Nucleic Acids Manipulation Methods

Question 1

What are the enzymes used to manipulate DNA?

Answer 1

1. Nucleases
2. Ligases
3. Modifying enzymes

Question 2

What are nucleases?

Answer 2

Enzymes that cleave nucleic acids by breaking phosphodiester bonds in DNA or RNA.

Question 3

What do most nucleases require for catalytic activity?

Answer 3

Metal ions.

Question 4

Which metal ions commonly act as cofactors for nucleases?

Answer 4

Mg²⁺ and Mn²⁺.

Question 5

What are exonucleases?

Answer 5

Nucleases that remove nucleotides one at a time from the ends of DNA or RNA strands.

Question 6

What biological processes involve exonucleases?

Answer 6

DNA replication, DNA repair, and nucleic acid degradation

Question 7

What are endonucleases?

Answer 7

Nucleases that cleave phosphodiester bonds within a nucleic acid chain.

Question 8

Where do endonucleases cut DNA or RNA?

Answer 8

Internally within the nucleotide chain rather than at the ends.

Question 9

Which molecular biology processes rely on endonucleases?

Answer 9

Restriction digestion and recombination.

Question 10

Why are nucleases important in molecular biology?

Answer 10

They are essential for genetic engineering, molecular cloning, and diagnostic procedures.

Question 11

What type of nuclease is Bal31?

Answer 11

An exonuclease.

Question 12

On what type of DNA does Bal31 act?

Answer 12

Double‑stranded DNA.

Question 13

Does Bal31 act on just one strand or both strands of DNA?

Answer 13

Both strands.

Question 14

What is a common laboratory application of Bal31?

Answer 14

Shortening a piece of DNA from both ends.

Question 15

What determines the final length of DNA fragments produced by Bal31 digestion?

Answer 15

The incubation time of DNA with Bal31 enzyme.

Question 16

How does Bal31 differ from restriction enzymes?

Answer 16

Bal31 removes nucleotides progressively from DNA ends, while restriction enzymes cut at specific internal sequences.

Question 17

Where does Bal31 cut, before or after the phosphate?

Answer 17

It will cut it so that the 5’ end still has the phosphate attached

Question 18

What type of nuclease is Exonuclease III?

Answer 18

An exonuclease

Question 19

From which end does Exonuclease III degrade DNA?

Answer 19

From the 3′ end.

Question 20

What type of DNA does Exonuclease III act on most efficiently?

Answer 20

Double‑stranded DNA.

Question 21

What type of DNA ends are resistant to Exonuclease III?

Answer 21

3′ overhangs longer than 4 bases (≥4 bases).

Question 22

Why are long 3′ overhangs resistant to Exonuclease III?

Answer 22

Exonuclease III requires a blunt or recessed 3′ end to initiate degradation.

Question 23

How can Exonuclease III be used to generate single‑stranded DNA?

Answer 23

By selectively degrading one strand from the 3′ end

Question 24

What are “nested deletions”?

Answer 24

A series of DNA fragments progressively shortened from one end.

Question 25

What type of nuclease is S1 nuclease?

Answer 25

An endonuclease that cuts single‑stranded DNA.

Question 26

Why is S1 nuclease used after Exonuclease III digestion?

Answer 26

To remove remaining single‑stranded DNA regions

Question 27

What are endonucleases?

Answer 27

Enzymes that break internal phosphodiester bonds within a DNA molecule.

Question 28

How do endonucleases differ from exonucleases?

Answer 28

Endonucleases cut within a nucleic acid chain, whereas exonucleases remove nucleotides from the ends.

Question 29

What is S1 nuclease?

Answer 29

A single‑strand‑specific endonuclease.

Question 30

Does S1 nuclease cut double‑stranded DNA?

Answer 30

No.

Question 31

What is DNase I?

Answer 31

A non‑specific endonuclease.

Question 32

What types of DNA can DNase I cleave?

Answer 32

Both single‑stranded and double‑stranded DNA.

Question 33

Does DNase I cut DNA at specific sequences?

Answer 33

No, it cuts DNA non‑specifically.

Question 34

What are the end products of DNase I digestion?

Answer 34

Mononucleotides and short oligonucleotides.

Question 35

What are restriction enzymes?

Answer 35

Endonucleases that cut double‑stranded DNA at specific recognition sequences.

Question 36

What are modifying enzymes in molecular biology?

Answer 36

Enzymes that modify DNA molecules by adding or removing specific chemical groups.

Question 37

What are the important modifying enzymes?

Answer 37

(a) Alkaline phosphatase (b) Polynucleotide kinase (c) Terminal deoxynucleotidyl transferase (d) Klenow fragment

Question 38

What does alkaline phosphatase do to DNA?

Answer 38

Removes phosphate groups from the 5′ ends of DNA, only at the ends, not in the middle

Question 39

Why is alkaline phosphatase used in cloning?

Answer 39

To prevent vector self‑ligation.

Question 40

How does alkaline phosphatase reduce non‑recombinant plasmids?

Answer 40

By removing 5′ phosphates required for DNA ligase activity.

Question 41

What is polynucleotide kinase (PNK)?

Answer 41

A DNA‑modifying enzyme that adds phosphate groups to the 5′ end of DNA (or RNA).

Question 42

What specific reaction does polynucleotide kinase catalyse?

Answer 42

Transfer of the γ (gamma) phosphate of ATP to the 5′‑OH end of a DNA chain. (gamma is the third one, at the end)

Question 43

How does polynucleotide kinase compare to alkaline phosphatase?

Answer 43

It has the opposite function—PNK adds 5′ phosphates, while alkaline phosphatase removes them.

Question 44

What is required on the DNA for PNK to act?

Answer 44

A free 5′‑hydroxyl (5′‑OH) group.

Question 45

Which enzyme is commonly used as polynucleotide kinase in molecular biology?

Answer 45

T4 polynucleotide kinase.

Question 46

Why is T4 polynucleotide kinase used before ligation?

Answer 46

To phosphorylate DNA fragments that lack 5′ phosphates so they can be ligated.

Question 47

What radioactive labelling application uses polynucleotide kinase?

Answer 47

Labelling DNA fragments with γ‑³²P‑ATP at the 5′ end.

Question 48

What is terminal deoxynucleotidyl transferase (TdT)?

Answer 48

A DNA‑modifying enzyme that adds nucleotides to the 3′ ends of DNA chains.

Question 49

What is unique about TdT compared to other DNA polymerases?

Answer 49

It is template‑independent.

Question 50

What type of nucleotides can TdT add?

Answer 50

Deoxynucleotides (e.g., dCTP, dATP, dGTP, dTTP).

Question 51

How is TdT used in cloning experiments?

Answer 51

To add homopolymeric tails to DNA fragments.

Question 52

Why are homopolymeric tails useful in cloning?

Answer 52

They allow complementary base‑pairing with similarly tailed DNA molecules. (can help make sticky ends)

Question 53

How is TdT used in DNA labelling?

Answer 53

To attach labelled nucleotides to the 3′ ends of DNA strands to prepare probes.

Question 54

What is the Klenow fragment?

Answer 54

A fragment of E. coli DNA polymerase I that lacks 5′→3′ exonuclease activity but retains 5′→3′ polymerase and 3′→5′ exonuclease activities.

Question 55

Which enzymatic activity is missing in the Klenow fragment?

Answer 55

5′→3′ exonuclease activity.

Question 56

Which enzymatic activities are retained in the Klenow fragment?

Answer 56

5′→3′ DNA polymerase activity

Question 57

How is the Klenow fragment used for DNA labelling?

Answer 57

It incorporates labelled nucleotides using its 5′→3′ polymerase activity.

Question 58

What is 5′→3′ polymerase activity?

Answer 58

When bases are added on at the 3' end to elongate the

Question 59

What is 3`-5` exonuclease activity?

Answer 59

3′→5′ exonuclease activity is the ability of certain enzymes to remove nucleotides one at a time from the 3′ end of a DNA strand, moving backward along the strand.

Question 60

Why is the Klenow fragment used in cloning?

Answer 60

To convert cohesive (sticky) ends into blunt ends.

Question 61

How does the Klenow fragment process 3′ overhangs?

Answer 61

It removes them using its 3′→5′ exonuclease activity.

Question 62

How does the Klenow fragment process 5′ overhangs?

Answer 62

It fills them in using its 5′→3′ polymerase activity.

Question 63

What is the final result of treating DNA with the Klenow fragment?

Answer 63

Blunt‑ended DNA fragments.

Question 64

What is a way to remember how Klenow fragments work?

Answer 64

If there is a 5' overhang, it is filled in by the polymerase; if there is a 3' overhang, it is chopped off by the exonuclease