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Direct sequencing of RAPD fragments using 3'-extended oligonucleotide primers and dye terminator cycle-sequencing.

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.

SUBMITTER: Mitchelson KR

PROVIDER: S-EPMC148638 | biostudies-other | 1999 Oct

REPOSITORIES: biostudies-other

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Direct sequencing of RAPD fragments using 3'-extended oligonucleotide primers and dye terminator cycle-sequencing.

Mitchelson K R KR Drenth J J Drenth J J Duong H H Chaparro J X JX

Nucleic acids research 19991001 19

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 e ...[more]

PMID: 10481040

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Project description:Third-generation DNA sequencing has enabled sequencing of long, unamplified DNA fragments with minimal steps. Direct sequencing of ssDNA or RNA gives valuable insights like base-level modifications, phosphoramidite synthesis yield estimates and strand quality analysis, without the need to add the complimentary strand. Direct sequencing of single-stranded nucleic acid species is challenging as they are non-compatible to the double-stranded sequencing adapters used by manufacturers. The MinION platform from Oxford Nanopore Technologies performs sequencing by passing single-strands of DNA through a layer of biological nanopore sensors; although sequencing is performed on single-strands, the recommended template by the manufacturer is double-stranded. We have identified that the MinION platform can perform sequencing of short, single-strand oligonucleotides directly without amplification or second-strand synthesis by performing a single annealing step before library preparation. Short 5' phosphorylated oligos when annealed to an adapter sequence can be directly sequenced in the 5' to 3' direction via nanopores. Adapter sequences were designed to bind to the 5' end of the oligos and to leave a 3' adenosine overhang after binding to their target. The 3' adenosine overhang of the adapter and the terminal phosphate makes the 5' end of the oligo analogous to an end-prepared dsDNA, rendering it compatible with ligation-based library preparation for sequencing. An oligo-pool containing 42,000, 120 nt orthogonal sequences was phosphorylated and sequenced using this method and ~90% of these sequences were recovered with high accuracy using BLAST. In the nanopore raw data, we have identified that empty signals can be wrongly identified as a valid read by the MinION platform and sometimes multiple signals containing several strands can be fused into a single raw sequence file due to segmentation faults in the software. This direct oligonucleotide sequencing method enables novel applications in DNA data storage systems where short oligonucleotides are the primary information carriers.

Dataset Information

Direct sequencing of RAPD fragments using 3'-extended oligonucleotide primers and dye terminator cycle-sequencing.

Publications

Direct sequencing of RAPD fragments using 3'-extended oligonucleotide primers and dye terminator cycle-sequencing.

Similar Datasets

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