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PETERIGARASHI
Procedure
1. Combine in a sterile 1.5 ml microcentrifuge tube on ice:
- 1-50 pmol Synthetic oligonucleotide
- 5 J..Ll 5x reaction buffer
- 10 J..Ll a[ 32 P]dATP (33 pmol)
- to 25 f. . . Ll sterile water
- 1 J..Ll Terminaltransferase (15 units)
2. Incubate at 37°C for 30 minutes.
3. Stop the reaction by heating to 65°C for 5 min. Remove unincorporated
nucleotides by centrifugation through a Sephadex G-25 spin column
(2000x g for 4 min) ( optional). Count 1 J..Ll of column eluate in a liquid
scintillation counter.
Note: dATP is the preferred nucleotide when preparing hybridization
probes in order to reduce non-specific binding.
Note: The number of dNTPs incorporated per molecule of substrate can be
adjusted by altering the duration of the reaction or the molar ratio of radionucleotide to oligonucleotide. Alternatively, the reaction mixture can be
"doped" with dideoxynucleotides (which arenot extended).
I Subprotocol 4
Random-Prime Labeling
The method of random primer extension has largely replaced the less efficient "nick-translation" as the preferred method for labeling doublestranded DNA (Feinberg and Vogelstein 1983). Template DNA is denatured
and annealed to hexamers of random sequence. The hexamers anneal at
various points along the template and are elongated by the Klenow fragment of E. coli DNA polymerase I in the presence ofthree unlabeled dNTPs
and one 32 P-labeled dNTP. The Klenow fragment possesses the 5'-----+3'
synthase activity but lacks the 5' -----+3' exonuclease activity so that newly
synthesized probe is not rapidly degraded. The substrate DNA is not itself
radiolabeled but serves as the template for DNA synthesis. The resultant
products range in size from 400-600 nucleotides and have typical specific
activities of 10 8 -10 9 cpm/J..Lg. Reagents are available in kit form from several
manufacturers ( e. g., Amersham).
PETERIGARASHI
Procedure
1. Combine in a sterile 1.5 ml microcentrifuge tube on ice:
- 1-50 pmol Synthetic oligonucleotide
- 5 J..Ll 5x reaction buffer
- 10 J..Ll a[ 32 P]dATP (33 pmol)
- to 25 f. . . Ll sterile water
- 1 J..Ll Terminaltransferase (15 units)
2. Incubate at 37°C for 30 minutes.
3. Stop the reaction by heating to 65°C for 5 min. Remove unincorporated
nucleotides by centrifugation through a Sephadex G-25 spin column
(2000x g for 4 min) ( optional). Count 1 J..Ll of column eluate in a liquid
scintillation counter.
Note: dATP is the preferred nucleotide when preparing hybridization
probes in order to reduce non-specific binding.
Note: The number of dNTPs incorporated per molecule of substrate can be
adjusted by altering the duration of the reaction or the molar ratio of radionucleotide to oligonucleotide. Alternatively, the reaction mixture can be
"doped" with dideoxynucleotides (which arenot extended).
I Subprotocol 4
Random-Prime Labeling
The method of random primer extension has largely replaced the less efficient "nick-translation" as the preferred method for labeling doublestranded DNA (Feinberg and Vogelstein 1983). Template DNA is denatured
and annealed to hexamers of random sequence. The hexamers anneal at
various points along the template and are elongated by the Klenow fragment of E. coli DNA polymerase I in the presence ofthree unlabeled dNTPs
and one 32 P-labeled dNTP. The Klenow fragment possesses the 5'-----+3'
synthase activity but lacks the 5' -----+3' exonuclease activity so that newly
synthesized probe is not rapidly degraded. The substrate DNA is not itself
radiolabeled but serves as the template for DNA synthesis. The resultant
products range in size from 400-600 nucleotides and have typical specific
activities of 10 8 -10 9 cpm/J..Lg. Reagents are available in kit form from several
manufacturers ( e. g., Amersham).