Labels/Reporter Molecules

Question 1

What are the characteristics of an ideal label?

Answer 1

• Incorporation into NA should be easy
• Detectable in low [C]s
• Should not affect the probe’s hybri. character
• Stable @ elevated T

Question 2

What is the unit of specic activity?

Answer 2

cpm/ug

Question 3

Choice for the label depends on?

Answer 3

Application

labeling process

sensitivity & resolution needed

Question 4

List some radio labels

Answer 4

32P, 3H, 125I

Question 5

32P

What does it emit?

Answer 5

beta particles

Question 6

Which label has the highest specific activity?

Answer 6

32P

Question 7

32P

Its t1/2? How long can it be stored? Drawbacks?

Answer 7

short t1/2

Week max

should be used immediately damages structural integrity of DNA

Question 8

35S characteristics?

Answer 8

less E

More half life

Less sensitivity

higher resolution

Question 9

Where the 35S bind in a nucleotide? afterwards the previously phophate grp is called?

Can it inhibit an enzymes?

Answer 9

at alpha & gamma P

phosphorothioate

Yes. Pol. 1 - reduced rate

Question 10

35S advantages?

Answer 10

• longer half life
• thiophosphodiester linkages are resistant to phosphotase enzymes
• Less radiation, little ext. hazard to user

Question 11

3H label characteristics

Answer 11

• Lowest specific activity
• Too weak for detection
• longest half life - 12yrs
• max resolution + low bg due to low energy

Question 12

125I & 131I (Iodine) characteristics

Answer 12

Hazardous

emit beta & gamma

high resolution, high sensitivity