Workshop V

Question 1

What are the 3 types of probes for hybridization?

Answer 1

cDNA, cRNA and oligonucleotide probes

Question 2

What compound is most often used as a radioactive label for probes?

Answer 2

32P or 33P

Question 3

What compounds are most often non-radioactive probe markers?

Answer 3

DIG, biotin, ECL

Question 4

Describe the process of random primer cDNA labeling.

Answer 4

1) DNA is denaturated to separate 2 strands
2) Primers, unlabelled & labeled dNTPs and Klenow polymerase are added
3) Primers anneal and polymerase extends the probes using the dNTPs, which label the synthesized probe
4) Denaturation to release the probe

Question 5

What is the preferred primer length in random primer cDNA labeling and why?

Answer 5

The primers are often hexamers, as they yield probes ~300bp-long, which is the optimal length for the kinetics of hybridisation reaction.

Question 6

Describe the process of nick translation cDNA labeling.

Answer 6

1) DNA polymerase I is added to dsDNA
2) polymerase nicks the DNA (3’-5’ exonuclease activity) and then a single/a few nucleotide is removed (5’-3’ exonuclease activity)
3) unlabelled & labeled dNTPs are added
4) missing nucleotides are replaced through 5’-3’ activity of DNA polymerase
5) Denaturation to separate probe

Question 7

Describe the process of in-vitro transcription cRNA labelling

Answer 7

1) a probe sequence is inserted into a phage
2) phage is linearised by a restriction enzyme
3) RNA polymerase is added and labeled and unlabeled dNTPs are added
4) RNA polymerase polymerase starte at the phage promoter and transcribes also the probe sequence with labeled dNTPs