification.

• A palindrome in DNA is a sequence of base pairs that reads the same on the two strands, when orientation of reading is kept the same.
• The palindromic sequence, which is recognised by the restriction enzyme, EcoRI is given below, along with the sites where it cuts the DNA strands.

• It is seen that the restriction enzyme cuts the two strands between the same two bases, a little away from the centre of the palindrome sequence.

1. Plasmid DNA: It is often used for constructing recombinant DNA, by ligating the gene of interest with it; it is used as the cloning vector.

2. Recognition sequences: These are the sequences of base pairs in DNA, which a restriction enzyme recognises and cuts the DNA
3. Gel electrophoresis: It is used to separate the DNA fragments, which separate/ resolve according to their size through the sieving effect of the gel; the DNA is isolated from the gel by the process of elution.

• To make E. coli cells competent to take up DNA, they should be treated with divalent cations or calcium.
• In 1970, Mandel and Higa found that E.coli cells become competent to take up DNA when they are suspended in cold calcium chloride.

1. Cloning sites are the recognition sites in the vector DNA, for the restriction enzymes; these are the sites where specific restriction enzymes cut the DNA strands.
2. When an alien DNA is ligated at a particular cloning/ recognition site, the recombinant plasmid loses its function of the gene, where the recognition sequence is present; for example if an alien DNA is ligated at a recognition site of an antibiotic resistance gene in E.coli, the recombinant will lose the resistance to the particular antibiotic.
3. The restriction sites in pBR 322 are:

• BamH I site of tetracyclin-resistance gene.
• Sal I site of tetracyclin-resistance gene
• Pst I site of ampicillin-resistance gene.

E. coli cloning vector pBR322 showing restriction sites (HindIII, EcoRI, BamHI, SalI, PvuII, PstI, ClaI), ori and antibiotic resistance genes (ampR and tetR). rop codes for the proteins involved in the replication of the plasmid.

2. Origin of replication is responsible for controlling the copy number of the DNA sequence inserted.

1. BamHI should be used, as restriction site for this enzyme is present in tet^R region.
2. PvuI will not be used, as restriction site for this enzyme is present in amp^R region (not in tet^R). EcoRI will not be used, as restriction site for this enzyme is not present in selectable marker tet^R.

1. The vector DNA is cut at a particular restriction site using a restriction enzyme (to cut the desired DNA segment). The alien DNA is then linked with the plasmid DNA using an enzyme called ligase to form the recombinant vector.
2. A restriction enzyme recognizes and cuts the DNA at a particular sequence, called recognition site. If more than one restriction enzymes are present, they will generate several segments and will complicate gene cloning.

1. 5' - GAATTC - 3'

2. EcoRI is the restriction endonuclease that recognizes this palindrome.
3. When the restriction enzyme cuts the DNA strands a little away from the centre of the palindromic sequence, between the same two bases on both the strands, sticky ends are produced.

• The stickiness of the ends facilitates the action of the enzyme DNA-ligase.

1. In the above bacterial cell, i.e. A is plasmid B is chromosomal DNA. Plasmid is used as a vector in rDNA technology.
2. If the enzyme EcoRI acts on both linear DNA and plasmid DNA, each having three recognition sites, the restriction enzyme will generate 3 fragments from plasmid DNA (as it is circular) and 4 fragments from linear DNA.

1. Salmonella tymphimurium was used in the lust instance of the construction of artificial recombinant DNA molecule because it has a plasmid which has an autonomously replicating circular extrachromosomal DNA- It a. Also has an antibiotic resistance gene.
2. Stanley Cohen and Herbert Boyer accomplished this in 1972. Antibiotic resistant gene was isolated using restliction enzymes and introduced into the plasmid of the bacterium, i.e. Salmonella tymphimurium. Later the recombinant plasmid was introduced into the bacterium-E coli so that it could make copies of gene.

• Taq polymerase is a thermostable enzyme that can withstand the high temperature used in the denaturation step, whereas ordinary DNA polymerase cannot withstand high temperature.
• It is obtained from the bacterium, Thermus aquaticus.

2. PCR is used for gene amplification, i.e., to make multiple copies of a gene or a DNA segment.

1. The recombinant/ transformaot can be selected out from the non-recombinants/ non-transformants by Plating the transformants on ampicilin-containing medium. The transformants will grow in it, while the non-transformants will not grow.
2. It acts as a selectable marker.

1. DNA is a hydrophilic molecule that cannot pass through cell membrane; so the bacterial cells must be made competent to take up the DNA.
2. The bacterial cells are made competent by treating them with a specific concentration of a divalent cation, such as calcium; this increases the efficiency with which DNA enters the bacterium.

1. Biolistics/ Gene gun.
2. Micro-injection.

1. Palindromic sequence and the site where EcoRI cuts it (indicated by the arrows).

2. Sticky ends

1. Origin of replication (ori): This is a sequence from where replication starts and any piece of DNA, when linked to this sequence can be made to replicate within the host cells. This sequence is also responsible for controlling the copy number of the linked DNA.
   
2. Selectable marker: It helps in identifying or selecting transformants and eliminating non-transformants by selectively permitting the growth of the transformants. 'Transformation is a procedure, through which a piece of DNA is introduced into the host bacterium. Normally, the genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, tetracycline, kanamycin, etc., are considered as useful selectable markers for E. coli. The normal E. coli cells do not carry resistance against any of these antibiotics.
   
3. Cloning (recognition) sites: These are generally required to link the foreign or alien DNA with the vector. For this, the vector requires very few or single recognition sites for commonly used restriction enzymes. If more than one recognition sites is present within the vector, it will generate several fragments that will lead to more complication in gene cloning.
   

1. Origin of replication (ori): This is a sequence from where replication starts and any piece of DNA, when linked to this sequence can be made to replicate within the host cells. This sequence is also responsible for controlling the copy number of the linked DNA.

2. Selectable marker: It helps in identifying or selecting transformants and eliminating non-transformants by selectively permitting the growth of the transformants. 'Transformation is a procedure, through which a piece of DNA is introduced into the host bacterium. Normally, the genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, tetracycline, kanamycin, etc., are considered as useful selectable markers for E. coli. The normal E. coli cells do not carry resistance against any of these antibiotics.

3. Cloning (recognition) sites: These are generally required to link the foreign or alien DNA with the vector. For this, the vector requires very few or single recognition sites for commonly used restriction enzymes. If more than one recognition sites is present within the vector, it will generate several fragments that will lead to more complication in gene cloning:

1. Exonucleases: remove nucleotides from the ends of the DNA (either 5' or 3’) in one strand of duplex.
2. Endonucleases: make cuts at specific position within DNA. The first restriction endonuclease named Hind II was isolated by Smith Wilcox and Kelley in 1968.

1. It is used to during IDNA for production of newer and desired DNA.
2. It is used for DNA sequencing.
3. It is used in DNA fingerprinting.

• Ethidium bromide used for staining DNA.
• Stained DNA becomes orange.