Single Stranded DNA Graph
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.

Selected References

  1. von Wintzingerode F. (2002). Base-specific fragmentation of amplified 16S rRNA genes analyzed by mass-spectrometry: A tool for rapid bacterial identification. PNAS 99(10):7039-7044.
  2. Pourmand N. (2002) Multiplex pyrosequencing. Nucleic Acids Res. 30(7):e31.
  3. Lindblad-Toh (2000) Large-scale discovery and genotyping of single-nucleotide polymorphisms in the mouse. Nature Genetics. 24:381-386.
  4. Hultman T. (1989) Direct solid phase sequencing of genomic and plasmid DNA using magnetic beads as solid support. Nucl. Acids Res. 17(13):4937-4946.
  5. Hultman T. (1991) Bidirectional solid-phase sequencing of in vitro-amplified plasmid DNA. BioTechniques 10(1):84-93.
  6. Dziembowski A. (2001) Analysis of 3' and 5' ends of RNA by solid-phase S1 nuclease mapping. Anal. Biochem. 294:87-89.
  7. Beulieu M. (2001) PCR candidate region mismatch scanning: adaptation to quantitative, high-throughput genotyping. Nucleic Acids Res. 29(5):1114-1124
  8. Hansen-Hagge TE. (2001) Identification of sample-specific sequences in mammalian cDNA and genomic DNA by the novel ligation-mediated subtraction (LIMES). Nucl. Acids Res. 29(4):e20.
  9. Pradel N. (2002) Genomic subtraction to identify and characterize sequences of Shiga toxin-producing Escherichia coli O91:H21. Appl. Env. Microbiol. 68(5):2316-2325.
  10. Laveder P. (2002) A two-step strategy for constructing specifically self-subtracted cDNA libraries. Nucleic Acids Res. 30(9):e38.