Applications of DNA 4 (MJC)
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Explain how electroporation can use used to introduce the recombinant DNA into a plant cell.
- -Enzymes are first introduced so as to digest the cellulose cell wall before electroporation
- -The plant cells are then stimulated with a brief shock from a weak electric current
- -This results in temporary pores to appear in the cell membrane, allowing the cell membrane to be more permeable to the recombinant DNA
State the differences between a genomic and a cDNA library.
- -A genomic library is made up of DNA extracted from any cell type of an organism while a cDNA library is obtained from mRNA extracted from specific cells or tissue-type that expresses the gene actively
- -A genomic library contains genetic information of the complete genome of an organism while the cDNA library is made up of only the expressed portion of the genome
- -A genomic library includes introns while a cDNA library do not have introns
- -A genomic library does not involve the use of reverse transcriptase as the DNA fragments are cloned directly into a vector while a cDNA library requires reverse transcriptase to convert mRNA to cDNA to be inserted into a vector
Explain the following terms
i) Transgenic plant
ii) Transformed cell
i) Transgenic plants are plants which carry a gene from other organisms using bacteria or viruses as vectors
ii) It is an animal or plant cell that has taken up extracellular foreign DNA from its surroundings and the foreign DNA is integrated into its genome
Explain why it is not possible to produce a transgenic plant by plant breeding.
- -It is not possible as the 2 plants are of different species and plant inter-species breeding is rare.
- -This inability is due to incompatible reproductive mechanisms such as different surface structure of pollen that is not recognised by the stigma of another species
- -In addition, the different species of plants may reach sexual maturation at different times of the year, hence decreasing the possibility of reproduction
- -The plants may also have incompatible chromosome number as the 2 species may have different number of chromosomes
Outline the possible hazards of genetically modifying organisms.
- -Microorganisms would produce many substances that they would not normally produce and without proper precautions, the escape of the microoragnisms may affect the general environment
- -GM crops are at a competitive advantage over the wild type. The GM crops may hybridise with the wild types and become weeds and invade natural habitats.
- -The use of herbicide resistant gene in crops will result in the increase in the usage of herbicides to climate weeds in farming which may leave toxic residues in the crops.
Describe 2 advantages of using liposome over adenovirus as a gene delivery system.
1) Liposomes do not trigger any immune response but adenovirus can trigger immune response from the body due to the expression of the viral genome which results in the formation of viral proteins
2) Liposome is not virulent but adenovirus can regain virulence and cause diseases in the host.
Outline the process of cloning plants using tissue culture.
- -Cells from the meristematic tissues/explants are first sterilised using sodium hypochlorite and asceptically transferred to a culture medium
- -Cytokinin, a plant hormone to stimulate cell division, is then added to the medium to stimulate mitosis to form a callus, which is a mass of undifferentiated, actively-growing cells
- -The callus is then sub-cultured in a medium containing auxin and giberellin acid to stimulate differentiation and development of root and shoot
- -The plantlets are then transferred to sterile soil and grown into a whole plant.
Explain the process of genetic fingerprinting.
- -A DNA sample is removed from the cells of the organism and digested with a restriction enzyme.
- -Restriction fragments are then subjected to gel electrophoresis where fragments are separated according to sizes.
- -The band patterns are then transferred via Southern Blot whereby a radioactively labelled probe, which is complementary to part of the gene of interest, is added to the DNA sample where it binds to the complementary gene of interest
- -Autoradiography is then carried out to determine where the gene of interest is
State an advantage and a disadvantage of using Taq DNA polymerase for PCR.
Advantage: It is thermostable whereby it only denatures at very high temperature, thus it allows for the automation of PCR
Disadvantage: Taq polymerase lacks a 3' to 5' exonuclease proofreading activity, hence its replication fidelity is very low
Describe how DNA is separated into bands using gel electrophoresis.
- -The DNA is first cleaved into DNA fragments of varying lengths using restriction enzymes
- -The fragments are then placed in the wells of the agarose gel which is then submerged into a buffer solution.
- -The agarose gel is then subjected to an electric field
- -As DNA is negatively charged due to its phosphate group, the DNA fragments will hence move to the positive electrode.
- -The longer fragments with higher molecular weight will travel more slowly through the agarose gel while shorter fragments will travel faster resulting in the separation of the DNA fragments into bands
Explain the following terms
i) RFLP analysis
ii) restriction site
i) RFLP analysis involves the analysis of restriction fragments created when DNA is cleaved with restriciton enzymes. The presence of different alleles would result in restriction fragments of varying lengths. This thus allows the analysis of band patterns after gel electrophoresis is performed on the fragments.
ii) A restriction site is a specific palindromic sequence of 4 to 8 nucleotide bases that is complementary to the active site of a restriction enzyme.
Explain why a particular primer will only bind to the DNA of one species of organism but not to others.
- -Each primer has a specific nucleotide sequence that is complementary to a particular DNA sequence
- -Different species of an organism have varying DNA base sequences, thus each primer is specific
State 2 limitations of RFLP method used for the determination of wheather a person has a genetic disorder
1) A human genetic disease is sometimes caused by more than 1 mutation in a single gene. For example, cystic fibrosis can arise due to different mutations of the CFTR gene.
2) A disease phenotype maybe a result of mutations to various genes, hence it is difficult to identify the occurrence of a genetic disorder based on RFLP.
Define genetic mapping and the principles in it.
- -A genetic map is an ordered list of genes along a chromosome and the relative distances between those genes
- -The order of genetic markers and the relative distances between them are based on recombinant frequencies, which is the frequency at which crossing over occurs between two chromosome loci
- -The frequency of recombination is a measure of the genetic distance between two loci on the DNA molecule.
- -The lower the frequency, the closer the two loci as they are not likely to become separated during crossing over and hence are inherited together
- -The frequency in which two alleles, where one of them is a RFLP marker, are inherited together is a measure of the closeness of 2 loci on a chromosome
Explain how the shotgun method is used to form a recombinant DNA.
- -DNA is isolated from an organism of interest and randomly fragmented into many pieces using a restriction enzyme
- -The vector is also cut using the same restriction enzyme to form complementary sticky ends
- -DNA ligase is then added to form phosphodiester bonds between the foreign DNA fragments and the vector genome to form the recombinant DNA
- -If a plasmid vector is used, the recombinant DNA is then introduced into bacterial cells and each transformed cell is plated on a agar plate
- -Each transformed cell will multiply to form its own colony of cells and individual colony will contain a different piece of DNA from the organism of interest
- -The colony containing the gene of interest can thus be identified using gene probing and a Southern Blot.
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