SCH2226 Human Molecular Genetics - Lecture 6: Recombinant DNA Technology

SCH2226 - Human Molecular Genetics

Focus Areas:   - Cloning Vectors   - Expression Vectors   - Plasmids   - Restriction Enzymes   - Genomic Library + cDNA library   - Southern Blotting   - Yeast 2 Hybrid

Cloning Vectors

  • Definition: Vectors that hold large fragments of DNA and increase the number of copies through replication.

  • Types of Cloning Vectors:   - Plasmid: Grows in bacteria; suitable for small DNA fragments.   - Bacteriophage: Grows in bacteria and destroys them.   - Yeast Artificial Chromosome (YAC): Suitable for very large inserts.

Expression Vectors

  • Definition: Vectors that contain promoters for DNA inserts, expressed only under certain conditions.

  • Purpose: To clone genes for the purpose of purifying proteins produced.

  • Special Features: Include components to control expression to avoid disruption of host cells.

  • Inducible promoters: Active only when an inducer molecule is present (e.g., IPTG with lac promoter from E. coli).

Cloning Vector pUC19

  • Structure Components:   - Contains an origin of replication (ori), ampicillin resistance marker (ampR), and a multiple cloning site (MCS).

  • Details:   - MCS within the β-galactosidase gene (lacZ) allows insertion of DNA fragments.   - Disruption of lacZ results in nonfunctional β-galactosidase in E. coli.

Blue-White Color Selection Test

  • Purpose: Select for vectors with or without inserts.

  • Mechanism:   - Functional β-galactosidase: IPTG broken down; no breakdown products yield blue colonies.   - Non-functional β-galactosidase: Presence of lacZ disruption; no IPTG breakdown leads to white colonies.

Restriction Enzymes

  • Definition: Molecular scissors that cut double-stranded DNA at specific recognition sequences (palindromes).

  • Source: Naturally found in various prokaryotes.

  • Functionality: Essential for manipulating DNA in recombinant technology.

Restriction Sites in DNA

  • Example Sequence: Palindrome structure (e.g., 5’-GAATTC-3’).

  • Occurrence Frequency:   - For EcoRI: Sequence 5’-GAATTC-3’ occurs once every 4,096 bp, calculated as rac1(1/4)6rac{1}{(1/4)^6} due to 6 bases in the recognition site.

Cloning DNA Using Restriction Enzymes

  • Process: Cut DNA with specific restriction enzymes (e.g., EcoRI) to produce sticky ends.

  • DNA Fragment Joining: Fragments can anneal and be sealed by DNA ligase to form recombinant DNA molecules.

Genomic Libraries

  • Definition: DNA fragments representing an organism's entire genome.

  • Creation: Produced by cutting genomic DNA into clonable-sized pieces using restriction endonucleases.

  • Contents: Whole genomic fragments, including exons, introns, promoters, etc.

cDNA Libraries

  • Definition: Libraries containing complementary DNA synthesized from mRNA.

  • Process: Reverse transcriptase uses mRNA as a template along with RNase H, DNA polymerase I, and DNA ligase to synthesize cDNA.

Southern Blotting

  • Definition: DNA analysis technique developed in 1975 by Edwin Southern.

  • Purpose: Confirm the presence of a specific DNA sequence inserted into the genome of a modified organism.

  • **Steps: **   1. DNA Digestion   2. Gel Electrophoresis   3. Blotting   4. Probe Labelling   5. Hybridisation and Washing   6. Detection

Protein-Protein Interactions (PPIs)

  • Definition: Physical interactions between two or more proteins. Classified as either stable or transient.

Yeast 2 Hybrid (Y2H)

  • Definition: A method to study protein interactions based on reconstituting a functional transcription factor when two proteins interact.

  • Mechanism:   - Involves genetically modified yeast strains where protein interaction leads to the expression of a reporter gene (commonly HIS3 or LacZ).

Limitations of Yeast Two-Hybrids

  • High false positives and negatives in screens.

  • Interactions must occur in the nucleus to be detected.

  • Proteins may fold or express differently in yeast than in humans.

Single Nucleotide Polymorphisms (SNPs)

  • Definition: Variations at a single base pair level in the genome.

  • Frequency: Occur once every 100-300 bases; contribute to 90% of human genome variation.

Identifying SNPs

  • Methods: Primers designed for PCR amplification followed by DNA sequencing.

Dideoxy DNA Sequencing Technique (Sanger Method)

  • Description: Uses dideoxynucleotides to terminate DNA strand elongation, allowing sequencing.

  • Components: Resemble normal nucleotides but lack a 3’ hydroxyl group.

Key Concepts Summary

  • Requirements in a Vector for DNA Manipulation.

  • Mapping and Generating Restriction Sites in Cloned DNA.

  • Expression of mRNA or Protein from Cloned Genes in Host Cells.

  • Finding Specific Genes in a DNA Library.

  • Identifying Protein Interactions.

  • Types of DNA Polymorphisms in the Genome.