Recombinant DNA Technology Summary PDF

Summary of Key Concepts in Recombinant DNA Technology and Genetic Manipulation Techniques. 1. Introduction to Recombinant DNA Technology Definition: Recombinant DNA (rDNA) technology involves combining DNA from different organisms to form new genetic combinations. History: De...

Summary of Key Concepts in Recombinant DNA Technology and Genetic Manipulation Techniques. 1. Introduction to Recombinant DNA Technology Definition: Recombinant DNA (rDNA) technology involves combining DNA from different organisms to form new genetic combinations. History: Developed in the early 1970s by Herbert Boyer, Stanley Cohen, and Paul Berg, foundational in biotechnology. Applications: Used in gene cloning, medicine (e.g., insulin production), agriculture (GMO crops), and industrial enzymes. Concept of Recombinant DNA rDNA combines DNA from two sources (e.g., human and bacterial) to create genetically modified organisms. Example: Human insulin genes placed in bacteria, leading to large-scale insulin production for diabetics. 2. Basic Steps of Recombinant DNA Technology 1. Isolation: o Obtain DNA from the organism containing the gene of interest and isolate vector DNA (e.g., plasmid). 2. Cutting (Cleavage): o Use restriction enzymes to cut DNA at specific sites, creating sticky or blunt ends. 3. Joining (Ligation): o DNA fragments are joined together with DNA ligase, forming recombinant DNA molecules. 4. Transformation: o Introduce recombinant DNA into host cells, often bacteria. 5. Cloning: o Host cells replicate, making multiple copies of the recombinant DNA. 6. Selection: o Identify successful recombinants using markers like antibiotic resistance. Restriction Enzymes (Restriction Endonucleases) Function: Cut DNA at specific sequences (restriction sites), creating sticky or blunt ends for cloning. DNA Ligase Function: Joins DNA fragments by forming phosphodiester bonds, stabilizing recombinant DNA. DNA Polymerase I Function: Synthesizes DNA by adding nucleotides complementary to a template strand, used in DNA replication and repair. Reverse Transcriptase Function: Synthesizes complementary DNA (cDNA) from an mRNA template, useful for creating cDNA libraries. Phosphatase Function: Removes 5' phosphate groups from DNA, preventing self-ligation of vectors. T4 DNA Ligase Function: A specific type of DNA ligase used to form phosphodiester bonds between DNA fragments, often in cloning. RNAse H Function: Degrades RNA in RNA-DNA hybrids, often used in cDNA synthesis after reverse transcription. Transposase Function: Catalyzes the movement of transposons (transposable elements) within the genome. 6. Transformation and Screening of Recombinants Transformation Methods: o Calcium Chloride Treatment: Makes bacterial cell walls permeable to DNA. o Electroporation: Uses electric pulses to introduce DNA into cells. Screening Techniques: Identifying cells with the desired recombinant DNA using markers such as: o Antibiotic Selection: Only cells with recombinant DNA survive on antibiotic- containing media. o Blue-White Screening: Uses the lacZ gene and X-gal to visually distinguish recombinants (white colonies) from non-recombinants (blue colonies). 7. Cloning Vectors: Types of Vectors: o Plasmids: Small, circular DNA molecules that replicate independently in bacteria. Contain an origin of replication (ori), selectable markers (antibiotic resistance), and multiple cloning sites (MCS) for DNA insertion o Bacteriophages: is a virus that infects E. coli, used to clone large DNA fragments (up to 18 kbp). It can introduce foreign DNA into bacteria more efficiently than plasmids. o Cosmids: Hybrid of plasmids and bacteriophage, for larger inserts (up to 45 kb). o BACs (Bacterial Artificial Chromosomes): Useful for cloning very large DNA fragments (up to 300 kbp), mainly for genome mapping o YACs (Yeast Artificial Chromosomes): Resemble normal yeast chromosome(telomeres and centromeres) Used for very large DNA fragments (up to 1,000 kb). 8. Expression Vectors Purpose: Designed to express the cloned gene as a protein. Features: Include promoter sequences to ensure transcription, ribosome binding sites for translation, and selectable markers. Expression in Bacteria vs. Eukaryotes: o Bacterial Systems: Quick, cost-effective but may lack post-translational modifications. o Eukaryotic Systems (Yeast, Baculovirus): Suitable for complex proteins needing modifications. 9. Gene Libraries Genomic Library: Collections of DNA fragments representing an entire genome, including non-coding regions. cDNA Library: Made from mRNA, containing only expressed genes without introns. Useful for studying gene expression under specific conditions. 10. Transposable Elements (Transposons) Definition: Segments of DNA that can move within the genome, often called "jumping genes." Types:  o Insertion Sequences (IS Elements): Simplest form, containing inverted repeats and a transposase gene. o Transposons: Larger, can carry antibiotic resistance genes or other traits. Mechanisms: o Replicative (Copy-and-Paste): Leaves original copy and inserts a duplicate at a new site. o Non-replicative (Cut-and-Paste): Moves original copy entirely to a new location. 11. Genetic Material Exchange in Bacteria Transformation: Uptake of free DNA from the environment. Transduction: Transfer of DNA via bacteriophages (viruses that infect bacteria). Conjugation: Direct transfer of DNA between bacteria through a pilus (e.g., R-plasmids). Transposition: Transposons can “hop” into chromosomes, spreading genes like antibiotic resistance.