Denaturation is the first step in many DNA-based techniques. In PCR, the denaturation step (~94–95°C) separates the double-stranded DNA into single strands by breaking hydrogen bonds between base pairs. This makes the single-stranded template available for primer annealing. In Southern blotting, DNA is denatured before transfer. In DNA hybridisation experiments, denaturation allows probe binding. The order in PCR is: Denaturation → Annealing → Extension.