Chapter 3

Question 1

5’ –> 3’ exonuclease

Answer 1

cleaves DNA in the 5’ to 3’ direction, this activity is required to remove RNA primers for DNA synthesis

Question 2

3’ –> 5’ exonuclease

Answer 2

cleaves DNA in the 3’ to 5’ direction; this activity is important for “proofreading” DNA synthesis

Question 3

Endonuclease

Answer 3

an enzyme that cleaves a DNA strand internally

Question 4

Bacteria produce two different enzymes to protect its genome

Answer 4

1) Site specific methylase - enzyme methylates specific sequences of DNA in bacterial genome
2) Restriction endonulcease - enzyme cleaved specific DNA sequence; however does not cleave ow DNA, because DNA is methylated by site-specific methylase

Question 5

Restriction endonuclease

Answer 5

enzyme cleaves specific DNA sequence; however does not cleave own DNA because DNA is methylated by site-specific methylase

Question 6

Type I restriction enzyme

Answer 6

Contain both methylase/restriction endonuclease activities
Methylate DNA at specific sites
Cleave DNA 400-1,000 nucleotides from recognition site

Question 7

Type II restriction enzyme

Answer 7

Restriction endonuclease activity only
Do not methylate DNA
Cleave DNA within or within a few nucleotides of recognition site - sequence is usually palindromic
Type of restriction enzyme typically used for cloning
Often work as a dimer

Question 8

Type III restriction enzyme

Answer 8

Contain both methylase/restriction endonuclease activities
Methylate DNA at specific sites
Cleave DNA 20-30 nucleotides from recognition site

Question 9

5’ Overhang

Answer 9

strands are cleaved asymmetrically so that overhanging ends contains 5’ P; refereed to as a sticky end

Question 10

3’ Overhang

Answer 10

strands are cleaved asymmetrically so that overhanging ends contains 3’ OH; refereed to as a sticky end

Question 11

Blunt

Answer 11

strands are cleaved symmetrically so that there is no overhang

Question 12

Isoschisomers

Answer 12

different restriction enzymes that can recognize the same sequence and cleave in the same location

Question 13

Neoschizomers

Answer 13

different restriction enzymes that can recognize the same sequence but cleave in different locations

Question 14

Factors influencing restriction enzyme efficiency

Answer 14

1) Buffer conditions - different restriction enzymes have different preferences for ionic strength and major cation
2) Temperature - most restriction enzymes work best at 37C, some exceptions
3) Methylation - some restriction enzymes will not cleave methylated DNA
4) ‘Star’ activity = cleavage in nonstandard conditions; this can lead to cleavage of sequences that differ from the normal recognition site by only a basepair

Question 15

Blunting DNA

Answer 15

1) fill in of 5’ overhangs using DNA polymerase and dNTPs

2) Removal of 3’ overhangs using nucleases (e.g., Mung bean nuclease

Question 16

DNA ligase

Answer 16

Absolutely essential for phosphodiester bond formation between 3’ OH and 5’ P. Ligates the pieces of DNA together

Question 17

Components necessary for genomic DNA replication

Answer 17

1) Helicase - unwinds double-stranded DNA
2) Single-stranded binding protein - binds single stranded DNA formed by unwinding strands
3) Primase - synthesizes short RNA primer with free 3’ OH
4) DNA polymerase III - synthesizes DNA from RNA primers; has ‘proofreading’ activity
5) Topoisomerases - necessary to remove supercoiling formed by unwinding strands
6) DNA polymerase I - has exonuclease activity to remove RNA primers and fill in the gap
7) DNA ligase - necessary to form phosphodiester bond between adjacent nucleotides

Question 18

Requirements for in vitro DNA replication

Answer 18

1) DNA template - need to have genetic information in which to duplicate
2) Free 3’ OH group - DNA synthesis cannot occur without the attachment of new nucleotides to the 3’ OH group of the preceding nucleotide. This requirement is met by using synthetic primers.
3) Deoxynucleotide triphosphates (dNTPs) - the building blocks of DNA
4) DNA polymerase - need enzyme to add ‘building blocks’ to the growing strand of newly synthesized DNA
5) Mg2+ - magnesium is a necessary cofactor for DNA polymerase

Question 19

Polymerase chain reaction (PCR)

Answer 19

in vitro DNA synthesis by Kary Mullis
Synthesis is repeated for 30-40 cycles to amplify a specific region of DNA
1 PCR cycle:
1) Denaturation - DNA is heated (92-95C) to separate DNA strands
2) Annealing - DNA is cooled (50-65C) to allow primers to anneal to DNA
3) Extension - DNA is heated (68-72C) to allow for optimal synthesis by polymerase

Question 20

Exponential amplification of DNA

Answer 20

(2^n -2(n))

Question 21

Processivity

Answer 21

rate at which the polymerase copies the template

Question 22

Fidelity

Answer 22

accuracy of the template copy being made

Question 23

Persistence

Answer 23

the stability of the enzyme at high temperature