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Flashcards in Enzymes Deck (33)
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1

What do nucleases do?

Cut, shorten, and degrade nucleic acid molecules

2

What do ligases do?

Join nucleic acid molecules together

3

What do polymerases do?

Make copies of nucleic acid molecules

4

What do modifying enzymes do?

Remove or add chemical groups to nucleic acid molecules

5

What do topoisomerases do?

Introduce or remove supercoils from covalently closed circular DNA

6

How do nucleases degrade DNA molecules?

Break the phosphodiester bonds that link one nucleotide to the next in a DNA strand

7

What are the 2 kinds of nucleases?

Exonucleases
Endonucleases

8

Describe how exonucleases work

Remove nucleotides one at a time from the end of a DNA molecule

9

Describe how endonucleases work

Break internal phosphodiester bonds within a DNA molecule

10

What are the 2 kinds of RNase?

RNase A
RNase H

11

Describe how RNase A works

Endoribonuclease that specifically degrades ssRNA

12

Describe how RNase H works

Endoribonuclease that digests the RNA of an RNA-DNA hybrid

13

How do ligases work?

Covalently links the free ends of DNA molecules and repairs the ss break in one of the strands of a ds molecule
Can also join together individual DNA molecules or the two cohesive ends of the same molecule
Catalyses the formation of a phosphodiester bond between adjacent 5'-P and 3'-OH termini in DNA
Can work on blunt or sticky ends
H bonding gives a stable structure for the enzyme to work on

14

How many types of polymerase are there and what are they?

4
DNA polymerase I
Klenow fragment DNA polymerase
Reverse transcriptase (RNA-dependant DNA polymerase)
Taq polymerase

15

Describe how DNA polymerase I works

Is a DNA dependant DNA polymerase
Has 3 different modes of actions;
5'-3' polymerase
5'3' exonuclease
3'-5' exonuclease

16

Where is DNA polymerase I most commonly used?

Nick translation
Probe preparation
Repairing DNA fragments
Producing blunt-end DNA from sticky-end DNA

17

Describe how Klenow fragment DNA polymerase works

Is a DNA dependant DNA polymerase
Has 2 different modes of action;
5'-3' polymerase
3'-5' exonuclease
Can only synthesise a complementary DNA strand on a single-stranded template

18

Where is Klenow fragment DNA polymerase most commonly used?

Sanger dideoxy sequencing
Synthesis of a synthesis of cDNA in cDNA cloning
Filling in the 3' recessed termini created by digestion of DNA with a restriction enzyme
Labelling of the termini of the DNA fragment

19

Describe how reverse transcriptase works

Is an RNA dependant DNA polymerase
# different modes of action;
5'-3' polymerase
5'-3' riboexonuclease
3'-5' exoribonuclease

20

Where is reverse transcriptase most commonly used?

Synthesis of cDNA for cloning
Labelling the termini of DNA fragments with protruding 5' ends

21

Describe how Taq polymerase works

5-3' polymerase ONLY

22

Where is Taq polymerase most commonly used?

Used in PCR but requires specific primers

23

What are the 4 types of DNA modifying enzymes?

Alkaline phosphatase
Terminal deoxynucleotide transferase
DNA methylase
Polynucleotide kinase

24

Describe alkaline phosphatase

Removes a phosphate group at the 5' terminus of a DNA molecule
Is used to prevent recircularisation of a plasmid during cloning work

25

Describe terminal deoxynucleotidyl transferase

Adds >1 deoxynucleotide onto the 3' terminus of DNA
Used for the 3' tailing reaction

26

Describe DNA methylase

Transfers a methyl group to internal A or C residues to produce methylated duplex DNA
Used to protect DNA from restriction enzymes

27

Describe polynucleotide kinase

Adds phosphate groups on to free 5' termini

28

What are the 3 classes of endonucleases?

Class I
Class II
Class III

29

What do class I endonucleases do?

Recognise some specific sequence but aren't useful in gene manipulation since their cleavage site is non-specific

30

What do class II endonucleases do?

Are Mg2+ dependant with a highly specific recognition site
Very useful for DNA manipulation
Are restriction enzymes
Used for cutting DNA in a very precise and reproducible way during molecular cloning work
They cut both strands of dsDNA within a palindromic recognition sequence
They hydrolyse the sugar-phosphate backbone to give a 5'- P on one side and a 3'-OH on the other
Give sticky or blunt ends