Lecture 1 Flashcards
What are the five broad classes of enzymes?
1) Nucleases
2) Ligases
3) Polymerases
4) Modifying enzymes
5) Topoisomers
What are the two kinds of nucleases?
Exonucleases and Endonucleases
What do endonucleases do?
Break internal phosphodiester bonds within a DNA molecule e.g. S1 Nuclease
What do exonucleases do?
Remove nucleases one at a time from the end of a DNA molecule
What is the mode of action for nucleases?
Degrade a DNA molecule by breaking the phosphodiester bonds that link one nucleotide to the next in a DNA strand.
What does RNAseA do?
Endoribonuclease that specifically degrades ss RNA
What does RNAseH do?
Endoribonuclease that digests the RNA of an RNA-DNA hybrid?
What is the mode of action for ligases?
Catalyses the formation of phosphodiester bond between adjacent 3’-OH and 5’-P termini in DNA
What do polymerases do?
Synthesise a new strand of DNA complementary to an existing DNA or RNA template.
What are the four types of DNA polymerases routinely used in molecular biology techniques?
1) DNA polymerase I
2) Klenow fragment DNA polymerase
3) RNA-dependent DNA polymerase
4) Taq DNA polymerase
What does DNA polymerase I do?
Usually from E.coli and T4 phage
- DNA dependent
- Has 5’-3’ action and 3’ to 5’ (so dual function DNA polymerisation and degradation)
- Commonly used in Nick Translation, Probe preparation, repairing DNA fragments, producing blunt end DNA from sticky end DNA.
What does Klenow Fragment DNA polymerase do?
- DNA dependent
- 5’-3’ polymerase activity
- 3’-5’ exonuclease activity
- Can only synthesise a complementary DNA strand on a single stranded template
- Used in Sanger dideoxy sequencing, filling of 3’ recessed termini created by digeston od DNA with RE/.
What does RNA-dependent DNA polymerase do?
- Needs RNA as template
- 5’-3’ polymerase
- 5-3’ riboexonuclease
- 3-5’ exoribonouclease activity
- used in synthesis of cDNA for cloning
What does TaqDNA polymerase do? (PCR enzyme)
- 5’-3’ polymerase activity
- NO 3’-5’ exonuclease (no proof reading)
- Widely used in PCR reaction
- High polymerase activity
- Latest version of Taq has proofreading activities with higher polymerisation activities.
What do DNA modifying enzymes do?
Can modify DNA molecules through addition or removal of specific chemical groups.
What are the four DNA modifying enzymes?
1) Alkaline phosphatase (AP)
2) Terminal deoxynucleotidyl transferase
3) DNA methylases (dam and dcm)
4) Polynucleotide Kinase
What does polynucleotide kinase do?
Adding phosphate groups on to free 5’ termini
reverse of AP
What does AP do?
Removes the phosphate group from 5’ terminus of DNA molecule
What does Terminal deoxynucleotidyl transferase do?
Adds 1 or more deoxynucleotides onto the 3’ terminus of a DNA molecule
What does DNA methylase do?
Transfer of methyl group to internal A or C residues in the specific sequences to produce methylated duplex DNA.
What do class I endonucleases do?
Recognise some specific sequences BUT are not useful in gene manipulation since their cleavage site is non-specific.
What do class II endonucleases do?
They are mg2+ dependant with a highly specific recognition site. Very useful for DNA manipulation
What do class III endonucleases do?
Recognition site not symmetrical, contain nuclease and methylase activity.
What do restriction enzymes do? (Class II endonucleases)
- Cut DNA in a very precise and reproducible manner
- They cut both strands of DS DNA within a recognition site
- Hydrolyse sugar phosphate backbone to give 5’-P on one side and 3’-OH on the other = sticky ends.
