Labeling Strategies

Question 1

List some labeling strategies

Answer 1

Nick translation
Random priming
End labeling
PCR-mediated labeling

Other-
AMV reverse transcriptase
T4 DNA polymerase
Klenow
TdT

Question 2

Advantages in nick translation?

Answer 2

Rapid
Easy
Commonly used
Inexpensive
Produce uniformly tagged probes with high specific activity

Question 3

Nick translation mechanism? What are the enzymes used?

Answer 3

DNAase I

• create random nicks by cleaving internal phosphodiester bonds
• create 2 termini. 3’-OH & P-5’

DNA pol I

• 5’-3’ exonuclease cleaves nts from 5’ end.
• 5’-3’ polymerase activity add nts(radiolabeled) to the 3’ end of the nick.
• causes lateral displacement of the nick

Question 4

Only ds NA can be used in nick translation. Why?

Answer 4

Need other strand as the template

Question 5

Explain the process of nick translation

Answer 5

DNAse I digestion

DNA pol 1 + dNTP* is added

Unincorperated dNTPs are removed by size exclusion chromatography

Denatured by boiling to make them ss

Labeled probes

Question 6

Optimization of DNAse activity, when too little and too much DNAse I is present?

Answer 6

Too lil - little nicking - inefficient incorporation

Too much - too short DNA probes

Question 7

To gain higher specific activity in nick translation what is used

Answer 7

2-labeled dNTPs are used

Question 8

What are the components used in random priming?

Answer 8

Random oligonucleotide primers(hexamers)

Klenow fragment of DNA pol 1

Question 9

Explain the procedure of random priming

Answer 9

Denature dna

Introduce random primers. They hybridize @ multiple sites

Add klenow fragment. 3’-end of primer is extended with 32P-dNTP*s

Denatured to separate

Question 10

Characteristics of klenow polymerase

Answer 10

Dont have 5’-3’ exonuclease activity - no loss of incorporated labels

3’-5’ exonuclease activity is greatly diminished(ph=6.6)

~60% radionts are incorporated

Question 11

Advantages of random priming?

Answer 11

Generate long, high specific activity probes

Both ss & ds DNA can be used

Probes can be used w/o removing unincorporated parts

Small amount of dna(<200ng) is sufficient

> 50% radiolabels are incorporated

Length of dna doesnt affect the rxn

Label is incorporated uniformly

Question 12

Disadvantages of random priming?

Answer 12

Circular dna is not efficiently labeled

Low quantities of probe

Question 13

End labeling components? What do they do?

Answer 13

32P-ATP*

T4 polynucleotide kinase

Transfer gamma P* group of 32P-ATP* to free OH grp of DNA

Question 14

What can be labeled by end labeling?

Answer 14

Oligonucleotides with free 5’-OH group

Dephosphorylated ds or ss DNA (by phosphotases)

DNA having 5’ P grp is labeled w/o the prior removal

Question 15

What are the 2 types of end labeling rxns? Explain each

Answer 15

Recall forward & exchange reactions

Question 16

Explain the process of fill-in labeling

Answer 16

DNA ends are cleaved by restriction enzymes and 5’ overhangs are formed

Klenow pol 1 fills in by polymerization by dNTP*
incorporation

Restriction nuclease cleave internal sites and form different sized fragments

Question 17

Examples for non-radioactive labeling? How they are introduced into DNA?

Answer 17

Biotin-11-dUTP

Nick translation, random priming

Question 18

How non-radio labels are detected? Examples?

Answer 18

By secondary labeling systems

Avidin, streptavidin, antibiotin

Question 19

Explain the process of using a secondary labeling system to detect biotin labeled hybridization

Answer 19

Avidin is used which has a high affinity to biotin

Avidin is conjugated with alkaline phosphotase(AP)

• Blot is incubated in avidin+AP conjugate containing buffer
• washed to remove excess & unbound conjugate
• colorless substrate for AP is added & incubated
  AP acts on the substrate & coloured parts can be detected

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Question 20

Other conjugates to use in avidin detection? But!

Answer 20

Fluorescently labeled peroxidase but sensitivity is low