Abstract
We report a simple and efficient method, which combines restriction endonuclease digestion and deoxynucleotide tailing, for cloning unknown genomic sequences adjacent to a known sequence. Total genomic DNA is partially digested with the frequent-cutting restriction enzymeNla III. A homo-oligomeric cytosine tail is added by terminal transferase. The tailed DNA fragments are used as the template for cloning flanking regions from all sequences of interest. A first round PCR amplification is performed with a gene-specific primer and the selective (modified polyguanine) anchor primer complementary to the cytosine tail and theNla III recognition site, with a universal amplification primer sequence at its 5′ end. This is followed by another PCR amplification with a nested gene-specific primer and the universal amplification primer. Finally, the amplified products are fractionated, cloned, and sequenced. Using this method, we cloned the upstream region of a salt-induced gene based upon a partial cDNA clone (RSC5-U) obtained from sunflower (Helianthus annuus L.).
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Abbreviations
- GSP:
-
gene specific primer
- RAGE:
-
rapid amplification of genomic ends
- RE:
-
restriction enzyme
- SAP:
-
selective anchor primer
- TdT:
-
terminal deoxynucleotide-transferase
- UAP:
-
universal amplification primer
References
Espelund M and Jakobsen KS (1992) Cloning and direct sequencing of plant promoters using primer-adaptor mediated PCR on DNA coupled to a magnetic solid phase. Bio Techniques 13: 74–81.
Frijters A, Pot J, Peleman J, Kuiper M and Zabeau M (1995) AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res 23: 4407–4414.
Iwahana H and Itakura M (1997) An end trimming method and its application to amplify adjacent cDNA and genomic DNA fragments by PCR. Methods Mol Biol 67: 247–260.
Kilstrup M and Kristiansen KN (2000) Rapid genome walking: a simplified oligo-cassette mediated polymerase chain reaction using a single genome-specific primer. Nucleic Acids Res 28: e55.
Moynihan TP, Markham AF and Robinson PA (1996) Genomic analysis of human multigene families using chromosome-specific Vectorette PCR. Nucleic Acids Res 24: 4094–4095.
Ochman H, Gerber AS and Hartl DL (1988) Genetic applications of an inverse polymerase chain reaction. Genetics 120: 621–623.
Qiang BQ and Schildkraut I (1986) Two unique restriction endonucleases fromNeisseria lactamica. Nucleic Acids Res 14: 1991–1999.
Parker JD, Rabinovitch PS and Burmer GC (1991) Targeted gene walking polymerase chain reaction. Nucleic Acids Res 19: 3055–3060.
Riley J, Butler R, Ogilvie D, Finniear R, Powell S, Anand R, Smith JC and Markham AF (1990) A novel, rapid method for the isolation of terminal sequences from yeast artificial chromosome (YAC) clones. Nucleic Acids Res 18: 2887–2890.
Rosenthal A and Jones DSC (1990) Genomic walking and sequencing by oligo-cassette mediated polymerase chain reaction. Nucleic Acids Res 18: 3095–3096.
Rudi K, Fossheim T and Jakobsen KS (1999) Restriction cutting independent method for cloning genomic DNA, segments outside the boundaries of known sequences. Bio Techniques 27: 1170–1174.
Sambrook J, Fritch EF and Maniatis T (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
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Liu, X., Vance Baird, W. Rapid amplification of genomic DNA ends bynla III partial digestion and polynucleotide tailing. Plant Mol Biol Rep 19, 261–267 (2001). https://doi.org/10.1007/BF02772898
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DOI: https://doi.org/10.1007/BF02772898