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YOSHIO KANEMITSU AND TAKEHIKO KOTI
probe made it much easier to prepare ss eDNA probes. We could
produce a sufficient amount of ss eDNA with good reproducibility, using ordinary PCR system. If only a target mRNA sequence is known, we can synthesize the complementary ss
eDNA to the arbitrary portion of the mRNA sequence by asymmetric PCR, following R T -PCR.
There are two types of asymmetric PCR; single-step method
and two-step method. As shown in Results, we found that twostep asymmetric PCR was superior to single-step method. Twostep asymmetric PCR constantly produced approximately ten
times more ss eDNA than ds eDNA in molarity, so the neutralizing effect of homologous strand included in the residual dsDNA
on hybridization was negligible and we did not need to remove
the ds eDNA.
There are a number of non-radioactive methods to label ss
eDNA. Most of the methods utilize haptenic nucleic acid analogues such as digoxigenin-ll-dUTP, and biotin-ll-dUTP which
are enzymatically incorporated during the generation of ss
eDNA. However, haptenic analogues spoil the efficacy of the
DNA production in some degree, or chemical labeling often
has the problem of instability of reaction. Among nonradioactive
ISH methods, T -T dimerization method which we originally developed is unique; T -T dimer, which is formed between adjacently located thymine molecules by UV irradiation, is used
as a hapten and subsequently detected immunohistochemically
by specific antibodies against T -T dimer. The haptenization is
performed after PCR, so no interference to PCR occurs. T -T dimer formation is a simple physical reaction and the formed T -T
dimers are chemically very stable. Thus we have practically no
need to check the labeling efficacy in each case, and no purification step is required after the haptenization of DNA.
Combination of two-step asymmetric PCR and T -T dimerization enables mass production of sensitive ss eDNA probe for ISH,
in a simple and reproducible way.
Outline
The entire protocol of in situ hybridization with T -T dimerized ss
eDNA probe is illustrated in Fig. 1.
YOSHIO KANEMITSU AND TAKEHIKO KOTI
probe made it much easier to prepare ss eDNA probes. We could
produce a sufficient amount of ss eDNA with good reproducibility, using ordinary PCR system. If only a target mRNA sequence is known, we can synthesize the complementary ss
eDNA to the arbitrary portion of the mRNA sequence by asymmetric PCR, following R T -PCR.
There are two types of asymmetric PCR; single-step method
and two-step method. As shown in Results, we found that twostep asymmetric PCR was superior to single-step method. Twostep asymmetric PCR constantly produced approximately ten
times more ss eDNA than ds eDNA in molarity, so the neutralizing effect of homologous strand included in the residual dsDNA
on hybridization was negligible and we did not need to remove
the ds eDNA.
There are a number of non-radioactive methods to label ss
eDNA. Most of the methods utilize haptenic nucleic acid analogues such as digoxigenin-ll-dUTP, and biotin-ll-dUTP which
are enzymatically incorporated during the generation of ss
eDNA. However, haptenic analogues spoil the efficacy of the
DNA production in some degree, or chemical labeling often
has the problem of instability of reaction. Among nonradioactive
ISH methods, T -T dimerization method which we originally developed is unique; T -T dimer, which is formed between adjacently located thymine molecules by UV irradiation, is used
as a hapten and subsequently detected immunohistochemically
by specific antibodies against T -T dimer. The haptenization is
performed after PCR, so no interference to PCR occurs. T -T dimer formation is a simple physical reaction and the formed T -T
dimers are chemically very stable. Thus we have practically no
need to check the labeling efficacy in each case, and no purification step is required after the haptenization of DNA.
Combination of two-step asymmetric PCR and T -T dimerization enables mass production of sensitive ss eDNA probe for ISH,
in a simple and reproducible way.
Outline
The entire protocol of in situ hybridization with T -T dimerized ss
eDNA probe is illustrated in Fig. 1.