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Semester 2: M.Sc. Biotechnology Syllabus 2023-2024
Gene cloning. Genetic engineering tools. Nucleic acid manipulating enzymes. Promoters, Selectable markers and reporters used in rDNA technology. Restriction digestion, Ligation, Transformation
Gene Cloning and Genetic Engineering Tools
Gene Cloning
Gene cloning is the process of isolating and making copies of a particular gene. It involves cutting DNA from an organism and inserting it into a vector, which is then introduced into a host cell where it can replicate.
Genetic Engineering Tools
Genetic engineering employs various tools such as plasmids, bacterial artificial chromosomes, and CRISPR-Cas9 to manipulate DNA. These tools facilitate the modification of genes for research, medicine, and agriculture.
Nucleic Acid Manipulating Enzymes
Essential enzymes in genetic engineering include restriction endonucleases, which cut DNA at specific sequences, and DNA ligases, which join DNA fragments together. These enzymes are crucial for creating recombinant DNA.
Promoters
Promoters are DNA sequences that initiate transcription of a gene. They dictate the spatial and temporal expression of genes, influencing how much protein is produced in a cell.
Selectable Markers
Selectable markers are genes that confer a trait suitable for artificial selection. Common markers include antibiotic resistance genes, which allow for the identification of successfully transformed cells.
Reporters
Reporter genes are used to monitor the activity of promoters and gene expression. Common examples include the green fluorescent protein (GFP), which fluoresces under specific light conditions.
Restriction Digestion
Restriction digestion involves using restriction enzymes to cut DNA at specific sites. This technique is essential for preparing DNA fragments for cloning and recombinant DNA technology.
Ligation
Ligation is the process of joining two DNA fragments together using DNA ligase. This step is critical after restriction digestion to form recombinant DNA molecules.
Transformation
Transformation is the process of introducing recombinant DNA into host cells. It can involve methods such as heat shock, electroporation, or viral vectors to achieve successful uptake and expression.
E.Coli vectors - pBR322 and its derivatives Cloning vectors for gram negative bacteria - ColE1, p15A, R1, IncPa, pSC101 Lambda bacteriophage vectors, filamentous phages, Cosmids, Phasmids, Phagemids. Cloning in gram-positive bacteria Bacillus subtilis
E.Coli Vectors and Cloning Techniques
pBR322 and its Derivatives
pBR322 is one of the first plasmid vectors used in E.Coli for cloning. It contains two antibiotic resistance genes, bla (for ampicillin resistance) and tet (for tetracycline resistance), which allow for the selection of transformed cells. Derivatives of pBR322 have been developed to increase cloning capacity and simplify screening processes.
Cloning Vectors for Gram-Negative Bacteria
Various vectors are available for cloning in gram-negative bacteria. ColE1, p15A, R1, and IncP vectors are examples that provide replication origins suitable for E.Coli. Each vector has unique features that facilitate cloning, such as distinct selectable markers and replication strategies.
Lambda Bacteriophage Vectors
Lambda phage vectors utilize the lambda bacteriophage to introduce foreign DNA into bacterial cells. These vectors can accommodate larger inserts than typical plasmids and are useful for genomic library construction.
Filamentous Phages
Filamentous phages such as fd and M13 allow for the packaging of cloned DNA into phage particles. They are particularly useful for displaying peptides and proteins on the phage surface for various applications.
Cosmids
Cosmids are hybrids of plasmids and lambda phage, designed to clone large fragments of DNA (up to 45 kb). They include cos sites from lambda phage for packaging into phage particles.
Phasmids and Phagemids
Phasmids are plasmids that contain a bacteriophage's replication origin, allowing them to be packaged into phage particles. Phagemids combine features of plasmids and phages for efficient cloning and expression.
Cloning in Gram-Positive Bacteria
Cloning in gram-positive bacteria, such as Bacillus subtilis, presents unique challenges due to differences in cell wall structure. Vectors for these organisms often include elements such as promoters recognized by gram-positive RNA polymerases and selection markers originally from gram-positive species.
Cloning in yeast Saccharomyces cerevisae. Life cycle and types of vectors Eukaryotic vectors. SV40 molecular genetics and expression Construction of gene libraries Genomic and cDNA library, Specialized cloning vector for cDNA Synthesis of specific RNA in vitro Vectors for cloning promoters and terminators vectors with adjustable copy number
Cloning in yeast Saccharomyces cerevisae
Introduction to Cloning in Yeast
Saccharomyces cerevisiae is a model organism in biotechnology and molecular biology. Cloning in yeast involves the introduction of foreign DNA into yeast cells to study gene function and protein expression.
Life Cycle of Saccharomyces cerevisiae
The life cycle of Saccharomyces cerevisiae includes both asexual and sexual reproduction. Asexual reproduction occurs through budding, while sexual reproduction involves the formation of spores.
Types of Vectors for Yeast Cloning
Common vectors used include plasmids, integrative vectors, and yeast artificial chromosomes. Each type serves different purposes based on the cloning goal.
Eukaryotic Vectors
Eukaryotic vectors are essential for gene expression in eukaryotic cells. These vectors provide necessary elements such as promoters and terminators specific to eukaryotic gene regulation.
SV40 Molecular Genetics and Expression
SV40 is a virus that serves as a valuable vector in gene cloning and expression systems. It contains origins of replication and promoters effective in eukaryotic cells.
Construction of Gene Libraries
Gene libraries can be genomic or cDNA types. Genomic libraries contain DNA fragments of an organism's genome, while cDNA libraries consist of DNA synthesized from mRNA.
Specialized Cloning Vectors for cDNA
Specialized vectors designed for cDNA cloning generally include features for efficient transcription and translation of the inserted cDNA.
Synthesis of Specific RNA In Vitro
In vitro transcription allows for the synthesis of specific RNA molecules using a DNA template, which can be utilized for functional studies.
Vectors for Cloning Promoters and Terminators
Vectors designed for cloning promoters and terminators ensure the regulation of gene expression. They contain sequences that facilitate proper initiation and termination of transcription.
Vectors with Adjustable Copy Number
Some vectors allow researchers to modify the copy number of plasmid DNA within the host cells, which can enhance protein production or gene expression.
Nucleic acid hybridization techniques Molecular probes Types of probes and its construction probe labeling. Nick translation, End labeling and Random primer labeling. Polymerase chain reaction and its variants DNA fingerprinting DNA sequencing first generation sequencing methods Maxam and Gilbert sequencing, Sangers Dideoxy sequencing, Pyrosequencing, PCR based sequencing and hybridization sequencing. Second generation sequencing methods
Nucleic acid hybridization techniques
Molecular probes
Molecular probes are fragments of nucleic acid used to detect the presence of complementary sequences by hybridization. They can be designed to bind specifically to a target DNA or RNA sequence, facilitating the identification and quantification of the target in various applications.
Types of probes and its construction
Probes can be categorized based on their design, labeling, and applications. Common types include DNA probes, RNA probes, and peptide nucleic acid (PNA) probes. The construction often involves synthesizing a sequence complementary to the target and incorporating labels for detection.
Probe labeling
Probe labeling is a process that attaches a detectable marker to a probe. Various methods include nick translation, end labeling, and random primer labeling. Each method modifies the probe to enhance its visibility in hybridization assays.
Nick translation
Nick translation is a technique that uses DNA polymerase to create nicks in a DNA strand while simultaneously incorporating labeled nucleotides. This method allows for the incorporation of a label into the probe, enabling its detection.
End labeling
End labeling involves adding a label to the 5' or 3' ends of a DNA fragment. This method typically uses enzymes like T4 polynucleotide kinase to add radioactive or fluorescent labels for visualizing the probe.
Random primer labeling
Random primer labeling utilizes short, random sequences to prime DNA synthesis, incorporating labeled nucleotides into the resulting probe. This approach is beneficial for amplifying fragments and increasing the sensitivity of detection.
Polymerase chain reaction and its variants
PCR is a technique that amplifies specific DNA sequences exponentially. Variants include quantitative PCR (qPCR) for measuring DNA concentration and reverse transcription PCR (RT-PCR) for obtaining cDNA from RNA.
DNA fingerprinting
DNA fingerprinting is a forensic technique used to identify individuals based on unique patterns in their DNA. It employs methods like PCR and gel electrophoresis to reveal variable number tandem repeats (VNTRs) and short tandem repeats (STRs).
DNA sequencing
DNA sequencing refers to determining the precise order of nucleotides. It encompasses various methods such as first-generation sequencing with Maxam-Gilbert and Sanger sequencing, and newer sequencing technologies.
First generation sequencing methods
First generation techniques like Maxam-Gilbert sequencing involve chemical cleavage of labeled DNA, while Sanger dideoxy sequencing uses chain-terminating nucleotides for sequence determination.
Pyrosequencing
Pyrosequencing is a method that detects the release of pyrophosphate during nucleotide incorporation, allowing for real-time sequencing. It is fast and suitable for high-throughput applications.
PCR based sequencing and hybridization sequencing
These methods combine PCR amplification with sequencing techniques to analyze specific regions of interest rapidly. Hybridization sequencing uses probes to enrich target sequences before sequencing.
Second generation sequencing methods
Second generation sequencing methods, often referred to as next-generation sequencing (NGS), include techniques like Illumina sequencing and 454 sequencing. These methods provide massively parallel sequencing capabilities, allowing for rapid and cost-effective sequencing of entire genomes.
Site directed mutagenesis DNA microarray, Molecular techniques in prenatal diagnosis gene therapy, Transgenic animals knockout mice and plants Flavr savr tomato, Pharmaceutical products Vaccine, Humulin, etc, Crop improvement. Pesticide resistance, herbicide resistance, transgenic animals and GM foods Modern Concepts in Genetic Analysis
Site Directed Mutagenesis
A technique used to make specific and intentional changes to the DNA sequence of a gene. It allows for the study of the effects of mutations on protein function.
DNA Microarray
A technology used to study gene expression and detect mutations by hybridizing DNA samples to a grid of known DNA sequences. It enables analysis of multiple genes at once.
Molecular Techniques in Prenatal Diagnosis
Techniques such as amniocentesis and chorionic villus sampling are used to detect genetic abnormalities in a fetus. They can provide valuable information about potential genetic disorders.
Gene Therapy
An experimental technique that uses genes to treat or prevent disease by inserting genes into a patient's cells instead of using drugs or surgery.
Transgenic Animals
Animals genetically engineered to express a gene from another species. They are used in research, agriculture, and producing pharmaceuticals.
Knockout Mice
Mice that have had one or more of their genes made inoperative. They are used to study the effects of specific genes and simulate human diseases.
Flavr Savr Tomato
The first commercially grown genetically engineered food. It was designed to have a longer shelf life and improved flavor.
Pharmaceutical Products
Products developed through genetic engineering for medical treatments. Examples include vaccines and insulin products such as Humulin.
Crop Improvement
Modification of crops through genetic techniques to improve yield, flavor, pest resistance, and adaptability to environmental conditions.
Pesticide Resistance and Herbicide Resistance
Genetic modifications that enable crops to withstand certain pesticides or herbicides, reducing the impact of pests and weeds without harming the crop.
Transgenic Animals and GM Foods
Transgenic animals are modified for specific traits. GM foods refer to crops that have been genetically modified for better yields or resistance to pests.
Modern Concepts in Genetic Analysis
The study of genetic variations and their implications in health and agriculture, utilizing advanced techniques and technologies to understand DNA functions.
