Biotechnology: Principles and Processes
What is the cell that receives a recombinant gene called ? (All India 2019)
Host cell is the cell that receives a recombinant gene.
Write the specific point in the palindrome and the bond that is cut by Eco Rl. (All Indio 2019)
Restriction endonuclease Eco RI cuts the DNA at the sequence known as palindromic sequence, i.e. GAATTC. The type of bond broken by Eco RI is phosphodiester bond between the G and A bases of the palindrome. This site is known as restriction site.
Why do DNA fragments move towards the anode during gel electrophoresis ? (Delhi 2011c)
DNA fragments are negatively charged molecules and hence, they move toward the positive charged anode during gel electrophoresis.
Suggest a technique to a researcher who needs to separate fragments of DNA. (Delhi 2016)
Mention the use of gel electrophoresis in biotechnology experiments. (Outside Delhi 2016C)
Gel electrophoresis is used to separate the fragments of DNA that were cut by restriction endonucleases.
Name the technique that is used to alter the chemistry of genetic material (DNA, RNA) to obtain desired result. (Delhi 2016C)
The technique used to alter the chemistry of genetic material to obtain desired result is called genetic engineering.
Why is it not possible for an alien DNA to become part of a chromosome anywhere along its length and replicate normally? (All India 2014)
The alien DNA itself cannot multiply and replicate but requires a specific sequence for initiating its replication called origin of replication in a chromosome ori acts as the starting point of replication as they it aids in binding of DNA polymerase.
Mention the type of host cells suitable for the gene guns to introduce an alien DNA. (Delhi 2014)
Plant host cells are suitable for the gene guns to introduce an alien DNA.
Write the two components of first artificial recombinant DNA molecule constructed by Cohen and Boyer. (Foreign 2014)
The two components of first artificial recombinant DNA molecule constmcted by Cohen and Boyer are
- Antibiotic resistance gene
- Plasmid of Salmonella typhimurium
Name the host cells in which microinjection technique is used to introduce an alien DNA. (Foreign 2014)
The microinjection technique is usually carried out in animal cell to inject alien DNA directly into the nucleus.
Name the material used as matrix in gel electrophoresis and mention its role. (All India 2014C)
The material used as matrix in gel electrophoresis is agarose.
This agarose gel acts as a sieve to separate the DNA fragments according to their size.
Write any four ways used to introduce a desired DNA segment into a bacterial cell in recombinant technology experiments. (All India 2013)
Ways to introduce desired DNA into bacterial cell are
- disarmed pathogen vectors
- treatment of host cell by bivalent cation such as calcium
- biolistic or gene gun
How can retroviruses be used efficiently in biotechnology experiments inspite of them being disease causing? (All India 2013C)
Retroviruses can be used in biotechnology experiments after being disarmed, i.e. removal of virulent gene so that it is unable to cause infection in hosts they are transferred to.
State what happens when an alien gene is ligated at Pvu I site of pBR322 plasmid. (All India 2013C)
An alien gene ligated at Pvu I site of pBR322 plasmid cause the transformant cell to loose the ampicillin-resistance as ampR gene becomes non-functional. Thus, the recombinant does not grow in the presence of ampicillin.
Why is ‘plasmid’ an important tool in biotechnology experiments? (All India 2013C)
Plasmid have the ability to replicate within bacterial cells independently of chromosomal DNA. They have high copy number, therefore an alien DNA ligated to it, will have equal copy number as that of plasmids. So, it is used as a vector in gene cloning experiments and thus acts as an important tool in biotechnology.
State what happens when an alien gene is ligated at Sal I site of pBR322 plasmid. (Delhi 2013C)
When an alien gene is ligated at Sal I site of tetracycline resistance gene in the vector pBR322, the recombinant plasmid lose its tetracycline resistance.
Mention the uses of cloning vector in biotechnology. (Delhi 2011)
Uses of Cloning Vector in Biotechnology
- Helps in linking the foreign/alien DNA with the host’s DNA.
- Helps in the selection of recombinants from the non-recombinants.
Biotechnologists refer to Agrobacterium tumefaciens as a natural genetic engineer of plants. Give reasons to support the statement. (All India 2011)
Agrobacterium tumefaciens is a pathogen of several dicot plants. It is used as a natural genetic engineer because it is able to deliver a piece of its DNA (called T-DNA) to transform normal plant cells into tumour cells. It direct the tumour cells to synthesise the chemicals required by the pathogen.
Why is it essential to have a selectable marker in a cloning vector? (All India 2011)
Selectable marker in cloning vector helps in identifying and selecting the recombinants and eliminating the non-recombinants.
In the year’1963, two enzymes responsible for restricting the growth of bacteriophage in E. coli were isolated. How did enzymes act to restrict the growth of the bacteriophage? (All India 2011C)
How is the action of exonuclease different from that of endonuclease? (All India 2010)
Two enzymes responsible for restricting the growth of bacteriophage in E. coli are Exonucleases Remove nucleotides from the ends of DNA. Endonucleases Cut DNA at specific points.
What is the host called that produces a foreign gene product? What is this product called? (Foreign 2010)
Transgenic organisms or genetically modified organisms are the host that produces a foreign gene product. Recombinant proteins are the products formed by these host cells.
ß-galactosides enzyme is considered a better selectable marker. Justify the statement. (Delhi 2019)
Coding sequence of p-galactosidase is a better maker, as the recombinants and non-recombinants are differentiated on the basis of their ability to produce colour in the presence of a chromogenic substrate, while the selection of recombinants due to inactivation of antibiotic resistant gene is a tedieus and time taking process to grow them simultaneously on two antibiotics containing media.
Explain the mode of action of Eco RI. (Delhi 2016C)
What is Eco RI? How does Eco RI differ from an exonuclease? (Delhi 2015C)
How does a restriction nuclease function? Explain. (All India 2014)
Restriction nucleases function by inspecting the length of DNA sequence and then binding to specific recognition sequences and cutting the strands at sugar phosphate backbones.
These nucleases are of two types depending on their mode of action
- Restriction exonucleases cut sequences at terminal ends of DNA.
- Restriction endonucleases, e.g. Eco RI, cut between the two bases of recognition sequence.
Write the role of ori and restriction site in a cloning vector pBR322. (Delhi 2014)
Ori is a sequence of DNA from where replication starts. Any piece of DNA that needs to replicate in the host cell has to be linked to it.
Restriction site is the recognition site made of palindromic sequence for restriction enzymes.
State how was Agrobacterium tumefaciens been made as a useful cloning vector to transfer DNA to plant cells. (Delhi 2014)
Agrobacterium infects plant tissues by transferring its plasmid T-DNA to the plant genome. This property of Agrobacterium was exploited to transfer desired gene to a particular plant. The desired gene is inserted in the plasmid T-DNA and the engineered Agrobacterium is allowed to infect that particular plant. Hence, it acts as a natural cloning vector.
Explain with the help of a suitable example the naming of a restriction endonuclease. (Delhi 2014)
Naming of restriction endonuclease involves the following rules
- The first letter of the enzyme comes from the genus and next two letters from species of the prokaryotic cell from where enzymes are extracted.
- The Roman numbers following the name shows the order in which the enzymes were isolated from the bacterial strain. For example, Eco RI is derived from Escherichia coli RY 13, Hind II from Haemophilus influenzae Rd, etc.
How are sticky ends formed on a DNA strand? Why are they called so? (Delhi 2014)
Sticky ends on DNA are formed by the action of enzymes restriction endonucleases. These enzymes cut the strand of DNA a little away from the centre of the palindrome sequence between the same two bases on both the strands.
This results in single-stranded stretches on both the complementary strands at their ends. These overhanging stretches are called sticky ends as they form hydrogen bonds with the complementary base pair sequences.
How is insertional inactivation of an enzyme used as a Selectable marker to differentiate recombinants from non-recombinants? (Foreign 2014)
The insertional activation of an enzyme, e.g. ß-galactosidase occurs by inserting the desired gene in the coding region of enzyme. It results in inactivation of ß-galactosidase gene in recombinants. Due to this, the recombinant or transformed hosts are unable to produce any colour when grown on chromogenic substrate. Thus, ß-galactosidase acts as a selectable marker to differentiate recombinants from non-recombinants.
Explain palindromic nucleotide sequence with the help of a suitable example. (Foreign 2014)
The palindromic nucleotide sequence is the sequence of base pairs in DNA that reads the same on both the complementary strands of DNA, with same orientation of reading.
Why are molecular scissors called so? Write their use in biotechnology. (Foreign 2014)
Molecular scissors are so called because they cut DNA at specific sequences between base pairs. Molecular scissors or restriction enzymes cut DNA at desired sequences and generate sticky ends that facilitate the cut DNA to join with host genome or vector DNA. They play an important role in genetic engineering or biotechnology. It is because with the help of these enzymes we can cut the desired gene and introduce into vectors for expression.
Why is making cells competent essential for biotechnology experiments? List any two ways by which this can be achieved. (Delhi 2014C)
Why and how bacteria can be made ‘competent’? Delhi 2013
Since, DNA molecules are hydrophilic, they cannot pass through cell membranes. For recombinant DNA to be integrated into vector or host genome, it is necessary for the DNA to be inserted in the cell. Therefore, making the host cells competent is necessary in biotechnology experiments.
The two ways by which cells can be made competent to take up DNA are
- Chemical action The host cell is treated with a specific concentration of divalent cation, i.e. calcium increases the pore size in the cell membrane. DNA is then incubated with treated bacterial cell at 42°C, thereby increasing the efficiency of DNA to enter it through pores in cell wall.
- Heat shock treatment Incubating the cells with recombinant DNA on ice, followed by brief treatment of heat at 42°C and again putting them back on ice.
How is an exonuclease functionally different from an endonuclease? Give an example of any two endonucleases other than Sal I. (Delhi 2013C)
Exonucleases are the enzymes which cleave base pairs of DNA at their terminal ends and act on single-strand of DNA or gaps in doublet stranded DNA. While, endonuclease cleaves DNA at any point except the terminal ends and can make cut on one strand or on both the strands of double-stranded DNA, e.g. Eco R1 and Hind II.
Explain the work carried out by Cohen and Boyer that contributed immensely in biotechnology. (Delhi 2012)
Stanley Cohen and Herbert Boyer constructed the first artificial recombinant DNA (rDNA) molecule.
They isolated the antibiotic-resistance gene by cutting out a piece of DNA from a plasmid with the help of restriction enzyme and linked it to a native plasmid of Salmonella typhimurium with the help of DNA ligase.
(i) A recombinant vector with a gene of interest inserted within the gene of α-galactosidase enzyme is introduced into a bacterium. Explain the method that would help in selection of recombinant colonies from non-recombinant colonies.
(ii) Why is this method of selection referred to as insertional inactivation? (All India 2012)
(i) The recombinant colonies can be differentiated from non-recombinant colonies by their inability to produce colour in the presence of a chromogenic substrate.
The recombinants do not produce any colour, while the non-recombinants produce a blue colour with chromogenic substrate in the medium. It occurs because of the presence of α-galactosidase in former and its absence in latter cells.
(ii) The enzyme α-galactosidase becomes inactivated on insertion of recombinant DNA, within the coding sequence of enzyme. Thus, the method is called insertional inactivation.
State the role of UV-light and ethidium bromide during gel electrophoresis of DNA fragments. (Delhi 2012c)
DNA fragments are observed only after staining with ethidium bromide followed by their exposure to UV radiation. This gives bright orange colour to DNA fragments.
Explain giving reasons why an alien piece of DNA needs to be integrated to a specific sequence of host DNA for its cloning. (All India 2011)
Refer to Answer No. 6
List the key tools used in recombinant DNA technology. (Delhi 2011)
Key tools used in recombinant technology are restriction enzymes, polymerases, ligases, cloning vectors and competent host organism or cells.
Explain the role of Ti plasmids in biotechnology. (Delhi 2011)
The Ti-plasmid isolated from Agrobacterium is responsible for the natural transformation of plant cells into tumours. So, it is modified into a non-pathogenic vector but still is able to deliver the DNA.
This disarmed plasmid of Agrobacterium is used as a vector for the transformation of plant cells, thus plays an important role in biotechnology.
How are recombinant vectors created? Why is only one type of restriction endonuclease required for creating one recombinant vector? (Foreign 2011)
Creation of recombinant vectors Vector DNA is cut at a particular restriction site by a restriction enzyme. The alien DNA is then linked with the vector DNA using enzyme ligase to form the recombinant vector.
A restriction enzyme recognises and cuts the DNA at a particular sequence called recognition site. The same restriction enzyme is used for cutting the DNA segment from both the vector and the other source, so as to produce same sticky ends in both DNA molecules to facilitate their joining. Question
Study the diagram given below and answer the following questions.
(i) Why have DNA fragments in band D moved farther away in comparison to those in band C? (ii) Identify the anode end in the diagram. (iii) How are these DNA fragments visualised? (Foreign 2011)
Answer: (i) In band D, DNA fragments are smaller than those on band C. The fragments separate according to their size through the sieving effect provided by the gel. So, the smaller fragments move farther away than the larger ones. (ii) B is anode end in the diagram as DNA fragments are moving towards this end. (iii) Gel containing DNA fragments are stained with ethidium bromide and exposed to UV radiation. Orange colour bands of DNA become visible.
Question 40. A recombinant DNA is formed when sticky ends of vector DNA and foreign DNA join. Explain how the sticky ends are formed and get joined? (All India 2010)
Answer: Refer to Answer No. 26.The sticky ends are joined via complementary factor of two polynucleotide strands bases and by the action of enzyme, DNA ligase.
Question 41. Explain the action of the restriction endonuclease Eco RI. Foreign 2010 Or (i) Illustrate the recognition sequence of Eco RI and mention what such sequences are called? (ii) How does restriction endonuclease act on a DNA molecule? (All india 2010c)
Answer: Restriction endonuclease Eco RI cuts the DNA strands a little away from the centre of the palindromic sequence, but between the same two bases on the two strands, i.e. G and A, on both the strands. The site of action of enzyme in palindrome sequence GAATTC is called recognition site.
(i) Due to this, single-stranded portions called sticky ends, overhang at the end of each strand. (ii) Because of the stickiness, they easily form hydrogen bonds with their complementary counterparts.
Question 42. How are the DNA fragments separated by gel electrophoresis visualised and separated for use in constructing recombinant DNA? (Foreign 2010)
Answer: The separated DNA fragments by gel electrophoresis are stained with ethidium bromide.
· By the exposure to UV radiation, the separated DNA fragments become visible as orange- coloured bands.
· The separated bands of DNA are cut out from the agarose gel and DNA is extracted from these gel pieces and this process is called elution.
Question 43. Expand ‘BAC’ and ‘YAC’. What are they and what is the purpose for which they are used? (All India 2019)
Answer: ‘BAC’ stands for Bacterial Artificial Chromosome and ‘YAC’ stands for Yeast Artificial Chromosome. These are vectors used in cloning DNA. For sequencing the total DNA from cell, the DNA is isolated and converted into relatively smaller size as fragments. DNA fragments are cloned in suitable host using specialized vectors, such as BAC and YAC. Fragments of DNA are then sequenced by automated DNA sequences.
Question 44. Explain how ‘sticky ends’ are obtained in a DNA segment. Write their importance in DNA technology year. (All India 2019)
Answer: Refer to Answer No. 26.
Role of the sticky ends The sticky ends are produced from hydrogen bonds with their complementary cut counterparts. The stickiness of the ends facilitates the action of the enzyme DNA ligase which helps to region the cut DNA.
Question 45. (i) Mention the importance of gel-electrophoresis in biotechnology. (ii) Explain the process of this technique. (All India 2019) Answer: (i) DNA fragments formed by the use of restriction endonucleases are separated by gel-electrophoresis. (ii) DNA fragments are negatively charged molecules. Thus, they move towards the positive charged anode under electric field through the medium. The smaller fragments move farther in the gel as compared to larger fragments due to the sieving effect of gel.
Question 46. How does p-galactosidase coding sequence act as a selectable marker? Why is it a preferred selectable marker to antibiotic resistance genes? Explain. (All India 2019) Answer: Selectable marker It helps in indentifying or selecting transformants and eliminating non-transformants and selectively permit the growth of the transformants.
ß-galactosidase acts as a selectable marker by inducing the property of insertional inactivation in transformed cells. In this process the recombinants and non-recombinants are differentiated on the basic of colour production in the presence of chromogenic substrate. A recombinant DNA is inserted within the coding sequence of an enzyme ß-galactosidase which results into inactivation of the enzyme. Therefore, the bacterial colonies having inserted plasmid, show no colouration while, thoseb without plasmid show blue colour.
Question 47. Give reason why (i) DNA cannot pass into a host cell through the cell membrane. (ii) Proteases are added during isolation of DNA for genetic engineering. (iii) Single cloning site is preferred in a vector. (All India 2019) Answer: (i) Hydrophobic molecules can diffuse through the lipid bilayer of the plasma membrane and not hydrophilic molecules. DNA being hydrophilic with the sugar-phosphate backbone, cannot pass through the cell membrane.
(ii) Proteases catalyze the breakdown of proteins present in the solution to its component amino acid. If the proteins are not removed from DNA preparation then, they could interfere with any downstream treatment of DNA (such as action of restriction endonuclease, DNA ligase, etc).
(iii) Single cloning sites are preferred as the the cloning of a sequence at more than one recognition sites within the vector would generate several fragments leading to complication in gene cloning.Question 48. Describe the formation of recombinant DNA by the action of Eco RI. (Delhi 2019) Or Prepare a flow chart information of recombinant DNA by the action of restriction endonuclease enzyme Eco RI. (Foreign 2015) Answer:
Role of Eco RI in Recombinant DNA (In detail) Refer to Answer No. 41.
Explain the roles of the following with the help of an example each in recombinant DNA technology.
(i) Restriction enzymes
(ii) Plasmids 2018
(i) The restriction enzymes are known as molecular scissors. These enzymes belong to a larger group of enzymes called nucleases, which are of following two types
- Exonucleases Those, who remove nucleotides from the ends of the DNA (either 5′ or 3′) in one strand of duplex.
- Endonucleases Those, who make cuts at specific position within the DNA. Each restriction endonuclease functions by ‘inspecting’ the length of a DNA sequence.
Role of Restriction Enzyme in Recombinant DNA Technology The restriction endonucleases are used to cut plasmid DNA as well as foreign DNA at desired sites. The foreign DNA is then inserted into plasmid DNA and the plasmid takes the foreign DNA into the desired host organism.
Example The first discovered restriction enzyme is Hind II. It was isolated from Haemophilus influenzae. always cuts DNA at 5 GT (Pyrimidine T Or C (Purine A or G) AC3′ and 3’CA (Purine A or G) (Pyrimidine T or C) TG5′.
produces DNA segments with blunt ends.
(ii) Plasmids These are extrachromosomal, self-replicating, double-stranded, closed and circular DNA molecules. It is found only in bacteria and few yeast cells.
Role of Plasmid in DNA Recombinant Technology These are used as vectors to carry the desired gene (foreign genes) into the desired host organisms.
For example, pBR322 is widely used plasmid vector. In its name P signifies plasmid, B is from Boliver and R is from Rodriguez. Boliver and Rodriguez were two scientists who developed pBR322 in 1977. This plasmid has genes for resistance against ampicillin and tatracycline. They have restriction sites for enzymes like PvuI, Pst I, Sal I, Bam HI.
Explain the role(s) of the following in biotechnology
(i) Restriction endonuclease
(iii) Selectable markers in pBR322 (Delhi 2017)
(i) Restriction endonucleases These are the bacterial enzymes that cut dsDNA into fragments after recognising and binding to the specific nucleotide sequences, known as recognition site. These enzymes are used to form recombinant molecules of DNA, composed of DNA from different sources.
(ii) Gel-electrophoresis is the technique which allows the separation and visualisation of fragments of DNA on an agarose gel matrix. Since, the DNA fragments are negatively charged molecules, they separate and move towards the anode (+ ve) under the influence of an electric field. DNA fragments are separated on the basis of their size through the sieving effect provided by the gel.
(iii) Selectable markers in pBR322 help in identification and selection of transformants. pBR322, an E. coli cloning vector has two antibiotic resistance genes, i.e. for ampicillin and tetracycline, which act as selectable marker. When a foreign DNA is ligated at the site of tetracycline resistance (tetR) gene in pBR322, the recombinant plasmid lose tetracycline resistance due to insertional inactivation of foreign DNA, but can still be selected out from non-recombinants by placing the transformants on ampicillin containing medium. The transformants growing on ampicillin containing medium are then transferred on tetracycline containing medium. The recombinants grow on ampicillin containing medium but not on tetracycline one whereas non-recombinants grow on both the media.
(i) Explain the significance of palindromic nucleotide sequences in the formation of recombinant DNA.
(ii) Write the use of restriction endonuclease in the above process. (All India 2017)
(i) Palindromic nucleotide sequences in the DNA are group of letters that form the same words when read both forward and backward. For example, the following sequence reads the same on the two strands in 5′ → 3’direction as well as 3′ → 5’direction.
5’— GAATTC —3′
Significance These sequences act as recognition sites which are recognised by specific restriction endonucleases.
(ii) Use of restriction endonuclease During recombinant DNA formation, these enzymes recognise and make a cut at specific positions within the DNA and vector. Due to this function, restriction endonucleases are also called as molecular scissors.
(i) Name the selectable markers in the cloning vector pBR322. Mention the role they play.
(ii) Why is the coding sequence of an enzyme p-galactosidase a preferred selectable marker in comparison to the ones named above? (All India 2016)
(i) Selectable markers in cloning vector pBR322 are ampicillin and tetracycline antibiotic resistance gene. They help in the selection of transformants and eliminating the non-transformants.
(ii) The selection of recombinants due to inactivation of antibiotics is a difficult process and requires simultaneous plating on two plates having different antibiotics. Thus, enzyme p-galactosidase is preferred as a selectable marker as it allows to differentiate non-recombinants from recombinants easily by insertional inactivations technique.
(i) Why must a cell be made ‘competent’ in biotechnology experiments? How does calcium ion help in doing so?
(ii) State the role of ‘biolistic gun’ in biotechnology experiments. (All India 2016)
(i) Refer to Answer No. 30.
(ii) Biolistic guns or gene guns are used to bombared rDNA loaded on gold or tungston particles with high velocity into host cells. In this way, the rDNA is delivered to the desired host cells.
How does Agrobacterium tumefaciens act as a suitable vector in the biotechnological experiments? Site an example where it has been successfully used as a vector. (Outside Delhi 2016C)
Refer to Answer No. 24.
Agrobacterium has been used as vector to introduce a gene from Tobacco Mosaic Virus (TMV) into tobacco plants.
Describe a palindrome with the help of an example. (Delhi 2016C)
Refer to Answer No. 51 (i).
(i) Why was a bacterium used in the first instance of the construction of an artificial recombinant DNA molecule?
(ii) Name the scientists who accomplished this and how. (Delhi 2016C)
(i) A bacterium Salmonella typhimurium was used in the first instance of construction of artificial recombinant DNA molecule because of the possibilities of linking a gene encoding antibiotic resistance with a native plasmid of the bacterium. This was made possible by the availability of restriction enzymes and the enzyme DNA ligase.
(ii) Stanley Cohen and Herbert Boyer accomplished this in 1972 by isolating the antibiotic resistance gene by cutting out a piece of DNA from a plasmid which was responsible for conferring antibiotic resistance. The cutting of DNA at specific sites was possible with the availability of restriction enzymes. The cut DNA was linked with the plasmid DNA using the enzyme DNA ligase. The plasmid DNA acts as vector to transfer the piece of DNA attached to it.
State the functions of the following in the cloning vector pBR322
(ii) rop and
(iii) Hind III sites (All India 2015C)
(i) Refer to Answer No. 23. (ori)
(ii) rop in pBR322 encodes for protein involved in plasmid replication.
(iii) Hind III is a restriction site in pBR322, where Hind III endonuclease makes a cut for the introduction of foreign DNA segment.
Name and explain the technique used for separating DNA fragments and making them available for biotechnology experiments. (Foreign 2015; All India 2014)
DNA fragments formed by the use of restriction endonucleases are separated by gel electrophoresis.
(i) DNA fragments are negatively charged molecules. Thus, they move towards the positive charged anode under electric field through the gel medium.
(ii) DNA fragments separate according to their size due to sieving effect of agarose gel.
(iii) The separated DNA fragments can be viewed by staining the DNA with ethidium bromide followed by exposure to UV radiation.
(iv) The separated bands of DNA are cut and extracted from gel piece. This is known as elution.
Draw a schematic diagram of the E. coli cloning vector pBR322 and mark the following in it
(iii) Ampicillin resistance gene
(iv) Tetracycline resistance gene
(v) Restriction site Bam HI
(vi) Restriction site Eco RI (Delhi 2015)
Draw a schematic sketch of pBR322 plasmid and label the following in it
(i) Any two restriction sites
(ii) ori and rop genes
(iii) An antibiotic resistant gene. (Delhi 2012)
E. coll cloning vector pBR 322.
Refer to figure 11.3 on page no. 278.
(i) Draw schematic diagrams of segments of a vector and a foreign DNA with the sequence of nucleotides recognised by Eco RI.
(ii) Draw the vector DNA segment and foreign DNA segment after the action of Eco RI and label the sticky ends produced. (Delhi 2014C)
(i) Refer to figure 11.1. on page no. 276.
(ii) Refer to figure 11.1 on page no. 276.
What are ‘cloning sites’ in a cloning vector? Explain their role. Name any two such sites in pBR322. (All India 2014C)
The cloning sites contain the specific unique recognition sequence for a particular restriction enzyme, so as to link the foreign DNA with the vector DNA and thus, create a recombinant DNA molecule.
These sites are important for joining the DNA fragments of vector and alien DNA. Multiple recognition sequences for a particular restriction enzyme within a DNA or vector complicate the process of gene cloning. The two cloning sites in pBR322 are Bam HI for tetracycline resistant gene and Pvu I for ampicillin resistant genes.
(i) Explain the basis on which the gel electrophoresis technique works.
(ii) Write any two ways the products obtained through this technique can be utilised. (Delhi 2013C)
(i) Refer to Answer No. 50 (ii).
(ii) Products obtained via gel electrophoresis can be utilised in following ways
(a) To construct a recombinant DNA molecule by joining them with cloning vector.
(b) For amplification of desired segment using Polymerase Chain Reaction (PCR).
How are the following used in biotechnology ?
(i) Plasmid DNA
(ii) Recognition sequence
(iii) Gel electrophoresis (All India 2011c)
(i) Plasmid DNA It is used for constructing recombinant DNA, by ligating the alien piece of DNA with it. It is used as a cloning vector and helps in the selection of recombinants from non-recombinants.
(ii) Recognition sequences These are specific base sequences of DNA, where restriction enzyme cuts the DNA. They are utilised to extract the desired gene or fragments from DNA molecules.
(iii) Gel electrophoresis It is a technique, used to separate the DNA fragments according to their size through seiviirg effect of the gel.
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