1 Introduction to the Detection of DNA Strand Breaks
5
the region of actively transcribed chromatin (Kerem et al. 1984)
and performed with radioactive compounds. However, the
radioactive ISNT is generally time-consuming and expensive,
and it is hard to localize the sites of nicks precisely on the subnuclear basis. That is why, more recently, the application of nonradioactive ISNT has become frequent (Gold et al. 1994; Hashimoto et al. 1995). Furthermore, the needs for such a technique
have recently increased rapidly in the particular field dealing
with apoptotic cell death, which is characterized by a special occurrence of DNA strand breaks in the early stage of cell death
commitment (Peitsch et al. 1993; Bortner et al. 1995). In addition,
SSB are also known to be closely associated with necrosis in the
cell death issues. Therefore, it now seems appropriate to give the
experimental details in this methodology, as a part of molecular
histochemical methods (Koji and Nakane 1996). In the following
part, I would like to show the basic principle of nonradioactive
ISNT and some examples in relation to the cell death subject.
"Nick translation" is a very common method of labeling
cDNA to produce molecular biological probes, in which
cDNA is nicked by DNase I digestion and at the nicked sites
a new DNA strand is synthesized using the intact strand as a template. Basically, ISNT is based on the same principle to that of
"nick translation". However, DNase I digestion is not needed.
The sites of SSB in nuclei can be first recognized by E. coli
DNA polymerase I, which has three enzymatic activities; 5' to
3' DNA polymerase activity, 3' to 5' exonuclease activity and
5' to 3' exonuclease activity. As shown in Fig. I, using the activities, the polymerase usually adds a new complementary deoxyribonucleotide to an intact free 3' -OH residue in the presence
of dATP, dGTP, dCTP and TTP, depending upon the base sequence of the intact template strand. At the time of elongation,
the newly synthesized DNA strand can be labeled with haptenic
nucleotide analogues such as biotin-ll-dUTP and digoxigeninII-dUTP when the one of substrates, TTP is replaced with these
analogues. Finally, the incorporated hapten molecules are reacted with horseradish peroxidase (HRP)-labeled anti-hapten
antibody and visualized enzyme-immunohistochemically. In
"nick translation", the polymerase reaction is usually conducted
around 15° C to ensure the high fidelity of the synthesized base
sequences to the template strand. In ISNT, however, such a high
fidelity in base complementarity between a newly synthesized
5
the region of actively transcribed chromatin (Kerem et al. 1984)
and performed with radioactive compounds. However, the
radioactive ISNT is generally time-consuming and expensive,
and it is hard to localize the sites of nicks precisely on the subnuclear basis. That is why, more recently, the application of nonradioactive ISNT has become frequent (Gold et al. 1994; Hashimoto et al. 1995). Furthermore, the needs for such a technique
have recently increased rapidly in the particular field dealing
with apoptotic cell death, which is characterized by a special occurrence of DNA strand breaks in the early stage of cell death
commitment (Peitsch et al. 1993; Bortner et al. 1995). In addition,
SSB are also known to be closely associated with necrosis in the
cell death issues. Therefore, it now seems appropriate to give the
experimental details in this methodology, as a part of molecular
histochemical methods (Koji and Nakane 1996). In the following
part, I would like to show the basic principle of nonradioactive
ISNT and some examples in relation to the cell death subject.
"Nick translation" is a very common method of labeling
cDNA to produce molecular biological probes, in which
cDNA is nicked by DNase I digestion and at the nicked sites
a new DNA strand is synthesized using the intact strand as a template. Basically, ISNT is based on the same principle to that of
"nick translation". However, DNase I digestion is not needed.
The sites of SSB in nuclei can be first recognized by E. coli
DNA polymerase I, which has three enzymatic activities; 5' to
3' DNA polymerase activity, 3' to 5' exonuclease activity and
5' to 3' exonuclease activity. As shown in Fig. I, using the activities, the polymerase usually adds a new complementary deoxyribonucleotide to an intact free 3' -OH residue in the presence
of dATP, dGTP, dCTP and TTP, depending upon the base sequence of the intact template strand. At the time of elongation,
the newly synthesized DNA strand can be labeled with haptenic
nucleotide analogues such as biotin-ll-dUTP and digoxigeninII-dUTP when the one of substrates, TTP is replaced with these
analogues. Finally, the incorporated hapten molecules are reacted with horseradish peroxidase (HRP)-labeled anti-hapten
antibody and visualized enzyme-immunohistochemically. In
"nick translation", the polymerase reaction is usually conducted
around 15° C to ensure the high fidelity of the synthesized base
sequences to the template strand. In ISNT, however, such a high
fidelity in base complementarity between a newly synthesized