L14 Classical Gene Cloning

Question 1

What does “cloning” mean in molecular biology?

Answer 1

copying or replicating DNA

Question 2

What is molecular cloning?

Answer 2

copying DNA in a living cell, possibly to express it

Question 3

What is a cloning vector?

Answer 3

a DNA molecule that can replicate in a host cell and carries the DNA of interest

Question 4

What is a recombinant vector?

Answer 4

a vector that has had foreign DNA inserted into it

Question 5

What is a host in gene cloning?

Answer 5

an organism used to replicate the recombinant DNA (e.g. E.coli)

Question 6

What are two outcomes of cloning DNA?

Answer 6

Replication and/or expression

Question 7

What is one reason to clone DNA in a cell instead of a test tube?

Answer 7

to express the DNA and produce a protein

Question 8

What is required for a host cell to replicate a DNA sequence?

Answer 8

a cloning vector with the proper signals for replication

Question 9

Name two things you can do with cloned DNA.

Answer 9

Alter its sequence or produce protein

Question 10

What must you know to perform PCR cloning?

Answer 10

the sequence at the ends of the DNA region

Question 11

What technique is used to amplify a DNA sequence?

Answer 11

PCR

Question 12

What is ligation in the context of cloning?

Answer 12

joining DNA fragments into a cloning vector

Question 13

Why is ligation essential in PCR cloning?

Answer 13

it physically joins the PCR product (the insert) to the DNA vector (like a plasmid), forming a recombinant DNA molecule

Question 14

Can mutations be introduced during PCR?

Answer 14

yes

Question 15

What is shotgun cloning used for?

Answer 15

to find unknown DNA sequences related to a phenotype or protein

Question 16

What do you create by inserting genome fragments into vectors?

Answer 16

a genomic library

Question 17

What is a recombinant clone?

Answer 17

a host cell containing a recombinant vector with an inserted genome fragment

Question 18

How is a genomic library made?

Answer 18

by cutting a genome, inserting fragments into vectors, and transforming a host

Question 19

what is the purpose of selecting recombinants in a genomic library?

Answer 19

to find the clone containing the DNA of interest

Question 20

What can you do after identifying the right recombinant in shotgun cloning?

Answer 20

purify and sequence the DNA, or introduce mutations

Question 21

What is subcloning?

Answer 21

moving a gene from one vector to another

Question 22

Why subclone a gene into another vector?

Answer 22

for expression or transfer into a new host

Question 23

What vectors might be used for subcloning?

Answer 23

Expression vectors, shuttle vectors, broad host range vectors

Question 24

what is a shuttle vector?

Answer 24

a vector that works in multiple species

Question 25

What is a broad host range vector (BHRV)?

Answer 25

a vector that replicated in many bacterial species

Question 26

When might you introduce mutations in subcloning?

Answer 26

before or after transferring the gene

Question 27

What is pUC18?

Answer 27

a plasmid cloning vector

Question 28

How big is pUC18?

Answer 28

2,600 base pairs

Question 29

What was pUC18 derived from?

Answer 29

the natural plasmid oMB1 from E.coli

Question 30

What function has pUC18 lost from its natural plasmid ancestor?

Answer 30

Mobilization and transfer to other bacteria - making it lab safe

Question 31

Why is pUC18 only able to replicate in E.coli?

Answer 31

it retains only the replication origin needed for E.coli

Question 32

Why is the small size of pUC18 advantageous?

Answer 32

it is easier to manipulate in the lab

Question 33

What is the selectable marker gene in pUC18?

Answer 33

bla (beta-lactamase gene)

Question 34

What does the bla gene provide resistance to?

Answer 34

ampicillin

Question 35

what gene is used in pUC18 for blue-white selection?

Answer 35

LacZ (beta-galactosidase gene)

Question 36

What is a MCS (multiple cloning stie)?

Answer 36

a region with many restriction sites for inserting DNA.

Question 37

Where is the lacZ gene located in pUC18?

Answer 37

inside the MCS

Question 38

What happens when DNA is inserted into the MCS?

Answer 38

the lacZ gene is disrupted

Question 39

Why are restriction sites only in the MCS and not elsewhere in the vector?

Answer 39

they were removed by mutagenesis and added only in the MCS for controlled insertion

Question 40

What is the advantage of clustering restriction sites in one region?

Answer 40

ensures insertions occur within the lacZ gene

Question 41

What does blue-white screening detect?

Answer 41

whether DNA has been inserted into the vector

Question 42

What substrate is used in blue-white screening?

Answer 42

X-gal

Question 43

What colour are colonies with functional lacZ?

Answer 43

blue

Question 44

What colour are colonies with disrupted lacZ?

Answer 44

white

Question 45

What colonies contain recombinant plasmids with inserts?

Answer 45

white colonies

Question 46

why are blue colonies not desired in cloning experiments?

Answer 46

they do not contain the inserted DNA

Question 47

What causes blue colour in in blue-white screening?

Answer 47

B-galactosidase breaks down XGAL and releases 5-bromo-4-chloro-3-indoyl which is blue

Question 48

What does ampicillin do in the screening process?

Answer 48

selects for cells that have taken up the plasmid

Question 49

What is a genomic library?

Answer 49

a collection of clones with the entire genome represented in fragments

Question 50

How is a genomic library stored for reuse?

Answer 50

by freezing transformed bacteria

Question 51

What organism is most commonly used for genomic libraries?

Answer 51

E.coli

Question 52

How is each clone in a genomic library different?

Answer 52

each has a different genome fragment

Question 53

what can be done once a desired clone is found in a genomic library?

Answer 53

grow, isolate, or further manipulate it

Question 54

Why is a dominant selectable marker important in cloning vectors?

Answer 54

to identify cells that have taken up the vector

Question 55

What is alpha-complementation?

Answer 55

a method used in blue-white selection to detect insertional inactivation of lacZ. a phenomenon in molecular biology where two inactive protein fragments, called alpha and omega peptides, are combined to create a functional enzyme, in this case, beta-galactosidase.

Question 56

Why are commonly used restriction enzymes clustered in MCS?

Answer 56

to simplify insertion and minimise unwanted cuts elsewhere.

Question 57

What ensures that a gene inserted into lacZ disrupts the gene?

Answer 57

the insertion occurs in the MCS within lacZ

Question 58

how does beta-lactamase provide antibiotic resistance?

Answer 58

breaks down ampicillin

Question 59

what happens if no DNA is inserted into the vector?

Answer 59

the lacZ gene remains functional, producing blue colonies.

Question 60

Why is site directed mutagenesis used in vector design?

Answer 60

to remove unwanted restriction sites and modify the vector precisely