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KAI-OLAF NETZER
Buffer conditions that influence hybridization are temperature, salt concentration, pH, and concentration of denaturants. The conditions are modified in order to achieve a certain Ievel of stringency, i.e. specificity of hybridization. Low stringency permits hybridization of imperfectly matched
sequences, whereas high stringency permits hybridization only of complementary strands. High temperature and low salt (NaCl) concentration create high stringency. This applies also to the post-hybridization washes.
Denaturation and melting point
The reverse of hybridization, denaturation, is accomplished by the disruption of double-stranded molecules. This is most simply achieved by boiling.
Denaturation Ieads to strand separation. At the melting point, 50% of all
possible double-stranded hybrids are formed. The melting point of a DNADNA hybrid in solution can be calculated according to the following estimation:
Tm[
0
C] = 81.5 + 16.6 log (MNa) + 0.41 (PcG) - PM - B/L - 0.65 (Pp)
MNa = molar concentration of monovalent cation (e. g., Na+)
PcG = percentage of guanine and cytosine in hybrid sequence
PM = percentage of mismatches ( on average, 1 o/o mismatch reduces Tm by
1 OC)
B = complexity ( 500 for hybrids Ionger than 100 nucleotides, 675 for hybrids
shorter than 100 nucleotides)
L = length in base pairs (bp)
Pp = percent formamide in the hybridization solution
The calculated melting point of hybrids may help to establish optimal
hybridization and washing conditions. In filter hybridizations, however,
the kinetics of hybrid formation and the Tm of hybrid molecules are different from those in solution hybridzation reactions. As a consequence, hybridization time and stringency for filter hybridizations must be determined mostly empirically.
For more details on nucleic acid hybridization, see Harnes and Higgins
(1985).
KAI-OLAF NETZER
Buffer conditions that influence hybridization are temperature, salt concentration, pH, and concentration of denaturants. The conditions are modified in order to achieve a certain Ievel of stringency, i.e. specificity of hybridization. Low stringency permits hybridization of imperfectly matched
sequences, whereas high stringency permits hybridization only of complementary strands. High temperature and low salt (NaCl) concentration create high stringency. This applies also to the post-hybridization washes.
Denaturation and melting point
The reverse of hybridization, denaturation, is accomplished by the disruption of double-stranded molecules. This is most simply achieved by boiling.
Denaturation Ieads to strand separation. At the melting point, 50% of all
possible double-stranded hybrids are formed. The melting point of a DNADNA hybrid in solution can be calculated according to the following estimation:
Tm[
0
C] = 81.5 + 16.6 log (MNa) + 0.41 (PcG) - PM - B/L - 0.65 (Pp)
MNa = molar concentration of monovalent cation (e. g., Na+)
PcG = percentage of guanine and cytosine in hybrid sequence
PM = percentage of mismatches ( on average, 1 o/o mismatch reduces Tm by
1 OC)
B = complexity ( 500 for hybrids Ionger than 100 nucleotides, 675 for hybrids
shorter than 100 nucleotides)
L = length in base pairs (bp)
Pp = percent formamide in the hybridization solution
The calculated melting point of hybrids may help to establish optimal
hybridization and washing conditions. In filter hybridizations, however,
the kinetics of hybrid formation and the Tm of hybrid molecules are different from those in solution hybridzation reactions. As a consequence, hybridization time and stringency for filter hybridizations must be determined mostly empirically.
For more details on nucleic acid hybridization, see Harnes and Higgins
(1985).