14
SATOSHI HASHIMOTO AND TAKASHI KANEMATSU
(1)
6'
3'
(2)
6'
3'
(3)
6'
3'
(4)
6'
3'
0000
* Biotin
(!) dUTP
OdNTP
°
000000000000000000000000
0000000000000000000000000000000
~o~ I
2
' ;' Ofr
°
0000
00000000000000000000000
00000000000000000000000000
Fig. 1. Principle of non-radioactive ISNT. (1) First nick contacts with NT buffer
and E. coli DNA polymerase 1. (2) E. coli DNA polymerase I first recognize "nick"
and connect with it. (3) The polymerase usually adds a new complementary
deoxynucleotide. Newly synthesized DNA strand can be labeled with haptenic
nucleotide analogues such as biotin-ll-dUTP. (4) Biotin are reacted with horseradish peroxydase (HRP)-labeled anti-biotin antibody and visualized enzymeimmunohistochemically. Pol: E. coli DNA polymerase I Ab: Anti-biotin antibody Inc., USA), HRP: Horseradish peroxydase
SATOSHI HASHIMOTO AND TAKASHI KANEMATSU
(1)
6'
3'
(2)
6'
3'
(3)
6'
3'
(4)
6'
3'
0000
* Biotin
(!) dUTP
OdNTP
°
000000000000000000000000
0000000000000000000000000000000
~o~ I
2
' ;' Ofr
°
0000
00000000000000000000000
00000000000000000000000000
Fig. 1. Principle of non-radioactive ISNT. (1) First nick contacts with NT buffer
and E. coli DNA polymerase 1. (2) E. coli DNA polymerase I first recognize "nick"
and connect with it. (3) The polymerase usually adds a new complementary
deoxynucleotide. Newly synthesized DNA strand can be labeled with haptenic
nucleotide analogues such as biotin-ll-dUTP. (4) Biotin are reacted with horseradish peroxydase (HRP)-labeled anti-biotin antibody and visualized enzymeimmunohistochemically. Pol: E. coli DNA polymerase I Ab: Anti-biotin antibody Inc., USA), HRP: Horseradish peroxydase