Vectors and Cloning of DNA

Question 1

What is vector DNA and why is it used in cloning?

Answer 1

Vector DNA carries and replicates foreign DNA in host cells, enabling gene cloning.

Question 2

What are common types of vectors used in cloning?

Answer 2

Plasmids, bacteriophages, artificial chromosomes (BACs/YACs), cosmids, and DNA libraries.

Question 3

What key features make plasmids useful cloning vectors?

Answer 3

Origin of replication, selectable marker (e.g., antibiotic resistance), and multiple cloning site (MCS).

Question 4

What is a DNA library?

Answer 4

A collection of DNA fragments cloned into vectors, representing either the whole genome or expressed genes (cDNA).

Question 5

Why is cDNA used instead of genomic DNA in cloning eukaryotic genes?

Answer 5

cDNA lacks introns, making it expressible in prokaryotes which can’t splice RNA.

Question 6

What are the advantages of artificial chromosomes like BACs and YACs?

Answer 6

They carry much larger DNA fragments than plasmids, useful for genome-scale projects.

Question 7

What is Gateway cloning and why is it used?

Answer 7

A recombination-based method for fast, enzyme-free gene cloning using site-specific recombination.

Question 8

What is a fusion protein and how is it made in cloning?

Answer 8

A combined protein made by cloning two coding sequences together without a stop codon between them.

Question 9

What is the role of the F factor in bacterial gene transfer?

Answer 9

It enables DNA transfer through conjugation and can be used as a cloning vector.

Question 10

What is blue-white screening?

Answer 10

A method using lacZ gene disruption to identify recombinant plasmids—white colonies have inserts.

Question 11

What is the difference between high-copy and low-copy plasmids?

Answer 11

High-copy plasmids replicate more frequently, yielding more DNA/protein; low-copy ones yield less but are more stable.

Question 12

Why use vectors to clone an entire pathway?

Answer 12

To express multiple genes in a host and reconstruct metabolic pathways for research or production.

Question 13

What is DNA cloning and how does it differ from organism cloning?

Answer 13

DNA cloning is inserting foreign DNA into a vector to replicate in a host cell, unlike organism cloning which creates whole identical organisms.

Question 14

Why is E. coli commonly used in DNA cloning?

Answer 14

It’s easy to manipulate, grows quickly, and supports plasmid replication, making it ideal for initial cloning steps.

Question 15

What types of host cells can be used for expressing cloned DNA?

Answer 15

Bacterial (E. coli), yeast, mammalian, insect, and plant cells, depending on the application.

Question 16

What are the essential components needed for DNA cloning?

Answer 16

Foreign DNA, a vector, a host cell, a method to insert DNA into the vector, a method to insert the vector into the host, and a selection method.

Question 17

What is the purpose of cloning DNA into a vector?

Answer 17

To produce multiple copies, study gene/protein function, sequence DNA, detect mutations, or create a DNA library.

Question 18

What is TA cloning and how does it work?

Answer 18

A method using Taq polymerase-generated A overhangs on PCR products and T overhangs on vectors to enable ligation via base pairing.

Question 19

How does Topo cloning improve on TA cloning?

Answer 19

It uses topoisomerase to recognize specific sequences and join DNA more efficiently without needing ligase.

Question 20

What is restriction enzyme digestion and ligation in cloning?

Answer 20

A method where restriction enzymes cut both vector and insert to create compatible sticky ends, which are then ligated.

Question 21

What are sticky ends and why are they important in cloning?

Answer 21

Single-stranded overhangs created by restriction enzymes that help the insert base pair with the vector for ligation.

Question 22

What is end-tailoring in cloning?

Answer 22

Adding restriction sites to PCR primers to create compatible ends for digestion and insertion into vectors.

Question 23

What does ligation do in DNA cloning?

Answer 23

DNA ligase forms covalent bonds to seal the phosphate backbone between vector and insert, stabilizing the recombinant DNA.

Question 24

What components are needed for a ligation reaction?

Answer 24

DNA fragments, a buffer with ATP, and DNA ligase.

Question 25

What is transformation in DNA cloning?

Answer 25

Introduction of recombinant DNA into a host cell, usually E. coli.

Question 26

What makes a bacterial cell competent?

Answer 26

Its ability to take up foreign DNA from the environment, often induced by heat shock or electroporation.

Question 27

How does heat shock transformation work?

Answer 27

Cells are rapidly heated (e.g., 0°C to 42°C), destabilizing their membranes and allowing DNA uptake.

Question 28

What is electroporation?

Answer 28

A method of using an electric field to open cell membranes for DNA entry.

Question 29

What are conjugation and transfection?

Answer 29

Conjugation is DNA transfer via a bacterial "sex pilus"; transfection uses bacteriophages to deliver DNA.

Question 30

What is a selectable marker and how is it used?

Answer 30

A gene (e.g., antibiotic resistance) that allows only transformed cells to survive on selective media.

Question 31

How does blue-white screening work?

Answer 31

insertional inactivation of the lacZ gene prevents β-galactosidase production, making colonies appear white (insert present) instead of blue.

Question 32

What do blue vs white colonies mean in blue-white screening?

Answer 32

Blue = functional lacZ, no insert. White = disrupted lacZ, insert present.

Question 33

What is a DNA vector?

Answer 33

A DNA molecule used to carry foreign genetic material into another cell for replication or expression.

Question 34

What are common types of DNA vectors?

Answer 34

Plasmids, cosmids, artificial chromosomes, and bacteriophages.

Question 35

What is the purpose of an antibiotic resistance gene in a plasmid?

Answer 35

To allow selection of cells that have successfully taken up the plasmid by growing only those that can survive the antibiotic.

Question 36

What is a multiple cloning site (MCS)?

Answer 36

A short region of DNA with many restriction sites, allowing easy insertion of foreign DNA.

Question 37

What is blue/white screening used for?

Answer 37

To distinguish between bacteria that have taken up plasmids with or without an insert.

Question 38

What gene is used in blue/white screening?

Answer 38

The lacZ gene, which encodes β-galactosidase.

Question 39

What causes a colony to be blue in blue/white screening?

Answer 39

The lacZ gene is functional, so β-galactosidase cleaves X-gal, producing a blue product.

Question 40

What causes a colony to be white in blue/white screening?

Answer 40

The lacZ gene is disrupted by the inserted DNA, preventing X-gal cleavage.

Question 41

What does it mean if a colony is white?

Answer 41

The bacteria contain the plasmid with the inserted DNA of interest.

Question 42

What happens to cells without a plasmid on an antibiotic plate?

Answer 42

They do not grow, as they lack antibiotic resistance.

Question 43

How are successful colonies preserved for future use?

Answer 43

By creating glycerol stocks, which are frozen at -80°C.

Question 44

What are the steps to expand a positive colony?

Answer 44

Pick a colony → inoculate liquid culture → grow bacteria → make glycerol stock or extract plasmid.

Question 46

What is Cas9?

Answer 46

A CRISPR-associated (Cas) protein that acts as a programmable endonuclease to cut DNA.

Question 47

What is the natural role of CRISPR in bacteria?

Answer 47

It functions as an adaptive immune system that stores fragments of viral DNA and uses them to recognize and destroy future viral invaders.

Question 48

What is a guide RNA (gRNA)?

Answer 48

A synthetic RNA combining crRNA and tracrRNA, directing Cas9 to a specific DNA sequence

Question 49

What is the PAM sequence?

Answer 49

Protospacer Adjacent Motif, typically NGG, required for Cas9 to bind and cut target DNA.

Question 50

Why doesn’t Cas9 cut the CRISPR array in the bacterial genome?

Answer 50

The CRISPR array lacks the PAM sequence, which is essential for Cas9 activity.

Question 51

What happens after Cas9 makes a double-stranded break?

Answer 51

The cell attempts repair via non-homologous end joining (NHEJ) or homology-directed repair (HDR), which can introduce mutations or insert new DNA.

Question 52

What is CRISPR-Cas9 and what does it do?

Answer 52

It’s a genome editing tool that acts like molecular scissors to cut DNA at specific sites.

Question 53

Who won the Nobel Prize for CRISPR-Cas9 and for what contributions?

Answer 53

Emmanuelle Charpentier (characterized CRISPR system) and Jennifer Doudna (recreated and reprogrammed Cas9 in vitro).

Question 54

How does CRISPR-Cas9 knock out a gene?

Answer 54

By introducing small insertions or deletions (indels) during repair of a Cas9-induced DNA cut, disrupting the gene.

Question 55

How can CRISPR-Cas9 be used for precise gene editing?

Answer 55

By providing a DNA template during repair to insert or replace specific sequences.

Question 56

What are CRISPRa and CRISPRi used for?

Answer 56

Activating (CRISPRa) or repressing (CRISPRi) gene expression without cutting the DNA.

Question 57

What is the PAM sequence?

Answer 57

A short DNA motif required for Cas9 to bind and cleave DNA.

Question 58

Why are controls essential in CRISPR experiments?

Answer 58

To account for off-target effects where Cas9 cuts unintended parts of the genome.

Question 59

What is DNA-free CRISPR-Cas9 editing?

Answer 59

Delivering Cas9 protein and guide RNA directly to cells without inserting any foreign DNA.

Question 60

What are some practical uses of CRISPR?

Answer 60

Gene knockout, crop improvement, disease research, gene regulation, and creating malaria-resistant mosquitoes

Question 61

What are the three natural components of a CRISPR locus?

Answer 61

CAS genes (coding Cas proteins), leader sequence (promoter), and repeat-spacer array (target memory).