Chapter-Recombinant DNA Technology

(a) Ligation absolutely requires the presence of a 5’ phosphate group at the DNA site to be ligated. If this phosphate group is removed, this DNA cannot be ligated. Alkaline phosphatase is an enzyme used to remove the phosphate group from the 5’ end of a DNA molecule, leaving a free 5’ hydroxyl group. Thus DNA cannot be ligated and this prevents self-annealing. So, the other three options are wrong.

(d) The application of electric current to a living surface (as the skin or plasma membrane of a cell) in order to open pores or channels through which a biologic material may pass (as a drug or DNA) is called electroporation. Biolistic is another method of gene delivery, which uses high-pressure helium gas to blast microscopic particles of gold coated with the material of interest into skin tissue. So, the option b is wrong. Transformation is also an approach used to introduce recombinant DNA into the host cell. But in this process the host cells take up DNA from their surrounding environment. So, the option a is wrong. Microinjection is the injection of DNA using a very fine needle into a cell. So, the option c is also wrong.

(d) This bacteria can be grown easily and its genetics are comparatively (of another three) simple and easily-manipulated, making it one of the best-studied prokaryotic model organisms, and an important species in biotechnology.

The work of Stanley Norman Cohen and Herbert Boyer in E. coli, using plasmids and restriction enzymes to create recombinant DNA, became a foundation of biotechnology.

(b) In this reaction several copies of genes of interest can be synthesized “in vitro” using two sets of primers and the enzyme DNA polymerase. Transcription involves synthesis of mRNA from DNA. Translation involves synthesis of proteins from mRNA. Duplication of DNA occurs in cell division. So, the other three options are wrong.

(b) Restriction enzymes are enzymes, which recognize a specific, short sequence of DNA and cut the DNA at that point. Different restriction enzymes recognize and cut different sequences. Restriction enzymes are a vital tool in genetics, since they allow cutting (and pasting) of DNA. Ligase is an enzyme, which can connect a DNA backbone. We rely on the sticky ends left from restriction enzyme cuts to line two pieces of DNA up, but the ligase is necessary to form the covalent bonds, which form the sugar-phosphate backbone.

(a) The simplest method for identifying the Transformants relies on the presence of an antibiotic resistance gene on the plasmid vector, for example ampicillin resistance gene. If the host cells transformed with this plasmid are grown on a medium containing ampicillin, only those cells which contain this plasmid will be able to grow and form colonies.

(c) Both the human DNA and the bacterial DNA must be first treated with the same restriction endonuclease, because the restriction site and recognition sequence is same restriction enzyme is same in both the organisms; by which they can join together.

(b) RFLP technique is based on the observation that there are variations in the DNA sequence of different individuals and even in same individual having genetic defects. In a DNA sample there are various restriction sites that can be recognized and digested by restriction enzymes. When the DNA sample is digested by the restriction enzyme, DNA fragments of varying lengths will be generated depending upon the length of the position of these sites on the DNA. If the DNA sequence of one or more than one of these sites is changed in a DNA sample from another, digestion of that sequence by the same restriction enzymes will generate different number and different size sequences.

(a) Plasmids are independent, free-floating circular piece of DNA in a bacterium, capable of making copies of itself in the host cell. Plasmids are used in recombinant DNA experiments to clone genes from other organisms by inserting the foreign DNA into itself and make large quantities of their DNA.

(d) Transgenic is an organism, which has a foreign gene (a transgene) incorporated into its genome or is an organism that has incorporated a functional foreign gene through recombinant DNA technology. The novel gene exists in all of its cells and is passed through to progeny.

(d) Normal E.coli cells do not carry genes that are resistant to antibiotics. It is due to this that E.coli cells are suitable as selectable markers for biotechnological synthesis of antibiotics.

(d) Restriction enzymes are used in genetic engineering because they can be used in vitro to recognize and cleave within specific DNA sequences.

(c) Escherichia coli can be grown easily and its genetics are comparatively simple and easily manipulated, making it one of the best-studied prokaryotic model organisms, and an important species in recombinant DNA technology.

Agrobacterium tumefaciens is known as the natural genetic engineer of plants. Its Ti plasmid is used as vector to transfer foreign genes of interest into the target plant cells.

(b) Restriction endonucleases are obtained from bacterial cells. These enzymes exist in many bacteria where they function as apart of their defense mechanism known as restriction –modification system.

(b) Southern blotting is a technique for transferring denatured DNA molecules that have been separated electrophoretically, from a gel to a matrix such as a nitrocellulose membrane on which a hybridization assay can be performed. In Northern blotting is transfer of RNA and in Western blotting transfer of proteins takes place.

(a) Site-directed mutagenesis is a technique by which it is possible to introduce mutations at precisely determined sites of genes.

It is a powerful technique where site specific changes in DNA sequence are produced in vitro - for instance to change an amino acid residue into another by changing the codon sequence within the gene sequence. Site-specific mutagenesis is a technique to change one or more specific nucleotides within a cloned gene in order to create an altered form of a protein with one or more specific amino acid changes. a.k.a. oligonucleotide-directed mutagenesis; oligonucleotide-directed site-specific mutagenesis.

These questions consist of two statements each, given as assertion and reason. While answering these questions you are required to choose any of the following four responses.

A. If both assertion and reason are true and the reason is a correct explanation of the assertion.

B. If both assertion and reason are true but reason is not a correct explanation of the assertion.

C. If assertion is true but the reason is false

D. If both assertion and reason are false.

(d) Both the statements are false. Restriction enzymes are found in bacterial cells not in eukaryotic cell and Restriction endonucleases cut the DNA molecule to form blunt as well as sticky ends.

(c) This is true that the cut pieces of DNA are linked with plasmid. Plasmid acts as a vector or cloning vehicle that is why second statement is false.

(b) Exonucleases can remove bases only from open ends of DNA. This means, it can cut DNA at the last nucleotide at either end only.

(a)The restriction enzymes acts as foundation of rDNA technology. These enzymes are present in bacteria and provide a type of defense mechanism called the ‘Restriction- Modification system’. They normally protect the host cell by destroying foreign DNA that enters into it, such as viral DNA.

(c) Given is the recognition sequence of BamHI.

(a) pUC19 possesses an ampr gene, lacZ gene (for b-galactosidase) and lacI gene (for production of repressor protein to regulate lacZ).

In pUC19, the multiple cloning sequences (MCS) is incorporated into lacZ gene without interfering the function of other genes.

(c) Cosmid is artificially constructed cloning vector containing the cos gene of phage lambda. Cosmids can be packaged in lambda phage particles for infection into E. coli; this permits cloning of larger DNA fragments (up to 45kb) than can be introduced into bacterial hosts in plasmid vectors.

(b) Those vectors that have the l phage cos sequences, e.g. cosmids, phasmids and l vectors, are generally packaged in vitro into specially produced empty l phage heads. The packaging procedure yields complete l particles. These phage particles are used to infect E.coli cells; this strategy is called transfection.

(b) In 1965, Robert Holley and his research group at Cornell University completely sequenced nucleotides of tRNAala (tRNA for yeast alanine).

(b) Several vectors have been constructed which exist both in E.coli and eukaryotic cells. Shuttle vectors contain two types of origin of replication, one specific for each host species (E.coli and eukaryotic cells).

(d) DNA Probes are small (20-50) nucleotide sequences used to detect the presence of complementary sequences in nucleic acid samples.

(c) Subtilisin is a bacterial serine-protease that can breakdown most proteins, which soil clothing and a major additive to modern 'bio' detergents. Before it became commercially useful, the recombinant protein was engineered ('improved') in order that it remained active in the presence of bleach. Bleach caused the oxidation of a methionine residue (position 222) - and the enzyme lost 90% of its activity. By replacing this amino acid with alanine, the engineered enzyme was no longer sensitive to oxidation.

(d) Alkaline phosphataseis the enzyme that is used to prevent unwanted self-ligation of vector DNA molecule during cloning procedures. When a vector molecule is cleaved with restriction enzymes, a major problem creeps in at that same time. The cohesive ends of the vector, instead of joining with foreign DNA, often join together resulting in the recircularization of the plasmid. The alkaline phosphatase enzyme overcomes this problem.

(c) The Sanger’s method utilizes 2’,3’-dideoxynucleoside triphosphates (ddNTPs) molecules that differ from deoxynucleotide triphosphates(dNTPs) by having a hydrogen atom attached at the 3’-carbon rather than hydroxyl group. The dideoxy method gets its name from the critical role played by synthetic nucleotides that lack the -OH at the 3′ carbon atom.

(b) Through polymerase chain reaction (PCR) one can generate a large amount of one of the strand of a DNA. It generates microgram (mg) quantities of DNA copies (upto billion copies) of the desired DNA segment, present even as a single copy in the initial preparation.

(b) Restriction enzymes cut DNA at both the antiparallel strands so that the DNA molecule is fragmented. This makes them act like molecular scissors. They first bind to the specific site of DNA and then break the sugar-phosphate backbone.

(d) In DNA fingerprinting sample analysis and comparison can be done on the basis of RFLP (restriction fragment length polymorphism), PCR (polymerase chain reaction), STR (short tandem repeats), AFLP (amplified fragment length polymorphisms), Y-chromosome variation and mitochondrial variations.

(c) Bacterial chromosome is generally circular and has an origin of replication, designated as ‘Ori’. This is a specific DNA sequence. Replication starts from here and ends near to it, as the DNA is circular. For a foreign DNA to be operative in a cell, it has to be associated with the ‘Ori’ sequence. Otherwise, it cannot be cloned and will be useless.

(a) Each plasmid is maintained in the bacterial cell at a characteristic copy number mainly due to its replication control system. In this context, the plasmids are of two types: single copy and multiple copy plasmids.

The replication control of single copy plasmids is the same as that of their bacterial host cells so that they replicate and segregate with the bacterial chromosome, which is called stringent replication. The replication control of multi copy plasmids is different from that of their bacterial host genome so that they undergo more than one replication for each replication of their host genome; which is called relaxed replication.

(b) Inducible promoter is the characteristic feature of operon where its function is to promote protein synthesis.

(b) DNA polymerase is isolated from a bacterium, Thermus aquaticus, which remain active during the high temperature induced denaturation of double stranded DNA. Bt toxin is produced by a bacterium called Bacillus thuringensis (Bt for short). Bt toxin gene has been cloned from the bacteria and been expressed in plants to provide resistance to insects. Haemophilus influenzae is the organism from which Hind III was isolated. pBR322 functions only in E.coli.

(a) For the PCR, it is important to have DNA, primers, Taq polymerase, ligase, dNTPs, MgCl­₂ and reaction buffer.

(d) Transformation, transfection and electroporation all the techniques can be used for transformation.

(c) In the process of insertional inactivation, a recombinant DNA is inserted within the coding sequence of an enzyme, â-galactosidase. This results into inactivation of the enzyme.

(d)

Explanation: A genetically modified organism (GMO) is an organism whose genetic material is altered using genetic engineering techniques. These techniques are commonly known as recombinant DNA technology.

(b)

Explanation: Hepatitis B vaccine is a vaccine developed for the prevention of hepatitis B virus infection. This vaccine is produced by yeast (Saccharomyces cerevisiae).

(c)

Explanation. In 1997, Shantha Biotechnic Ltd. (Hyderabad) produced India’s first genetically engineered vaccine, Guni for hepatitis B virus.

(c)

Explanation: Human genome project or HGP was aimed to sequence all genes in the total human genome. It was completed in 13 years.

(d)

Explanation. The culmination of genetic engineering is seen in animal cloning.

The scientists of ‘Human Cloning Society’, of France, made a claim for the birth of a clone baby ‘Eve’ on 27 December 2002.

(a)

Explanation: Bacterial chromosome is generally circular and has an origin of replication, designated as ‘Ori’. This is a specific DNA sequence. Replication starts from here and ends near to it, as the DNA is circular.

(b)

Explanation: Bacterial chromosome has an origin of replication, designated as ‘Ori’. For a foreign DNA to be operative in a cell, it has to be associated with the ‘Ori’ sequence. Otherwise, it cannot be cloned and will be useless.

(c)

Explanation: Plasmids are circular pieces of DNA with closed ends. Plasmids are potentially useful tools for the multiplication of foreign genes. They are hence known as vectors.

(b)

Explanation: Nucleases cut DNA at both the antiparallel strands so that the DNA molecule may be fragmented. This makes them act like molecular scissors. They first bind to the specific site of DNA and then break the sugar-phosphate backbone.

(a)

Explanation: Arthur Cornberg and his colleagues isolated DNA polymerase in 1955. DNA polymerase forms the DNA chain during DNA replication or any other DNA synthesis process.

(b)

Explanation: Bernerd Weiss and Charles Richardson isolated DNA ligase in the year 1966. DNA ligase joins cut ends of any DNA.

(c)

Explanation: Hamilton Smith, K. W. Wilcox and T. J. Kelley had isolated the first restriction endonuclease in the year 1968. Restriction enzymes are peculiar enzymes which cut DNA anywhere in between at specific sequences.

(d)

Explanation: Exonucleases can remove bases only from open ends of DNA. This means, it can cut DNA at the last nucleotide at either end only. It may have 5’ or 3’ activity. DNA polymerases can also have exonuclease activity at times.

(c)

Explanation: Endonucleases can nick the DNA at any point in between. When derived from certain prokaryotes, they are termed restriction endonucleases because they cut DNA at ‘restricted’ sequences only.

(d)

Explanation: Plasmids are potentially useful tools for the multiplication of foreign genes. They are hence also known as vectors.

(c)

Explanation: Many endonucleases cleave DNA molecules at random sites, but a class of endonucleases cleaves DNA only within or near those sites, which have specific nucleotide sequences; are called restriction endonucleases or restriction enzymes.

(a)

Explanation: The restriction enzymes act as foundation of rDNA technology. These enzymes are present in bacteria (prokaryotes) and provide a type of defense mechanism called the ‘Restriction- Modification system’.

(c)

Explanation: Restriction enzymes provide a type of defense mechanism called the ‘Restriction- Modification system’. They normally protect the host cell by destroying foreign DNA (such as viral DNA) that enters into it.

(a)

Explanation: The first letter of the name represents the genus of the bacterium from where it is derived. The next two letters denote the species of the bacterium. E.g., BamHI is derived from Bacillus amyloliquefaciens.

(b)

Explanation: The next two letters after first letter denote the species of the bacterium. E.g., BamHI is derived from Bacillus amyloliquefaciens. The next letter in upper case represents the strain of the bacterium. E.g., EcoRI is derived from the R-strain of E. coli.

(b)

Explanation.This bacterium is also known as natural genetic engineer of plants due to its natural ability of DNA transfer. Gene transfer through Agrobacterium is achieved by its co- culture with tissue explants

(b)

Explanation: Palindromic sequences are recognized by restriction endonucleases. The enzyme either cuts DNA at exactly middle position of the sequence or at two further ends of the sequences in two strands.

(a)

Explanation: Electrophoresis is done generally in a solid support provided by a gel. The gel is made up of agarose. Electrodes connected to the gel provide current. DNA being slightly negative will migrate towards the positive electrode.

(a)

Explanation.Agrobacterium tumefaciens is a natural genetic engineer of plant that has the natural ability to transfer T-DNA of its plasmid.

(c)

Explanation: Ethidium bromide is a fluorescent dye used for staining any nucleic acid (DNA or RNA).

(a)

Explanation: Restriction fragment length polymorphism (RFLP) is a variation in the DNA sequence of a genome that can be detected by breaking the DNA into pieces with restriction enzymes and analyzing the size of the resulting fragments by gel electrophoresis.

(c)

Explanation. Agrobacterium tumefaciens consists of Ti-plasmid, which is directly responsible for tumour induction. Transfer of small DNA segment (T-DNA) from Ti- plasmid and its integration into host genome, induces tumour formation in plants.

(a)

Explanation: The cohesive ends of the vector, instead of joining with foreign DNA, often join together resulting in the recircularization of the plasmid. The alkaline phosphatase enzyme overcomes this problem.

(c)

Explanation. Agrobacterium tumefaciens bacteria is also known as natural genetic engineer of plants due to its natural ability of DNA transfer.

(a)

Explanation: Agrobacterium tumefaciens bacterium is also known as natural genetic engineer of plants due to its natural ability of DNA transfer.

(d)

Explanation: The Roman numbers indicate the serial number of discovery of the enzyme from that particular strain. E.g., HaeIII is the third restriction enzyme isolated from Haemophilus aegyptius.

(b)

Explanation: Electrophoresis is done generally in a solid support provided by a gel. The gel is made up of agarose. It is not soluble in water, but it will dissolve in boiling water and then settle as a jelly-like solid as water-cools.

(b)

Explanation.Agrobacterium tumefaciens consists of Ti-plasmid, which is directly responsible for tumour induction. Transfer of small DNA segment (T-DNA) from Ti plasmid and its integration into host genome induces tumour formation in plants.

(d)

Explanation: DNA Ligase seals nicks between adjacent nucleotides in a double-stranded DNA molecule. T4 DNA ligase is the enzyme that is used most often in cloning experiment, which is encoded by phage T4.

(a)

Explanation: Vectors are carriers of the gene to be inserted into a cell. They are vehicles of gene transfer. A vector is efficient when it can transfer the gene of interest without alteration of its size and effect to the target cell.

(b)

Explanation: Bacteriophage is a virus, which attacks bacteria and infects them by inserting their DNA into it. The DNA if manipulated with foreign genes can be inserted into bacterial host.

(a)

Explanation: Plasmids are extra-chromosomal, self-replicating circular double stranded DNA molecules that often confer traits like antibiotics resistance or toxins.

(d)

Explanation: Origin of replication or ‘Ori’ is the sequence from where replication starts. A DNA with ‘Ori’ linked to it can control the number of DNA replicas. So, for large-scale production of DNA, it should be linked to an ‘Ori’ with high copy number.

(b)

Explanation: The vector must contain atleast a unique recognition site for restriction enzyme, when the desired foreign DNA should be inserted. Presence of multiple cloning site (MCS) or polylinker provides flexibility in the choice of restriction enzymes that can be used in cloning.

(b)

Explanation: pBR322 has two selectable markers (tetracycline, tet^r and ampicillin, amp^r, resistance genes), and only single or unique recognition site for 12 different restriction enzymes (two, PstI and PvuI, located within the amp^r gene, and 4, BamHI, SalI, etc., within tet^r gene).

(a)

Explanation: Plasmids can be put into two major classes:

1. Conjugative plasmids are of high molecular weight and have low copy number (1-3/chromosome).

2. Non-conjugative plasmids are smaller and exist in multiple copies per cell.

(d)

Explanation: Col plasmid contains genes that code for bacteriocins, which are the proteins that can kill other bacteria.

(a)

Explanation: The plasmid pBR322 is one of the most commonly used E. coli cloning vectors. The name pBR322 denotes - p signifies plasmid, B is Bolivar and R is Rodriguez- the name of the scientists who developed pBR322, and 322 is the numerical designation.

(c)

Explanation: A plasmid is an extra-chromosomal DNA molecule, which is separated from the chromosomal DNA and is capable of replicating independently of the chromosomal DNA.

(b)

Explanation: pUC19 is identical to pBR322 except that it contains multiple cloning sites (MCS) arranged in opposite orientations. The MCS has the unique sites for several restriction endonucleases. This vector also replicate only in E. coli.

(d)

Explanation: Shuttle vectors contain two types of origin of replication, one specific for each host species (E. coli and eukaryotic cells). These are constructed by recombinant DNA techniques e.g., YEp (yeast episomal plasmid).

(a)

Explanation: These recombinant Ti plasmids can then be used for transformation of higher plants.

(b)

Explanation: A virus, which has RNA instead of DNA as a genetic material, is called retroviruses. It has the ability to transform human cells into cancerous cells. These can also be used as vectors of genes.

(a)

Explanation: Expression of cloned gene is carried out by inserting a ‘promotor sequence’ (signal for initiation of transcription), and a ‘terminator sequence’ (that provide signal for termination of transcription).

(c)

Explanation: The M13 phage’s DNA enters the bacterial cell; it is converted into a double- stranded molecule known as the replicative form or RF, which replicates until there are 100 copies in the cell. Finally, single stranded DNA molecules are produced from RF DNA and extrude from the cell as M13 particles.

(a)

Explanation: M13 is a filamentous phage which infects E. coli having F pili. It consists of single stranded circular DNA molecule having 6407 base pairs and 10 genes in the genome.

(d)

Explanation: The simplest cosmid vector contains a plasmid’s origin of replication, a selectable marker, suitable restriction enzyme sites and the lambda ‘cos’ site.

(b)

Explanation: The yeast artificial chromosome (YAC) vector is capable of carrying a large DNA fragment (up to 2 Mb), but its transformation efficiency is very low. These vectors contain telomeric sequences, the centromere and independently replicating sequences.

(c)

Explanation: They contain suitable restriction enzyme sites and genes, which can act as selectable markers in yeast.

(d)

Explanation: This mutant strain of E. coli is missing the normal restriction and modification system.

(a)

Explanation: Gemini viruses have single stranded DNA as genetic material, which causes important diseases in cassava, maize, and other cereals.

(c)

Explanation: The virus spreads systematically throughout the plant in a very high copy number reaching up to 105 virus particles per cell. These features make CaMV a suitable vector for transformation of higher plants.

(b)

Explanation: A vector based on Simian Virus 40 (SV40) was used in the first cloning experiment involving mammalian cells in 1979. Presently, retroviral vectors are the most commonly used vectors for cloning genes in mammalian cells.

(d)

Explanation: A vector based on Simian Virus 40 (SV40) was used in the first cloning experiment involving mammalian cells in 1979. Since 1979, a number of vectors based on other types of viruses like Adenovirus and Papilloma virus have been used to clone genes in mammals. Presently, retroviral vectors are the most commonly used vectors for cloning genes in mammalian cells.

(a)

Explanation: The gene coding for alpha-galactosidase produces blue colors in bacterial cells. When a foreign gene in inserted into this gene, it is inactivated. This is known as insertional inactivation.