Enzymes in Med lab

Question 1

Define star activity

Answer 1

The cutting of DNA at sites other than the enzyme’s restriction site = unwanted outcome.

Question 2

Function of protinase K

Answer 2

• break down proteins

* inc activity w/ presence of : SDS or chaotrophic salt; ~56ºC; Ca2+

Question 3

Function of reverse transcriptase

Answer 3

converts RNA to complimentary DNA

Question 4

Function of DNA polymerase

Answer 4

> produce new strands of DNA from dNTP & template strand
• dependent on Mg2+
Other functions:
3-5exonuclease activity (proof reading) - except Taq
OR terminal transferase activity : add A to 3’ DNA strand

Question 5

Function of restriction endonucleases

Answer 5

• cleaves DNA at/near recognition site (sequ)

* create sticky or blunt ends

Question 6

Function of DNA ligase

Answer 6

joins 2 pieces of DNA tog by forming P-O bond

• ATP & Mg2+ dependent

Question 7

Function of alkaline phosphatase

Answer 7

remove 5’ phosphate group from DNA = prevent self ligation

Question 8

Star activity can be caused by

Answer 8

• hi [glycerol]
• Hi [enzyme]
• incorrect buffer
• Prolonged incubation
• rganic contaminants

Question 9

Proteinase K cleaves proteins*

Answer 9

keratin*

Question 10

Which type of restriction enzyme is most commonly used in the laboratory

Answer 10

Type II

Question 11

An oligo d(T) primer is often used in the production of cDNA. Which region of RNA does this primer bind to?

Answer 11

poly A tail (acting as arvs primer for reverse transcriptase)

Question 12

Proteinase K activity is greatest in the presence of Ca2+ ions. When used for the lysis of tissues, why is it used in lysis buffers which contain EDTA?

Answer 12

accelerate sample lysis during NA isolation

Question 13

Trypsin cleaves….

Answer 13

after Lys or Arg residues, except when followed by Pro