Direct sequencing of RAPD fragments using 3'-extended oligonucleotide primers and dye terminator cycle-sequencing
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Updated: Oct 19, 2026

Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo
Published on: October 31, 2014
K R Mitchelson1, J Drenth, H Duong
1ForBio Research Pty Ltd, 52 Douglas Street, Milton, Queensland 4064, Australia. keithmitchelson@hotmail.com
Abstract:
Random amplified polymorphic DNA (RAPD) markers are used widely to develop high resolution genetic maps and for genome fingerprinting. Typically, single oligomers of approximately 10 nucleotides are used to PCR amplify characteristic RAPD marker fragments. We describe an efficient method for the direct end-sequencing of gel-purified RAPD fragments using one primer from a set of four 3'-terminal extended (A, T, C or G) oligonucleotides, identical to the RAPD primer but for the single nucleotide extension. Strand-specific DNA sequence could be independently read from each of the RAPD fragments without recourse to strand separation or fragment cloning. Informative RAPD fragments could be readily converted into mapped STS or SCAR loci using this technology. The 3'-extended primers may also be used to amplify independent genomic RAPD markers.
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