Recombinant DNA and Protein Expression

Gene of Interest and Protein Expression

• Genes of interest can be linked to specific diseases, with associated defects.
• Copies of these genes are used in basic research and various applications, leading to protein harvesting.
• Organisms can be engineered for purposes like insulin production and insect resistance, referred to as "pharm" animals.

Recombinant DNA Products

• Notable recombinant DNA products include:
  • Human Insulin
  • Human growth factor
  • Hepatitis B virus vaccine
  • Streptokinase, which dissolves blood clots
  • Recombinant antibodies and enzymes

Regulations for Recombinant DNA

• International and national regulations oversee organisms that use recombinant DNA (rDNA).
• Special permissions and facilities are required for the use of rDNA organisms and products.

DNA Cloning Process

• DNA cloning involves several steps:
  • Extraction of DNA from blood or tissue.
  • PCR/RE digestion of DNA and vector.
  • Ligation of DNA into the vector.
  • Transformation into a suitable host.
  • Screening for positive clones.
  • Expression of desired products in the host.

Nucleic Acids Isolation

• Genomic DNA is the complete DNA content used for full coding regions of prokaryotes.
• cDNA is synthesized from mRNA, essential for eukaryotic coding regions.

Genomic DNA Isolation

• Involves lysis of cells with detergent, protein digestion, and DNA precipitation.
• Key steps:
  • Use of Proteinase K to digest proteins.
  • DNA is purified using phenol, chloroform, or salt, followed by isopropanol precipitation.

cDNA Synthesis

• Begins with purification of mRNA followed by synthesizing first and second strands of cDNA.
• Cloning of cDNA is the final step for applications.

Transcription and RNA Processing

• Exons represent coding regions separated by non-coding introns.
• Processed mRNA results from intron splicing, leading to the final transcript.

Molecular Scissors: Restriction Endonucleases

• Discovery in 1962 by Werner Arber, who identified enzymes that cut DNA.
• Restriction endonucleases (REs) recognize specific sequences to cleave DNA.
• REs are critical for recombinant DNA technology, categorized into:
  • Type I: Multifunctional, random cutting.
  • Type II: Specific, used in cloning.
  • Type III: Recognizes non-palindromic sequences, cut near recognition sites.

Characteristics of Type II Restriction Enzymes

• Single subunit enzymes requiring Mg2+ and recognizing palindromic sequences (4-8 nucleotides).
• Specific cleavage closely at recognition sites, facilitating gene cloning.

Nomenclature of Restriction Enzymes

• Named after the genus and species of the host organism, with modifiers for strains.
• Prefix 'R' for REs and 'M' for modification enzymes.

DNA Ligases in Cloning

• Used to join DNA fragments, with T4 phage ligase being commonly utilized.
• Ligases enable the formation of continuous DNA from cut fragments, essential for creating recombinant DNA.

DNA Polymerase Function

• DNA polymerase adds nucleotides in a 5’-3’ direction and requires a primer for initiation.
• Proofreading mechanisms are in place for error correction during DNA strand synthesis.

This quiz covers the essentials of recombinant DNA technology, including gene manipulation, protein expression, and the applications of genetically engineered organisms. Additionally, it explores notable recombinant products and the regulations surrounding their use. Test your knowledge on DNA cloning processes and the significance of these biotechnological advancements.