5 Introduction to the Detection of Specific DNA and RNA Sequences
51
and digoxigenin (Dig). Among various nonradioactive labels,
thymine-thymine (T -T) dimer method developed by us previously (Koji and Nakane 1990) is unique because T-T dimer
can be easily introduced by ultraviolet light (UV) irradiation
on a DNA solution and it does not require any purification of
labeled probe from unreacted compounds. In this chapter, I
will use T -T dimer method as well as Dig method (Koji and Brenner 1993; Koji et al. 1994) for example, because of the close sensitivity between them. The overview will include recent progress
in optimization of the protocol and enhancement of signal intensity.
Principle of in situ hybridization
DNA consists of 4 bases, i.e. adenine (A), guanine (G), cytosine
(C) and thymine (T) and in the case of RNA thymine is replaced
with uracil (U). A can form a stable complex with T or U through
two hydrogen bonds and G can bind to C through three hydrogen
bonds, as shown in Pig. 1. Therefore, A to T or U and G to C, vice
versa, are regarded as complementary bases. This is a basic principle for nucleic acid hybridization. If a base sequence on a single
nucleic acid strand is complementary to that of the other strand,
they can form stable molecular hybrids to be double-stranded.
The stability of molecular hybrids highly depends upon temperature, a base sequence and a salt concentration. As a physicochemical parameter of the stability, Tm (melting temperature)
is often used, showing a temperature where a half of bases are
complex, if simplified. Tm between DNA and DNA can be calculated by the following empirical equations; Tm=8l.5 + 16.6
log[Na+]+0041(O/OGC)-820Ibases-0.61(O/OP) (for a DNA probe
with more than 100 bases) (Thomas et al. 1973; Meikoth et
al. 1984), Tm=8l.5 + 16.6 10g[Na+ ]+0041 (%GC)-675/bases0.61(%P) (for an oligodeoxynucleotide (oligo-DNA) probe)
(Henderson et al. 1991). Here, [Na+] ; a concentration of salt
in the reaction mixture, %GC; total number of G and C/total
number of bases, bases; total number of bases in the probe,
%P; a concentration of formamide, which is thought to work
as a hydrogen-bond blocker like urea and decreases the Tm value. When there are some base mismatches between the probe
and target sequences, 104 x (%mismatch) should be subtracted
51
and digoxigenin (Dig). Among various nonradioactive labels,
thymine-thymine (T -T) dimer method developed by us previously (Koji and Nakane 1990) is unique because T-T dimer
can be easily introduced by ultraviolet light (UV) irradiation
on a DNA solution and it does not require any purification of
labeled probe from unreacted compounds. In this chapter, I
will use T -T dimer method as well as Dig method (Koji and Brenner 1993; Koji et al. 1994) for example, because of the close sensitivity between them. The overview will include recent progress
in optimization of the protocol and enhancement of signal intensity.
Principle of in situ hybridization
DNA consists of 4 bases, i.e. adenine (A), guanine (G), cytosine
(C) and thymine (T) and in the case of RNA thymine is replaced
with uracil (U). A can form a stable complex with T or U through
two hydrogen bonds and G can bind to C through three hydrogen
bonds, as shown in Pig. 1. Therefore, A to T or U and G to C, vice
versa, are regarded as complementary bases. This is a basic principle for nucleic acid hybridization. If a base sequence on a single
nucleic acid strand is complementary to that of the other strand,
they can form stable molecular hybrids to be double-stranded.
The stability of molecular hybrids highly depends upon temperature, a base sequence and a salt concentration. As a physicochemical parameter of the stability, Tm (melting temperature)
is often used, showing a temperature where a half of bases are
complex, if simplified. Tm between DNA and DNA can be calculated by the following empirical equations; Tm=8l.5 + 16.6
log[Na+]+0041(O/OGC)-820Ibases-0.61(O/OP) (for a DNA probe
with more than 100 bases) (Thomas et al. 1973; Meikoth et
al. 1984), Tm=8l.5 + 16.6 10g[Na+ ]+0041 (%GC)-675/bases0.61(%P) (for an oligodeoxynucleotide (oligo-DNA) probe)
(Henderson et al. 1991). Here, [Na+] ; a concentration of salt
in the reaction mixture, %GC; total number of G and C/total
number of bases, bases; total number of bases in the probe,
%P; a concentration of formamide, which is thought to work
as a hydrogen-bond blocker like urea and decreases the Tm value. When there are some base mismatches between the probe
and target sequences, 104 x (%mismatch) should be subtracted