Chapter 9
Radioactive labeling of DNA and RNA Probes
PETER IGARASHI
Subprotocol 1
End-Labeling Using T 4 Polynucleotide Kinase
Description End-labeling is most commonly utilized for synthetic oligodeoxyribonucleotides, although double-stranded DNA may also be labeled (particularly
if protruding 5' ends are present). End-labeled oligonucleotides are used for
hybridization probes, primer extension analysis, or DNA sequencing using
the Maxam-Gilbert chemical cleavage method. End-labeled doublestranded DNA is used in nuclease protection assays and other applications
requiring probes of defined length.
5' ends ofDNA (or RNA) are labeled with y[ 32 P]ATP using bacteriophage
T4 polynudeotide kinase (see also Chap. 2). Polynucleotide kinase transfers
the y-phosphate group from ATP to the free 5' hydroxyl group ofDNA (or
RNA) (Chaconas and van de Sande 1980). For DNA containing a 5' phosphate group, dephosphorylation with calf intestinal alkaline phosphatase
is required prior to labeling. Alternatively, an exchange kinase reaction
(Berkner and Folk 1977) may be performed in which polynucleotide kinase
transfers the 5' phosphate group from the DNA substrate to an excess of
AD P. The dephosphorylated substrate is then rephosphorylated by transfer
of y-phosphate from y[ 32 P]ATP.
3' ends of DNA are labeled with terminal transferase (terminal deoxynucleotidyl transferase) which transfers dNTPs to free hydroxyl groups at
the 3' ends ofDNA (Deng and Wu 1983). End-labeled probes generated with
polynucleotide kinase have relatively low specific activity because only one
mole of 32 P is incorporated per mole of template. Since terminal transferase
can add many nucleotides to each molecule of template, this enzyme is frequently preferred when higher specific activity is required ( e.g., in situ hybridization) (Biroc et al. 1993).
Peter Igarashi, Yale University Medical School, Division ofNephrology, 333 Cedar Street,
New Haven, Connecticut, USA
Radioactive labeling of DNA and RNA Probes
PETER IGARASHI
Subprotocol 1
End-Labeling Using T 4 Polynucleotide Kinase
Description End-labeling is most commonly utilized for synthetic oligodeoxyribonucleotides, although double-stranded DNA may also be labeled (particularly
if protruding 5' ends are present). End-labeled oligonucleotides are used for
hybridization probes, primer extension analysis, or DNA sequencing using
the Maxam-Gilbert chemical cleavage method. End-labeled doublestranded DNA is used in nuclease protection assays and other applications
requiring probes of defined length.
5' ends ofDNA (or RNA) are labeled with y[ 32 P]ATP using bacteriophage
T4 polynudeotide kinase (see also Chap. 2). Polynucleotide kinase transfers
the y-phosphate group from ATP to the free 5' hydroxyl group ofDNA (or
RNA) (Chaconas and van de Sande 1980). For DNA containing a 5' phosphate group, dephosphorylation with calf intestinal alkaline phosphatase
is required prior to labeling. Alternatively, an exchange kinase reaction
(Berkner and Folk 1977) may be performed in which polynucleotide kinase
transfers the 5' phosphate group from the DNA substrate to an excess of
AD P. The dephosphorylated substrate is then rephosphorylated by transfer
of y-phosphate from y[ 32 P]ATP.
3' ends of DNA are labeled with terminal transferase (terminal deoxynucleotidyl transferase) which transfers dNTPs to free hydroxyl groups at
the 3' ends ofDNA (Deng and Wu 1983). End-labeled probes generated with
polynucleotide kinase have relatively low specific activity because only one
mole of 32 P is incorporated per mole of template. Since terminal transferase
can add many nucleotides to each molecule of template, this enzyme is frequently preferred when higher specific activity is required ( e.g., in situ hybridization) (Biroc et al. 1993).
Peter Igarashi, Yale University Medical School, Division ofNephrology, 333 Cedar Street,
New Haven, Connecticut, USA