Genetic Engineering

Genetic Engineering Overview

  • Genetic Engineering: The process of intentionally altering an organism's DNA to change its form or function.

Modern Biotechnology

  • Biotechnology: A collection of techniques that use science and engineering to manipulate living organisms or their parts.

Plasmids

  • Plasids: Small circular DNA molecules that replicate independently of chromosomal DNA in cells.

Gene Cloning

  • Gene Cloning: A molecular biology technique that creates copies of a specific gene.

DNA Toolbox

  • Human Genome: Sequencing completed by 2007; DNA sequencing advances relied on techniques such as recombinant DNA.

  • Recombinant DNA: Combining nucleotide sequences from different sources in vitro to create new DNA molecules.

Methods of Genetic Engineering

  • Methods for Recombinant DNA: Central to genetic engineering; direct gene manipulation for practical purposes.

  • DNA Technology: Revolutionizes biotechnology for manipulating organisms or their genetic components.

  • Microarray Technology: Measures gene expression levels of thousands of genes concurrently.

Concept of DNA Cloning

  • DNA Cloning: Yields multiple copies of a gene segment; scientists prepare gene-sized pieces of DNA in identical copies.

DNA Cloning Techniques and Applications

  • Cloning Methods: Most methods utilize bacteria and plasmids; cloned genes enable gene and protein product creation.

  • Process: Foreign DNA is inserted into a plasmid, creating a recombinant plasmid, which is then inserted into a bacterial cell for reproduction.

  • Cloning Process Summary:

    • Gene of interest is isolated and inserted into a plasmid.

    • Plasmid is transformed into a bacterium, allowing for cloning.

Restriction Enzymes and Cloning

  • Restriction Enzymes: Cut DNA at specific sequences called restriction sites, producing fragments with "sticky ends".

  • DNA Ligase: Enzyme that seals bonds between restriction fragments.

Cloning Eukaryotic Genes in Bacterial Plasmids

  • Cloning Vector: The original plasmid that carries foreign DNA into host cells and replicates.

  • Steps to Clone Genes:

    • Isolation of genomic and plasmid DNA.

    • Digestion with the same restriction enzyme; bonding fragment sticky ends using DNA ligase.

Amplifying DNA

  • Polymerase Chain Reaction (PCR): Produces multiple copies of a specific DNA segment through a three-step cycle (denaturation, annealing, and extension).

Screening and Identifying Clones

  • Nucleic Acid Probes: Used for identifying clones carrying a gene of interest; hybridization detects complementary sequences.

DNA Libraries

  • Genomic Library: Collection of recombinant vector clones from an entire genome; can be constructed using bacteria or bacteriophages.

  • BACs and cDNA Libraries: Bacterial artificial chromosomes (BACs) can carry large inserts; cDNA libraries are derived from mRNA via reverse transcription, reflecting expressed genes.

Challenges in Cloning and Expression

  • Expression Systems: Bacterial systems face difficulties in expressing eukaryotic genes, requiring specially designed expression vectors.

  • Eukaryotic Systems: Utilize cultured cells and yeast artificial chromosomes (YACs) to manage gene expression and protein modifications.

Gene Cloning Process Steps

  1. Isolation of plasmid.

  2. Isolation of foreign DNA/gene of interest.

  3. Cutting plasmid with restriction enzymes.

  4. Cutting foreign DNA with the same enzyme.

  5. Insertion of foreign DNA to plasmid.

  6. Joining the DNA with DNA ligase.

  7. Transformation into a host cell.

  8. Reproduction and harvesting of proteins.