Molecular Biology Techniques I: DNA Cloning and Vectors

Lecture by Prof Guy Tear

Learning Outcomes

• Understand recombinant DNA
• Steps of molecular cloning
• Function and use of restriction enzymes
• Properties and uses of plasmids
• Production of genomic libraries
• Nature and production of cDNA and cDNA libraries
• Difference between cloning and expression plasmids
• Principles of nucleic acid hybridization
• Usage and labeling of DNA probes
• Southern blot method and its application

Genes to DNA

• DNA as genetic material
• Isolation and manipulation of DNA
  • Understand gene function
  • Manufacture proteins (e.g., insulin)
  • Create transgenic organisms
  • Diagnose genetic diseases
  • Develop gene therapy

Recombinant DNA Technology

• Allows isolation and manipulation of DNA
• Gene Cloning creates multiple copies of DNA sections
• Enable understanding and manufacturing using DNA

DNA Cloning

• Methods:
  • Copy DNA using cloning vectors (e.g., plasmids)
  • Cutting DNA with restriction enzymes
• Molecular Cloning:
  • DNA fragments are inserted into vectors to create recombinant DNA
  • Introduction into host organisms for replication
  • Resulting in identical DNA or molecular clones

Restriction Enzymes

• Function as molecular scissors cutting DNA at specific sequences
• Recognize specific palindromic nucleotide sequences
• Cut to leave cohesive (sticky) or blunt ends
• Used in mapping and analyzing DNA

Recombinant DNA

• Amplification using bacteria or yeast
• Plasmids as common vectors:
  • Contain origin of replication and antibiotic resistance for selection
  • Include multiple cloning sites (MCS)

Cloning DNA

• DNA fragments and vectors cut with the same restriction enzymes
• Cohesive ends anneal and are ligated by DNA ligase
• E. coli used for plasmid amplification

Uses of Cloned DNA

• Sequencing genomes to map and understand gene organization
• Genetic engineering and protein expression

cDNA Libraries

• Creation from mRNA (introns removed)
• Reflect genes expressed in particular tissues
• cDNAs used in expression vectors for eukaryotic systems

Expression Plasmids

• Designed for gene expression in bacteria or eukaryotic cells
• Include sequences to enable transcription and translation

Nucleic Acid Hybridization

• Identification of specific nucleic acid sequences
• Utilizes complementarity of DNA/RNA strands
• Probes used to identify sequences

Southern Blot

• Identify specific DNA fragments post-restriction digestion
• DNA transferred to membrane, probed with labeled DNA
• Used to identify genomic or cloned fragments

Summary

• Techniques discussed are foundational for modern molecular biology and applications in research, therapeutics, and biotechnology.