DNA Cloning

Question 1

What is cloning?

Answer 1

A sequence of DNA into a plasmid vector. this vector can then be put into a cell

Question 2

What is the aim of genes cloning?

Answer 2

the aim of gene cloning is to obtain isolated and purified copies of specific gene sequences

Question 3

cloned genes allow us to:

Answer 3

• determine the sequence of the gene (and protein)
• obtain leads into the function of the gene
• manipulate the gene

Question 4

What are the two pathways for gene cloning to take?

Answer 4

1) to understand the gene / protein
2) exploit the gene

Question 5

clone

Answer 5

a large number of identical cells or molecules with a single ancestral cell or molecule

Question 6

DNA cloning vector

Answer 6

a carrier DNA molecule that allows attached DNA to be replicated in a cell

e.g. plasmids or viruses

Question 7

Restriction enzymes

Answer 7

an enzyme that recognises and cleaves DNA at specific sequences

e.g. EcoRI cleaves the palindrome GAATTC

Question 8

DNA ligase

Answer 8

an enzyme that can covalently link DNA molecules

Question 9

What are Plasmids?

Answer 9

Double stranded DNA molecules that replicate in cells independently of the host chromosome. They are inherited by daughter cells and are no-essential for growth

Question 10

Why are plasmids an ideal cloning vector to use?

Answer 10

• it is well characterised (sequence ad function of genes known)
• small and easy to purify and manipulate

Question 11

A plasmid vector must contain:

Answer 11

• an origin of replication (Ori)
• a selectable marker
• unique restriction enzyme cleavage sites

Question 12

The Four steps that is involved in cloning a piece of DNA

Answer 12

1) Restriction
2) Ligation
3) Transformation
4) Selection

Question 13

Restriction

Answer 13

the plasmid vector and the DNA fragment of interest must be digested with a restriction enzyme to produce sticky end

Question 14

Ligation

Answer 14

the DNA fragment of interest must be ligated into a plasmid vector

Question 15

Transformation

Answer 15

the ligated plasmid vector must be transformed into a bacterial host (usually E.coli)

Question 16

Selection

Answer 16

bacterial colonies carrying the plasmid with the DNA fragment must be selected from those that do not

Question 17

What enzyme does the lacZ alpha gene encodes?

Answer 17

beta-galactosidase alpha peptide

Question 18

If Beta-galactosidase is functional?

Answer 18

a bacterial colony that contains an active beta-galactosidase will cleave x-gal which means that it will produce blue colonies

Question 19

If Beta-galactosidase is non-functional?

Answer 19

a bacaterial colony that does not have an active beta-galactosidase means that it cannot cleave the x-gal which means that it will not produce any colour change so the colonies are white

Question 20

IPTG relation with lac Z gene?

Answer 20

if IPTG is added means that the lacZ is always being made

Question 21

what advantage does adding a promoter in front of the MCS?

Answer 21

this would allow for expression of the gene we have cloned in

Question 22

control of expression

Answer 22

Ptac promoter - induced by IPTG

Question 23

ccdB

Answer 23

toxic cells

Question 24

Amplification

Answer 24

a process by which nucleic acid molecule is enzymatically copied to generate a progeny population with the same sequence as the parental one

Question 25

PCR

Answer 25

to amplify a specific sequence of a DNA

Question 26

Genomic library

Answer 26

contains DNA fragments representing the entire genome of an organism

Question 27

cDNA library

Answer 27

contains cloned cDNA (reversed-transcribes mRNA), representing the transcriptome of an organism

Question 28

DNA inserts are stored in plasmids or BACs:

Answer 28

Plasmids
- insert size <20kb

Bacterial artificial chromosomes
- insert size 75-300kb (fewer clones required)