W1L2 - Enzymes Used in Molecular Laboratory

Question 1

Proteinase K

Answer 1

Serine proteinase
Relatively non-specific
Digests keratin
Greatest activity in presence of 
- SDS or chaotropic salt
- increased temp
- Ca2+
Often used in buffers containing EDTA
Used to accelerate lysis during NA isolation or anytime that proteins must be degraded while retaining NA

Question 2

Trypsin

Answer 2

Serine endopeptidase
Cleaves after Lys or Arg residues
Used for prep of samples requiring cytogenetic analysis

Question 3

DNase

Answer 3

Endonuclease
Cleaves single and double stranded DNA
Used to remove contaminating DNA that would interfere with RNA analysis

Question 4

RNase

Answer 4

Endonuclease that cleaves bonds between adjacent nucleotides in RNA
May need to be removed from sample

Question 5

Reverse Transcriptase

Answer 5

RNA must be converted to DNA sometimes
Reverse transcriptase used to produce complementary DNA from RNA
2 most commonly used
- Moloney Murine Leukaemia Virus (MMLV)
- Avian Myeloblastosis Virus (AMV)

Question 6

DNA Polymerase

Answer 6

Enzyme that produces new strands of DNA from dNTP’s and a template strand
Can possess two other enzymatic activities:
1. 3’-5’ Exonuclease Activity
- sometimes, polymerase inserts nucleotide that’s not complementary
- this activity allows polymerase to correct this mistake
2. Terminal Transferase Activity
- adds adenine base to 3’ end of newly replicated DNA strand

Question 7

4 Types of Restrition Enzymes

Answer 7

Type 1 - cleaves at random sites at least 1kb from recognition site
Type 2 - cleave at their recognition site
Type 3 - cleave 20-30 bp after recognition site
Type 4 - cleave methylated DNA
Type 2 most commonly used

Question 8

DNA Ligase

Answer 8

Enzyme which joins ss breaks in ds DNA

Forms phosphodiester bond between 5’ phosphate and 3’ hydroxyl of two DNA strands

Question 9

Alkaline Phosphates

Answer 9

Used to removes 5’ phosphate group
Removal of 5’ phosphate prevents self ligation
Useful when DNA ends are blunt ended