Restriction Digestion & Ligation; Vectors

Question 1

Restriction enzymes

Answer 1

Type of endonuclease that cuts double-stranded DNA at a specific sequence of bases (i.e the recognition site)

(4,6,8, and >8 base cutters)

Question 2

Why do the enzymes not cut up their own cell’s DNA?

Answer 2

The enzymes are methylation sensitive
(if one of the nucleotide bases in the recognition sequence is methylated then the restriction enzyme can not bind therefore it can not be cut)

Question 3

What determines the type of restriction enzyme?

Answer 3

Differences in the cleavage site

Question 4

Type 1 restriction enzymes

Answer 4

They cut the DNA strand 1000 or more base pairs from the recognition site

Question 5

Type 2 restriction enzymes

Answer 5

cut in the middle of the recognition sequence and are the most useful for genetic engineering

Question 6

What two ends do Type 2 restriction enzymes leave?

Answer 6

Blunt ends
Sticky ends

Question 7

Blunt ends

Answer 7

Cutting both strands of the double helix at the same point

Question 8

Sticky ends

Answer 8

Cutting at different sites on each strand leaving single-stranded ends

Question 9

What results when two different DNA samples are cut with the same sticky-end restriction?

Answer 9

All the fragments will have identical overhangs which allow the two DNA fragments to be linked together

Question 10

How are fragments linked together?

Answer 10

DNA Ligase

Question 11

Where does the ligase catalyze linkage?

Answer 11

Between 3’-OH of one strand and 5’-PO4 of the other strand

Question 12

What type of end does ligase attach more efficiently?

Answer 12

Sticky ends
(it can do blunt ends just much more slowly)

Question 13

Cloning Vectors

Answer 13

specialized plasmids that will hold any piece of foreign DNA for further study or manuiplation

Question 14

What are the three most common requirements a cloning plasmid has?

Answer 14

1. Small in size
2. Easy to transfer from cell to cell
3. Easy to isolate from host

Question 15

Plasmids vary in their _____ _________

Answer 15

Copy Number

Question 16

Copy Number

Answer 16

The number of copies of a plasmid found within a single host cell

Question 17

What controls the copy number and why?

Answer 17

The type of origin of replication
(because this region on the plasmid determines how often DNA polymerase binds and induces replication)

Question 18

Multiple cloning site (MCS)

Answer 18

A stretch of artificially synthesized DNA
that contains cut sites for seven or eight widely used restriction enzymes

Question 19

Construct

Answer 19

any rDNA molecule that has been assembled by genetic engineering

Question 20

What happens if both the vector and insert are cut with the same restriction enzyme

Answer 20

The two pieces will have complementary ends and require only ligase to link them

Question 21

How do you attach DNA pieces without complementary ends (2 ways)

Answer 21

1. Add short oligonucleotides (called linkers) onto the insert to make it compatible with the vector
2. They can also be made compatible by adding a vector multi-cloning site by PCR amplification

Question 22

Shuttle Vector

Answer 22

A vector that can survive in and be moved between more than one type of host cell

(contains origins of replication for two organisms plus any other sequences necessary to survive in either organism)

Question 23

Bacteriophage Vectors

Answer 23

viral genomes that have been modified so that large pieces of nonviral DNA can be packages in the virus particle

Question 24

How are smaller genes studied?

Answer 24

using bacterial plasmids or shuttle vectors

Question 25

How are larger genes studied?

Answer 25

using bacteriophage vectors, cosmids, and artificial chromosomes

Question 26

EcoRI

Answer 26

6 base cutter Restriction Endonuclease that recognizes GAATTC (palindromic)

Question 27

What bond do Restriction Enzymes break?

Answer 27

RE’s catalyze the breakage of sugar-phosphate bonds in the backbone by hydrolysis. (phosphodiester bonds)