2018 Solved Old Paper (BOT - 402) New OK
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Gene:-
Definition:-
“A functional segment of DNA which manufacture protein, regulate gene expression and renowned as a hereditary unit is known as a gene.”
History:-
- Mendel first discovered the concept of the “inheritance of traits”, despite, he fails to describe it.- The term “gene” was coined and studied by Wilhelm Johannsson. But he was unable to describe the chemical structure of it. In 1953, James Watson and Francis Crick defined the chemical structure of the DNA viz gene.
Structure of gene:-
- Genes are actually DNA segment thus are made up of the nucleotide chain. The chemical structure of a gene comprises nucleotides.
- Genes are made up of A, T, G and C nucleotides. With the nucleotides of the opposite strand, it binds with hydrogen bonds and with the adjacent nucleotide, it binds with phosphodiester bonds.
- The nucleotides are the combination of nitrogenous bases (A, T, G and C), phosphate and pentose sugar.
- In general, the gene structure consists of two types of components: core components and regulatory components.
- The core components or sequences actually take parts in protein formation. While the regulatory components maintain gene expression.
- Exons are core elements. Sequences on the other side like promoters, enhancers and silencers are regulatory elements of a gene.
- The third type of element called maintenance elements possesses information for DNA repair, modification and replication. The functional or physical structure of a gene comprises introns, exons, promoters, enhancers and UTRs.
- Introns are non-coding sequences removed from the final transcript.
- Exons are coding part of a gene which are joined after splicing and constructs the final transcript.
- Regulatory components are located on the extreme ends of a gene.
- Promotes are non-coding sequences but facilitates binding sites for enzymes and transcriptional factors to work. The promoter consists of TATA box and CCAAT sequences for enzyme binding.
- The entire promoter region is located on the 5’ end and made up of core promoter and proximal promoter sequences.
Functions of gene:-
- The main function of a gene is to form or manufacture a protein.
- Some genes can’t form protein, although they transcribe into mRNA. For instance, the microRNAs are the type of tiny ribonucleic acid formed from some genes but it doesn’t undergo protein formation. It helps in gene regulation instead.
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Principles of Genetic Engineering:-
1. Introduction:-
> The technology of recombinant DNA was developed in 1973 by Boyer and Cohen.
> It is popularly known as genetic engineering.
> Recombinat DNA:- When foreign gene is inserted into a vector, then it is called as recombinant DNA.
> Objective:- This is the natural mathod of amplification of gene of interest.
2. Process of Recombinant DNA Technology:- The complete process of recombinant DNA technology includes multiple steps-
Step-1. Isolation of Genetic Material:- The first and the initial step in Recombinant DNA technology is to isolate the desired DNA in its pure form i.e. free from other macromolecules.
Step-2. Cutting the gene at the recognition sites:- The restriction enzymes play a major role in determining the location at which the desired gene is inserted into the vector genome. These reactions are called ‘restriction enzyme digestions’.
Step-3. Ligation of DNA Molecules:- In this step of Ligation, the joining of the two pieces – a cut fragment of DNA and the vector together with the help of the enzyme DNA ligase.
Step-4. Insertion of Recombinant DNA Into Host:- In this step, the recombinant DNA is introduced into a recipient host cell. This process is termed as Transformation.
Step-5. Amplifying the gene copies:- Once the recombinant DNA is inserted into the host cell, it gets multiplied. As a result the inserted gene of interest is also multiplied.
Application of recombinant DNA technology:-
1. Medicines:-
> By 2001 over 80 recombinant DNA based products had been approved for treating disease and for vaccination.
> A further 350 recombinant DNA-based drugs were being tested for safety and efficacy.
> Human insulin was one of the first therapeutic proteins that was genetically cloned.
2. Genetic mapping (linkage mapping):- It is a process or method of discovering the location of genes on a chromosome.”
> It helps to identify:
- that a disease transmitted from parent to child is linked to one or more genes.
- which chromosome contains the gene and precisely where the gene lies on that chromosome.
- the gene responsible for relatively rare, single-gene inherited disorders such as cystic fibrosis and Duchenne muscular dystrophy.
- the genes that are believed to play a role in the development of common disorders such as asthma, heart disease, diabetes, cancer, and psychiatric conditions.
> Genetic maps are species-specific and comprised of genomic markers and/or genes and the genetic distance between each marker.
3. Gene therapy:-
> Diseases caused by deficiency of a gene product are amenable to replacement therapy.
> The strategy is to clone a gene into a vector that will readily be taken up and incorporated into genome of a host cell.
> Adenosine deaminase deficiency has been treated successfully with gene replacement therapy.
> For many sickle cell anaemia, thalassaemias, and various other metabolic disorders Gene therapy is under trial.
4. Transgenesis:-
> The somatic gene replacement therapy can not pass on to the offspring.
> Transgenesis refers to the transfer of genes into fertilised ovum which can be found in somatic as well as germ cells and passed on to the successive generations.
5. Gene Disruption (knockout genes):-
> A gene's function can also be probed by inactivating the gene and looking for resulting abnormalities.
> Powerful methods have been developed for accomplishing gene disruption (also called gene knockout) in organisms such as yeast and mice.
> Specific genes can be targeted if their nucleotide sequences are known.
6. Manufacture of proteins / hormones:-
> By inserting the gene for a rare protein into a plasmid and expressing it in bacteria, large amounts of the recombinant protein can be produced. Many proteins have been produced e.g., human growth hormone, insulin, interferons and blood clotting factors.
> Another approach to producing proteins via recombinant DNA technology is to introduce the desired gene into the genome of an animal, engineered in such a way that the protein is secreted in the animal’s milk, facilitating harvesting.
> Tissue plasminogen activator, which is administered to a patient after a heart attack, is made in large quantities in mammalian cells. A new pharmacology, using proteins produced by recombinant DNA technology as drugs, is beginning to significantly alter the practice of medicine.
7. Site specific mutagenesis:-
> New genes with designed properties can be constructed by making three kinds of directed changes:
i. deletions
ii. insertions
iii. substitutions
> Substitutions:- Oligonucleotide- Directed Mutagenesis. A primer containing a mismatched nucleotide is used to produce a desired change in the DNA sequence.
> Insertions:-
- In cassette mutagenesis, plasmid DNA is cut with a pair of restriction enzymes to remove a short segment
- A synthetic double-stranded oligonucleotide (the cassette) with cohesive ends that are complementary to the ends of the cut plasmid is then added and ligated.
- Each plasmid now contains the desired mutation.
- It is convenient to introduce into the plasmid unique restriction sites spaced about 40 nucleotides apart so that mutations can be readily made anywhere in the sequence.
8. Designer Genes:-
> Novel proteins can also be created by splicing together gene segments that encode domains that are not associated in nature.
> For example, a gene for an antibody can be joined to a gene for a toxin to produce a chimeric protein that kills cells that are recognized by the antibody.
> These immunotoxins are being evaluated as anticancer agents.
> Entirely new genes can be synthesized de novo by the solid-phase method. Furthermore, noninfectious coat proteins of viruses can be produced in large amounts by recombinant DNA methods.
> They can serve as synthetic vaccines that are safer than conventional vaccines prepared by inactivating pathogenic viruses.
> A subunit of the hepatitis B virus produced in yeast is proving to be an effective vaccine against this debilitating viral disease.
9. Diagnosis of Infectious disease:-
Diagnosis of HIV infection:- The widely used methods for diagnosing HIV infection have been developed using recombinant DNA.
> The antibody test (ELISA or western blot) uses a recombinant HIV protein to test for the presence of antibodies.
> The DNA test detects the presence of HIV genetic material using reverse transcriptase polymerase chain reaction (RT PCR).
> Development of the RT-PCR test was made possible by the molecular cloning and sequence analysis of HIV genomes.
10. Diagnosis of molecular diseases:-
> Many genetic diseases that yield developmental abnormalities can be detected by characteristic patterns in DNA primary structure.
> Such mutational changes in DNA sequences are identified by restriction fragments analysis and Southern blotting, using appropriate DNA probes.
> Analysis of this type could be done in understanding the molecular basis of diseases like sickle cell anaemia, thalassaemias, familial hypercholesterolaemia, cystic fibrosis, etc.
11. Prenatal diagnosis:-
> In diseases where the genetic defect is known and a specific probe is available, prenatal diagnosis can be made.
> DNA from cells collected from as little as 10 ml of amniotic fluid or by chorionic villi biopsy can be analysed by Southern blot transfer.
12. Forensic medicine:-
> Advances in genetic engineering have greatly helped to specifically identify criminals and settle the disputes of parenthood of children.
> Based on the basis of Restriction fragment length polymorphism, the identity of a person can be confirmed.
13. Agriculture:-
> Genetically engineered plants have been developed to resist drought and diseases.
> Good quality of food and increased yield of crops can be possible by applying this technology.
> Incorporation of nif genes to cereals has given higher yield of the crops.
14. Industries:-
> Enzymes synthesised by this technology are used to produce sugars, cheese and detergents.
> Certain protein products produced by this technology are used as food additives to increase the nutritive value, besides imparting flavour.
> Ethylene glycol is in great demand for industry.
> Preparation of ethylene glycol from ethylene is made possible by this technology.
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