Lecture 5 Flashcards
What is a restriction digest?
A process where bacterial restriction enzymes cut DNA at specific sequences (e.g., EcoRI recognizes GAATTC).
What are restriction enzymes?
Bacterial defense proteins that cut foreign DNA at palindromic sequences (e.g., GAATTC for EcoRI).
What is the difference between sticky and blunt ends?
Sticky ends have single-stranded overhangs (e.g., EcoRI); blunt ends lack overhangs (e.g., SmaI).
What conditions do restriction enzymes require?
37°C, Mg²⁺ ions, specific pH, and salt concentrations.
How was DNA fingerprinting used in the Caulerpa taxifolia study?
To confirm Mediterranean samples matched aquarium strains via restriction digest and Southern blotting.
What role did Southern blotting play in the Caulerpa study?
Transferred DNA to a membrane for radioactive probe hybridization, confirming clonal identity.
What is the basis of Sanger sequencing?
Chain termination using ddNTPs lacking a 3’-OH group, halting DNA synthesis.
How do ddNTPs differ from regular NTPs?
ddNTPs lack 3’-OH, terminating elongation; NTPs (dATP, dTTP, etc.) enable strand extension.
How was the original Sanger method performed?
Four reactions, each with one ddNTP (ddATP, ddTTP, etc.), separated by gel electrophoresis.
How is modern Sanger sequencing optimized?
Fluorescent ddNTPs in a single reaction, analyzed via capillary electrophoresis and laser detection.
List key applications of DNA sequencing.
Primer design, cloning, species ID, phylogenetics, polymerase production, and coral reef monitoring.
What is NGS?
High-throughput methods (e.g., Illumina) for rapid, cost-effective whole-genome sequencing.
How is sequencing used in marine biology?
Detecting invasives (e.g., Caulerpa), studying coral-algae symbiosis, and conservation genetics.
What was the zooxanthellae study’s aim?
To analyze genetic diversity in corals and anemones after aquarium culturing.
What did the zooxanthellae study find?
Some algae strains were replaced by stress-tolerant variants under controlled conditions.
How is sequencing used in recombinant protein studies?
To express and analyze marine proteins (e.g., coral fluorescent proteins).
How do these tools aid conservation?
Tracking genetic diversity, monitoring reef health, and informing restoration strategies.
Why do bacteria produce restriction enzymes?
To degrade viral DNA as a defense mechanism.
How does gel electrophoresis work?
Separates DNA fragments by size using an electric field; smaller fragments migrate faster.
What is molecular cloning?
Using restriction enzymes and ligases to insert DNA into vectors for replication.
How does sequencing aid evolutionary biology?
By comparing DNA sequences to infer phylogenetic relationships (e.g., coral speciation).
How is sequencing applied to coral reefs?
Identifying heat-tolerant genes, tracking bleaching responses, and assessing biodiversity.
Name a biotech application of sequencing.
Producing polymerases (e.g., Taq polymerase) for PCR via recombinant DNA technology.
How does sequencing study coral symbiosis?
Identifies zooxanthellae strains and their adaptation to environmental stress.
What is recombinant DNA (rDNA)?
Artificially engineered DNA combining genetic material from different sources, creating sequences not found in nature.
List four applications of recombinant DNA.
DNA cloning, protein expression (e.g., Pfu polymerase), genetic engineering, and producing therapeutics (e.g., insulin).
What are the five steps in DNA cloning?
Isolation of target DNA, ligation into a vector, transformation, selection, and verification.
How is target DNA isolated for cloning?
Extracted from a source organism and cut with restriction enzymes for insertion into a vector.
What is ligation, and which enzyme facilitates it?
Joining DNA fragments into a vector using DNA ligase, forming covalent bonds between strands.
Name two methods to introduce plasmids into bacteria.
Heat shock (temperature change) and electroporation (electric pulses).
Why is selection necessary after transformation?
To identify bacteria with recombinant plasmids using antibiotic resistance or blue-white screening.
How does antibiotic resistance screen for transformed bacteria?
Bacteria with plasmids (containing resistance genes) survive on antibiotic-laced agar (e.g., ampicillin).
How does blue-white screening work?
Uses X-gal and lacZ gene disruption: white colonies (insert present) vs. blue colonies (no insert).
How are positive clones verified?
Colony PCR checks for inserts; DNA sequencing confirms the sequence.
What are plasmids, and why are they used?
Small circular bacterial DNA; they autonomously replicate and carry foreign DNA.
List five key features of plasmids.
Replication origin, MCS (cloning sites), selectable markers (e.g., antibiotic resistance), promoter, and polyhistidine tags.
How do restriction enzymes aid cloning?
Cut DNA at specific sites (sticky/blunt ends) for insertion into vectors.
What is the role of DNA ligase?
Catalyzes phosphodiester bonds between vector and insert DNA during ligation.
Describe the heat shock transformation process.
Bacteria + plasmid exposed to sudden temperature change, creating membrane pores for DNA uptake.
How does electroporation work?
High-voltage pulses create temporary membrane pores, allowing plasmid entry into bacteria.
What is the purpose of selectable markers in plasmids?
To ensure only bacteria with the plasmid survive (e.g., ampicillin resistance gene).
Why do white colonies indicate successful cloning?
Insert disrupts lacZ gene, preventing β-galactosidase from cleaving X-gal into blue pigment.
What is colony PCR used for?
Amplifies plasmid DNA from bacterial colonies to confirm insert presence.
Why is DNA sequencing used post-cloning?
To validate the inserted DNA sequence matches the target gene.
Give a biotech example of recombinant DNA.
Producing Taq polymerase for PCR via bacterial expression systems.
Name a protein produced using recombinant DNA.
Insulin (medicine) or Pfu polymerase (PCR).
Why do plasmids have a Multiple Cloning Site (MCS)?
Contains restriction sites for flexible insertion of foreign DNA.
Match techniques to purposes: Recombinant DNA, DNA Ligase, Blue-White Screening.
Recombinant DNA: Genetic modification; DNA Ligase: Joins DNA fragments; Blue-White Screening: Confirms cloning success.
How is recombinant DNA used in marine research?
Studying coral symbiosis, engineering stress-tolerant algae, and producing marine proteins (e.g., fluorescent proteins).
