E3

Recombinant DNA Technology

Overview

• Recombinant DNA: artificially manipulated DNA formed from the joining of DNA from different biological sources.

• Applications of Recombinant DNA Technology:

  • Isolate and manipulate genes of interest.

  • Clone DNA to make many copies.

  • Analyze cloned DNA and its gene products.

Key Components

Restriction Enzymes

• Cut DNA at specific sequences, typically palindromic.

• Creates restriction fragments that are utilized for cloning.

Vectors

• Vectors: plasmids used as carrier molecules for DNA cloning.

  • Must have:

    • Replication origin: allows for independent replication.

    • Polylinker: region containing several restriction sites for insertion of DNA.

    • Selectable marker gene: identifies host cells containing recombinant DNA.

• Example: R plasmid that contains an antibiotic resistance gene.

Cloning Process

1. DNA purification: Isolate DNA from cells/tissues.

2. Restriction enzyme digestion: Cut DNA into specific fragments using restriction enzymes.

3. Joining insert to vector: ligate the DNA fragment of interest (insert) to a plasmid (vector).

• Resulting in recombinant DNA.

4. Transformation: Transfer recombinant DNA into host cells (e.g., bacteria) for replication.

5. Replication: As bacteria replicate, the recombinant DNA is also replicated.

6. Recovery: Cloned DNA can be recovered, purified, and analyzed.

7. Expression: Cloned DNA can be transcribed and translated in the host to study mRNA and proteins.

Polymerase Chain Reaction (PCR)

• Technique for amplifying DNA sequences rapidly, producing billions of copies in hours.

• Requirements:

  • Template DNA: little quantity needed (e.g., genomic DNA).

  • Primers: short oligonucleotides that flank the target region.

  • DNA polymerase and nucleotides (dNTPs).

• Cycle:

  1. Denaturation: Heat to break hydrogen bonds, separating strands at ~95°C.

  2. Primer Annealing: Cool to allow primers to bind (45-65°C).

  3. Extension: Raise temperature to ~72°C for DNA synthesis, repeated 25-40 times.

DNA Libraries

• Library: a collection of DNA clones from a single source.

• Types:

  • Genomic Library: contains clones representing all genomic sequences.

  • Chromosome Library: similar to genomic but for a specific chromosome.

  • cDNA Library: contains clones of expressed genes at a specific time.

  • Screening: retrieving specific clones using labeled probes.

Gene Editing: CRISPR-Cas9

• Utilizes guide RNA (sgRNA) and Cas9 enzyme to create double-stranded breaks in specific DNA regions.

• Enables precise edits through non-homologous end joining (NHEJ) or homology-directed repair (HDR).

Cancer Genetics

Overview

• Cancer defined by:

  • Abnormal proliferation of cells and metastasis.

  • Different cancers (e.g., breast, colon) show varied properties and behaviors.

Genetic Mechanisms

• Cancer arises from mutations in specific genes:

  • Proto-oncogenes: regulate cell growth and division; mutated versions can lead to cancer.

  • Tumor suppressor genes: normally inhibit cell division; mutations lead to unchecked growth.

  • Example: p53 protein regulates response to DNA damage; mutations in 50% of cancers.

Treatment Approaches

• Traditional methods target rapidly dividing cells (chemotherapy/radiation).

• Newer approaches involve genetic profiling to personalize treatment, such as TPMT genotype for 6MP dosing in leukemia.

Translation

Major Steps

1. Initiation: mRNA binds to the ribosomal subunit, initiator tRNA binds to start codon (AUG).

2. Elongation: tRNAs bring amino acids; peptide bonds form, and the ribosome moves along mRNA.

3. Termination: Release factors bind to stop codons, releasing the newly synthesized polypeptide.

tRNA and Ribosomes

• tRNA: unique structure with an anticodon, allows specific pairing during protein synthesis.

• Ribosome: complex of rRNA and proteins, varies in size between prokaryotes (70S) and eukaryotes (80S).

Gene Regulation in Bacteria and Eukaryotes

Bacterial Regulation

• Operons control gene expression (Lac operon for lactose metabolism).

• Default behaviors:

  • Inducible: turned on when substrates are present.

  • Repressible: turned off when products accumulate.

Eukaryotic Regulation

• More complex regulation at multiple levels (transcription, post-transcription).

• Chromatin remodeling: influences access to DNA, involves histone modifications and DNA methylation.

Post-Transcriptional Regulation

• Includes alternative splicing, which leads to different protein isoforms based on tissue type or conditions.

• RNA interference (RNAi) can silence gene expression post-transcriptionally.