Week 4 - Recombinant DNA Techniques & DNA Sequencing

Enzymes purified from bacteria that cut DNA at specific sites; act as molecular scissors to defend against foreign DNA.
Type II restriction endonucleases: cleavage site is within or close to the recognition site; most recognition sites are $4$–$8$ nucleotides long and palindromic; require Mg}^{2+}$ for activity; can cut to blunt ends or generate sticky ends.
Recognition sites are sequence-specific and often palindromic; EcoRI example creates sticky ends from GAATTC / CTTAAG with 5′ overhangs; cleavage pattern yields complementary, directional ends.
Host bacteria protect their own DNA by methylating recognition sites; EcoRI will not cleave methylated DNA (restriction–modification system).
Common enzymes include EcoRI, HindIII, PstI, HpaI, HhaI; named after the source organism.

Fragment genomic DNA into manageable pieces using restriction enzymes; generates reproducible fragments for analysis.
Fragments are ligated into plasmid vectors cut with the same enzymes by DNA ligase to form recombinant DNA.
Competent bacteria are transformed with recombinant plasmids; selective antibiotics enable growth of transformed cells.
Directional cloning: use different restriction enzymes at each end to control orientation of the insert in the vector.
Blue–white screening (LacZ disruption): insert disrupts β-galactosidase; white colonies indicate presence of insert; blue colonies indicate no insert.

Components: promoter, ribosome-binding site (RBS), transcription terminator, multiple cloning site (MCS)/polylinker, selectable antibiotic resistance marker, and optional purification tag.
Inducible expression: regulatory elements (repressor/operator) allow controlled expression.
Directional cloning via MCS ensures correct orientation for transcription.

Expression hosts: Escherichia coli, yeast, insect, or mammalian cells; choice affects yield, folding, and activity.
Considerations: promoter strength, ribosome binding, folding chaperones, post-translational modifications, and potential need for purification tags.
Applications: production of vaccines/therapeutics (e.g., insulin, HBV/HPV vaccines, antibodies), enzymes, biomaterials, and gene therapy vectors.

Site-directed mutagenesis: PCR with modified primers to introduce mutations; template DNA is degraded with methylation-dependent endonuclease (e.g., DpnI).
Gene replacement/knockout/addition: replace a normal gene, inactivate a gene, or insert a mutant gene to study function.
Conditional mutations: regulatory DNA sequences enable selective control of gene expression; tissue-specific activation possible.
Genome editing tools: CRISPR-Cas9, TALENs, Zinc Finger Nucleases (ZFNs) for targeted genome modification.

Principle: DNA synthesis terminates at ddNTPs lacking 3′-OH; four reactions with ddATP, ddTTP, ddCTP, ddGTP in separate tubes produce fragments terminating at each nucleotide.
Separation by gel electrophoresis; labeled fragments detected to infer sequence complementary to the template strand.
Automation: fluorescently labeled ddNTPs in a single-tube reaction; four colors detected by a laser; readout is the sequencing trace.
Limitations: short read lengths; large genomes require fragmentation and overlap assembly (shotgun sequencing).

Shotgun sequencing: long DNA broken into random fragments; sequence reads are assembled by overlaps to reconstruct the genome.
Next-Generation Sequencing (NGS): sequencing by synthesis on beads/slides; massively parallel sequencing; depth/reads define coverage and accuracy; up to billions of clusters.
Read depth (coverage) is the average number of reads covering a position; higher depth increases confidence.

Genome annotation relies on sequence homology (comparative genomics) and genetics/biochemistry to assign function to genes.
Gene families: many genes are related; some essential genes are highly conserved across species.
Noncoding DNA: introns, regulatory elements, and repetitive sequences; important for regulation and genome organization.
Costs of sequencing have fallen dramatically over time: $(3 imes 10^{8})$ USD (early estimates) in 2003 to ≲ $(1 imes 10^{3})$ USD per genome by 2020; Archon X Prize highlighted rapid genome sequencing.
Model organisms (yeast, nematode, fruit fly, zebrafish, mouse) have revealed fundamental cellular principles transferable to humans.

Diagnostics: identify disease-causing genes and genetic variants.
Personalized medicine: sequencing informs targeted therapies and risk assessment.
Biotechnology: transgenic organisms, production of vaccines, enzymes, and biofuels; gene therapy vectors.
Population genetics & evolution: compare genomes across populations to trace history and variation.

Genome organization includes coding and noncoding regions; many regulatory elements control gene expression.
Protein-coding genes vs. noncoding DNA; conservation helps predict gene function.
Tools and databases support annotation (e.g., model organism resources, genome browsers).

Model organisms reveal core cellular processes that are conserved across species.
Examples: Yeast, Worm (C. elegans), Flies (Drosophila), Zebrafish, Chickens, Mouse.
Studies in models underpin understanding of human biology and disease.

Restriction enzymes generate reproducible fragments; blunt vs sticky ends affect ligation strategy.
Cloning couples restriction digestion with ligation and transformation, followed by selection and screening.
Expression vectors require proper regulatory elements for controlled protein production.
Site-directed mutagenesis and genome editing enable precise genetic changes.
Sanger sequencing laid the groundwork; NGS enables large-scale genome projects with high depth and speed.
The genome projects inform diagnostics, therapeutics, and our understanding of biology across species.