Lecture 5

Question 1

What is a restriction digest?

Answer 1

A process where bacterial restriction enzymes cut DNA at specific sequences (e.g., EcoRI recognizes GAATTC).

Question 2

What are restriction enzymes?

Answer 2

Bacterial defense proteins that cut foreign DNA at palindromic sequences (e.g., GAATTC for EcoRI).

Question 3

What is the difference between sticky and blunt ends?

Answer 3

Sticky ends have single-stranded overhangs (e.g., EcoRI); blunt ends lack overhangs (e.g., SmaI).

Question 4

What conditions do restriction enzymes require?

Answer 4

37°C, Mg²⁺ ions, specific pH, and salt concentrations.

Question 5

How was DNA fingerprinting used in the Caulerpa taxifolia study?

Answer 5

To confirm Mediterranean samples matched aquarium strains via restriction digest and Southern blotting.

Question 6

What role did Southern blotting play in the Caulerpa study?

Answer 6

Transferred DNA to a membrane for radioactive probe hybridization, confirming clonal identity.

Question 7

What is the basis of Sanger sequencing?

Answer 7

Chain termination using ddNTPs lacking a 3’-OH group, halting DNA synthesis.

Question 8

How do ddNTPs differ from regular NTPs?

Answer 8

ddNTPs lack 3’-OH, terminating elongation; NTPs (dATP, dTTP, etc.) enable strand extension.

Question 9

How was the original Sanger method performed?

Answer 9

Four reactions, each with one ddNTP (ddATP, ddTTP, etc.), separated by gel electrophoresis.

Question 10

How is modern Sanger sequencing optimized?

Answer 10

Fluorescent ddNTPs in a single reaction, analyzed via capillary electrophoresis and laser detection.

Question 11

List key applications of DNA sequencing.

Answer 11

Primer design, cloning, species ID, phylogenetics, polymerase production, and coral reef monitoring.

Question 12

What is NGS?

Answer 12

High-throughput methods (e.g., Illumina) for rapid, cost-effective whole-genome sequencing.

Question 13

How is sequencing used in marine biology?

Answer 13

Detecting invasives (e.g., Caulerpa), studying coral-algae symbiosis, and conservation genetics.

Question 14

What was the zooxanthellae study’s aim?

Answer 14

To analyze genetic diversity in corals and anemones after aquarium culturing.

Question 15

What did the zooxanthellae study find?

Answer 15

Some algae strains were replaced by stress-tolerant variants under controlled conditions.

Question 16

How is sequencing used in recombinant protein studies?

Answer 16

To express and analyze marine proteins (e.g., coral fluorescent proteins).

Question 17

How do these tools aid conservation?

Answer 17

Tracking genetic diversity, monitoring reef health, and informing restoration strategies.

Question 18

Why do bacteria produce restriction enzymes?

Answer 18

To degrade viral DNA as a defense mechanism.

Question 19

How does gel electrophoresis work?

Answer 19

Separates DNA fragments by size using an electric field; smaller fragments migrate faster.

Question 20

What is molecular cloning?

Answer 20

Using restriction enzymes and ligases to insert DNA into vectors for replication.

Question 21

How does sequencing aid evolutionary biology?

Answer 21

By comparing DNA sequences to infer phylogenetic relationships (e.g., coral speciation).

Question 22

How is sequencing applied to coral reefs?

Answer 22

Identifying heat-tolerant genes, tracking bleaching responses, and assessing biodiversity.

Question 23

Name a biotech application of sequencing.

Answer 23

Producing polymerases (e.g., Taq polymerase) for PCR via recombinant DNA technology.

Question 24

How does sequencing study coral symbiosis?

Answer 24

Identifies zooxanthellae strains and their adaptation to environmental stress.

Question 25

What is recombinant DNA (rDNA)?

Answer 25

Artificially engineered DNA combining genetic material from different sources, creating sequences not found in nature.

Question 26

List four applications of recombinant DNA.

Answer 26

DNA cloning, protein expression (e.g., Pfu polymerase), genetic engineering, and producing therapeutics (e.g., insulin).

Question 27

What are the five steps in DNA cloning?

Answer 27

Isolation of target DNA, ligation into a vector, transformation, selection, and verification.

Question 28

How is target DNA isolated for cloning?

Answer 28

Extracted from a source organism and cut with restriction enzymes for insertion into a vector.

Question 29

What is ligation, and which enzyme facilitates it?

Answer 29

Joining DNA fragments into a vector using DNA ligase, forming covalent bonds between strands.

Question 30

Name two methods to introduce plasmids into bacteria.

Answer 30

Heat shock (temperature change) and electroporation (electric pulses).

Question 31

Why is selection necessary after transformation?

Answer 31

To identify bacteria with recombinant plasmids using antibiotic resistance or blue-white screening.

Question 32

How does antibiotic resistance screen for transformed bacteria?

Answer 32

Bacteria with plasmids (containing resistance genes) survive on antibiotic-laced agar (e.g., ampicillin).

Question 33

How does blue-white screening work?

Answer 33

Uses X-gal and lacZ gene disruption: white colonies (insert present) vs. blue colonies (no insert).

Question 34

How are positive clones verified?

Answer 34

Colony PCR checks for inserts; DNA sequencing confirms the sequence.

Question 35

What are plasmids, and why are they used?

Answer 35

Small circular bacterial DNA; they autonomously replicate and carry foreign DNA.

Question 36

List five key features of plasmids.

Answer 36

Replication origin, MCS (cloning sites), selectable markers (e.g., antibiotic resistance), promoter, and polyhistidine tags.

Question 37

How do restriction enzymes aid cloning?

Answer 37

Cut DNA at specific sites (sticky/blunt ends) for insertion into vectors.

Question 38

What is the role of DNA ligase?

Answer 38

Catalyzes phosphodiester bonds between vector and insert DNA during ligation.

Question 39

Describe the heat shock transformation process.

Answer 39

Bacteria + plasmid exposed to sudden temperature change, creating membrane pores for DNA uptake.

Question 40

How does electroporation work?

Answer 40

High-voltage pulses create temporary membrane pores, allowing plasmid entry into bacteria.

Question 41

What is the purpose of selectable markers in plasmids?

Answer 41

To ensure only bacteria with the plasmid survive (e.g., ampicillin resistance gene).

Question 42

Why do white colonies indicate successful cloning?

Answer 42

Insert disrupts lacZ gene, preventing β-galactosidase from cleaving X-gal into blue pigment.

Question 43

What is colony PCR used for?

Answer 43

Amplifies plasmid DNA from bacterial colonies to confirm insert presence.

Question 44

Why is DNA sequencing used post-cloning?

Answer 44

To validate the inserted DNA sequence matches the target gene.

Question 45

Give a biotech example of recombinant DNA.

Answer 45

Producing Taq polymerase for PCR via bacterial expression systems.

Question 46

Name a protein produced using recombinant DNA.

Answer 46

Insulin (medicine) or Pfu polymerase (PCR).

Question 47

Why do plasmids have a Multiple Cloning Site (MCS)?

Answer 47

Contains restriction sites for flexible insertion of foreign DNA.

Question 48

Match techniques to purposes: Recombinant DNA, DNA Ligase, Blue-White Screening.

Answer 48

Recombinant DNA: Genetic modification; DNA Ligase: Joins DNA fragments; Blue-White Screening: Confirms cloning success.

Question 49

How is recombinant DNA used in marine research?

Answer 49

Studying coral symbiosis, engineering stress-tolerant algae, and producing marine proteins (e.g., fluorescent proteins).