Chapter 20 Molecular Technologies

Question 1

gene cloning

Answer 1

the procedure of isolating and making many copies of a gene

Question 2

One typically clones chromosomal DNA into a

Answer 2

DNA vector

Question 3

what serves as the source of the DNA segment of interest

Answer 3

chromosomal dna

Question 4

Describe vector DNA

Answer 4

• serves as the carrier for the DNA segment that is to be cloned
• can replicate independently of the host chromosomal DNA

Question 5

vectors used in gene cloning are usually derived from:

Answer 5

naturally occurring plasmids, but sometimes viruses

Question 6

describe plasmid vectors

Answer 6

• High copy number bacterial plasmids give higher DNA yields when DNA is purified (limit: ~20-kb inserted)
• Low copy number bacterial plasmids can handle having larger DNA fragments inserted into them when a bacterial chromosomal origin of replication is used: 100-kb to 200-kb can be inserted.

Question 7

viruses

Answer 7

have sequences removed or mutated so that it is no longer a pathogen but can still function as a vector

Question 8

commercially available plasmids have

Answer 8

selectable markers

Question 9

genes conferring antibiotic resistance to the host cell most commonly include

Answer 9

Ampicillin resistance (AmpR)
Chloramphenicol resistance (CanR)
Kanamycin resistance (KanR)

Question 10

Typical bacterial plasmid vector design

Answer 10

usually grown in E. coli

Question 11

host cell

Answer 11

the cell that harbors the vector
- DNA the vector carries gets replicated

Question 12

bacterial host cell

Answer 12

• The host cell is usually a non-pathogenic strain of the bacterium E. coli
• The bacterial host cell lacks the antibiotic resistance that the plasmid has so the bacterium only survives an antibiotic if a plasmid is present

Question 13

Yeast Host Cell

Answer 13

Yeast can be used as a eukaryotic host cell but yeast has a tendency to rearrange (alter) the cloned DNA

Also: antibiotics cannot be used to retain plasmids in yeast. Instead, people have an essential gene exclusively on the plasmid

Question 14

to prepare chromosomal DNA; the scientist has to:

Answer 14

• obtain cellular tissue and break open the cells
• extract and purify DNA using a variety of biochemical techinques
• obtain the DNA fragment of interest

Question 15

restriction enzymes

Answer 15

• used by bacteria as a defense against viruses
• many bacteria methylate their own DNA at specific sites
• the bacteria also express restriction enzymes that would cleave the DNA if it were unmethylated
• viruses that infect the bacteria have unmethylated DNA and can get cleaved (unless virus found a way around this problem)

Question 16

Many cloning experiments involve restriction enzymes (a.k.a. restriction endonucleases)

Answer 16

• restriction enzymes bind to specific DNA sequences and then cleave the DNA at two defined locations, one on each strand
• some restriction enzymes produce “blunt ends” while others produce “sticky ends”
  Ex. BamHI or Hpal

Question 17

using restriction enzymes that produce sticky ends to cleave and ligate

Answer 17

1. DNA from two different sources incubate both with EcoRI which cuts the DNA
2. incubating together H bonds the two sticky ends
3. add DNA ligase which covalently links the DNA backbones

Question 18

typical bacterial plasmid vector design

Answer 18

LacZ encodes a protein (beta-galactoside) that can cleave a chemical called “X-gal”
- intact X-gal is colorless
- cleaved X-gal is blue

Question 19

Four ways to clone a gene

Answer 19

1. Shotgun clone genomic DNA into a vector and identify gene of interest (best way, but most work)
2. Make a cDNA library and shotgun clone this into a vector and then identify gene of interest
3. PCR amplify the gene and clone it into a vector
4. RT-PCR a gene and clone it into a vector

Question 20

Describe shotgun clonning

Answer 20

Ex. human b-globin gene
1. cut the human and plasmid DNAs with the same restriction enzyme
2. mix DNAs together
3. sticky ends base pair
4. DNA ligase covalently links
5. mix DNA with E. coli cells that have been treated with chemicals to make them permeable
6. plate cells on media containing X-gal, IPTG (allolactose mimic), and ampicillin and incubate overnight
7. each colony is derived from a single cell with a different plasmid
8. filter is gently laid onto the master plate and lifted so the colonies stick
9. filter is treated with detergent to permeabilize the bacteria & DNA is fixed to the filter with UV light
10. NaOH is added to denature the DNA
11. a probe is added that is complementary to the b-globin gene
12. filter is washed to remove unbound probe and placed next to x-ray film
13. identify colonies from orientation of the master plate

Question 21

blue colony

Answer 21

recircularized plasmid without insert

Question 22

white colony

Answer 22

hybrid vector with an insert

Question 23

disadvantages of genomic DNA cloning

Answer 23

1. gene may be too large for a plasmid (BACs may be used instead)
2. more difficult to clone large DNA fragments

Question 24

advantages of genomic DNA cloning

Answer 24

1. also have the promotor
2. gene in genomic DNA often is more accurately expressed if make transgene from it (alternatives)