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Molecular Biology and Genetics |
| Electronic Journal of Biotechnology ISSN: 0717-3458 |
Vol. 8 No. 2, Issue of August 15, 2005 |
| © 2005 by Pontificia Universidad Católica de Valparaíso -- Chile |
Received July 2 , 2004 / Accepted March 29, 2005 |
LaNe RAGE: a new tool for genomic DNA flanking sequence determination
Daniel J. Park
Department of Zoology
The University of Melbourne
Gate 12, Royal Parade
Victoria 3010, Australia
Tel: 61 3 83444351
Fax: 61 3 83447909
E-mail: d.park@unimelb.edu.au
Financial support: This work was supported by the National Health and Medical Research Council of Australia grant no. 148630 to Professors J.A.M. Graves and M.B. Renfree, Associate Professor V.R. Harley and Dr. A.J. Pask.
Keywords: cloning, DNA fingerprinting, genomic DNA walking, PCR, sequencing, transposon mutagenesis.
| Abbreviations: |
GAPDH: glyceraldehyde-3-phosphate dehydrogenase;
gDNA: genomic DNA;
LaNe RAGE: lariat-dependent nested PCR for rapid amplification of genomic DNA ends;
PCR: polymerase chain reaction;
PGK1: phosphoglycerate kinase 1;
RAPD: randomly amplified polymorphic DNA.
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The determination of genomic DNA sequence flanking a known region is often problematic. Existing technologies depend on multiple, efficient enzyme-catalysed preparative processing steps and/or rely on relatively inefficient 'one-sided' PCR mechanisms. I demonstrate the application of a simple 'two-sided' PCR-based approach, lariat-dependent nested PCR for rapid amplification of genomic DNA ends (LaNe RAGE), applied to the mouse GAPDH and PGK1 gene flanking sequences. This demonstration offers great promise in applications such as genome walking, transposon mutagenesis mapping and DNA fingerprinting.
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