Preparing Single-Stranded DNA Templates
Many applications require the use of single-stranded DNA templates. The immobilization of a double-stranded PCR product for preparation of two single-stranded DNA fragments is probably the most widely used protocol for Dynabeads® M-280 Streptavidin. A PCR product is first generated by having one of the two PCR-primers biotinylated. The PCR product is immobilized onto the beads for separation of the biotinylated and non-biotinylated strands. Elution conditions can be heating or alkali denaturation. All excess primers, nucleotides and buffer components are removed, without any need for centrifugation or precipitation.
The streptavidin-coupled Dynabeads® will not inhibit any enzymatic activity. This enables further handling and manipulation of the bead-bound DNA directly on the solid-phase.
Both the immobilized and the eluted single-stranded DNA template can be used in downstream applications such as MALDI-MS (1), pyrosequencing technology (2) and SNP analysis (3), as well as single-strand conformation polymorphism (SSCP), solid-phase DNA sequencing (4,5), DNA chips & microarrays, allele-specific extension and primer extension. Single-stranded DNA templates are also used for in vitro mutagenesis, nuclease S1 mapping (6), probe preparation and labelling (7), subtractive hybridization (8,9,10) and a number of other molecular techniques.
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