Info

Watson-Crick base pairing mediates the mutual recognition of two complementary nucleic acid strands [1]. It is the molecular basis of life and

Fig. B.17.1. Hybridization leads to the formation of the PNA double helix.

of most current approaches in oligonucleotide-based diagnostics. The hybridization event anneals two single-stranded nucleic acids to form a duplex molecule with helically stacked nucleobases (Figure B.17.1). Nucleic acid hybridization is reversible and can be regarded as an association-dissociation or renaturation-denaturation process. The stability of a duplex molecule toward thermal denaturation depends on (a) base composition, (b) base sequence, (c) oligomer length, and (d) cation concentration.

Fig. B.17.2. Real melting curve of a duplex with TM = 65.8 °C. The duplex comprised 5'-GGGCGCTGGAGGTGTG-3' and the complementary strand.

At high temperatures duplex dissociation occurs. The temperature at which 50% of the duplexes are dissociated is called the melting temperature Tm. The TM value is, hence, a measure of the thermal stability of a duplex. How are TM values determined?

Nucleic acids absorb UV light with an absorption maximum at approximately 260 nm. This absorption mainly results from the nucleobases. Importantly, double-stranded DNA has a lower molar absorption coefficient, e, than calculated for the sum of the molar absorption coefficients of the bases. Accordingly, duplex denaturation is accompanied by increased UV absorption [2]. This effect is termed "hyperchromicity" and originates from the disruption of base stacking.

The thermal denaturation of a DNA duplex is also called ''DNA melting''. The melting behavior of a duplex can be conveniently studied by measuring the temperature dependence of UV absorption. The resulting plot is known as the ''melting curve'' (Figure B.17.2). Typically, melting curves are sigmoid in form, which is indicative of co-operative base stacking. The inflection point is known as the ''melting temperature, TM''.

0 0

Post a comment