Enzymes In RDT

Question 1

What do enzymes do? What do enzymes do in NA?

Answer 1

Break

Mend

Synth

Add/remove P

Question 2

List RDT enzymes types & examples

Answer 2

Polymerases

Ligases

Modifying enzymes

Nucleases

Question 3

Show how DNA ligase mend a nick using its mechanism

Answer 3

ATP > Enz–AMP > 5’P–AMP > -OH attacks >

Question 4

What are the applications of DNA ligase?

Answer 4

In cloning - join the DNA fragment to the vector

In labeling - seal nicks

Question 5

Show the mechanism of polymerases? Give the reaction for NA with n # of nucleotides.

Answer 5

dNTP + (dNMP)n —> (dNMP)n+1 + PPi

Question 6

List the types of polymerases

Answer 6

T7 RNA Polymerase

Klenow fragment

DNA pol 1

Reverse transcriptase

taq polymerase

Question 7

Explain the activities of DNA polymerase 1. Why is it accurate? Drawbacks? Applicrjtthehu4hrhy😍😂😃🤣😋😊🤣😁😊🔈🎼🔇🎼🎶🔉📻🔊🎵🎶🔥🤙😊ations in RDT?

Answer 7

5’-3’ & 3’-5’ exo

polymerase

Question 8

How the Klenow fragment is made? activities? What are its applications?

Answer 8

3’-5’ exo

polymerase

synth, labeling

Question 9

What does reverse transcriptase do?

Applications?

Answer 9

Quantification of mRNA

Cloning intron less genes

Question 10

What are the 2 types of T7 polymerase and their activity?

Answer 10

S

Question 11

Taq polymerase short note

Answer 11

Ss

Question 12

What does nucleases do?

Answer 12

break phosphodiester bonds

Question 13

Explain the types of nucleases

Answer 13

multi subunit complex

groups of separated proteins - defined cuts

2 subunits

cleaves modified DNA

Question 14

What does restriction endonucleases do in bacteria to viruses? Main advantage of these?

Answer 14

restrict the host range of viruses

Question 15

How the name comes? (ECoRI)

Answer 15

R - strain

I - First enzyme to be identified - order of discovery

Question 16

Show the cleavage by Eco RI of methylated & non-methylated DNA

Answer 16

methylated - no cleavage

Question 17

Design a method to identify if a restriction site in methylated

Answer 17

hint: Hsp I

Question 18

types of restriction cut ends?

Answer 18

blunt

3’ protruding

5’ protruding

Question 19

Relationship bw RRS length and occurrence of cleavage sites

Answer 19

4^n

Question 20

What are the necessary conditions for use of RE

Answer 20

Star activity - ph, salt, T, high glycerol and dmso can allow cleavage at diff. sites

end effects - cut poorly @ ends

supercoiled vs linear dna - poorly

Question 21

What is 1 unit of RE activity?

Answer 21

Amount of RE needed to completely digest 1 ug of substrate DNA

Question 22

What does DNAse I do under different conditions?(give conditions)

Answer 22

mn2+

Mg2+

Question 23

What are the uses of DNAse I?

Answer 23

in labeling - nick formation - nonspecific cleavage

removal of dna prior to RT PCR

Question 24

What does RNA do? Uses?

Answer 24

remove rna contaminants

Question 25

List the differences bw RNAse A & RNAse H and their uses

Answer 25

A - rna sequencing, removing rna

H - removing poly a tail
quantifying poly a tail

Question 26

Explain S I nuclease activity in low conc & high conc. And list its uses.

Answer 26

low - ss dna/rna
high - ds dna/rna/ dna-rna hybrids

s1 nuclease mapping
obtaining cdna from mrna

Question 27

Bal 31 activity & uses?

Answer 27

digest from both ends. active at nicks/ds both ends & ss dna at 3’-OH

obtain large deletions

Question 28

Exo I activity & use?

Answer 28

ds DNA ,, 3’ – 5’

no action on sticky ends/ss

Question 29

Activity of Exo iii ?

Answer 29

same as exo I but works on ss, blunt, nicks

Question 30

Activity of mung bean nuclease and its speciality?

Answer 30

cleave ss until it meets ds

make blunt ends
hair pin loop cleavage

Question 31

why terminal transferases are needed?

Answer 31

blunt end ligations are inefficient

Question 32

Show the activity of terminal transferase. List its uses

Answer 32

3’ + dATP — 3’-AAAAA

homopolymer tail/ single nt attaching

Question 33

Activity of kinases & phosphotases?

Answer 33

5’-OH-NNNN + P – P-NNNN

P-NNN – OH-NNN

Question 34

Activity and uses of polynucleotide kinase?

Answer 34

5’-P —> 5’-P*

lacks - 5’-OH —> 5’-P