Auszug
Wenn man schließlich einen neuen Klon gefunden hat, ist der nächste Schritt die Analyse. Die kann man unterschiedlich weit treiben, je nachdem, wie viel Zeit und Energie zu investieren man gewillt ist. Die einfachste Variante ist die Kartierung mittels Restriktionsfragmentanalyse (Restriktionskartierung). Die DNA wird dazu mit verschiedenen Restriktionsenzymen — einzeln und paarweise — verdaut, im Agarosegel getrennt und die resultierenden Fragmentgrößen bestimmt (s. Abb. 45). Die einzelnen Fragmente miteinander zu kombinieren, bis man eine verlässliche Restriktionskarte seines Klons in Händen hält, gleicht einem Puzzle, und bei DNAs von über 10 kb Länge bedarf es schon einiger kombinatorischer Intelligenz, um die Stückchen zu einem sinnvollen Ganzen zusammenzusetzen, aber die Analyse ist recht flott — Verdau und Gel lassen sich problemlos in einem halben Tag erledigen — und verschafft einem für den Anfang eine gute Vorstellung davon, womit man es überhaupt zu tun hat.Wenn man das Gel zusätzlich blottet und mit einer passenden, bekannten Sonde hybridisiert, kann man sich weitere Informationen verschaffen, anhand derer man auch längere Klone charakterisieren kann, das dauert dann allerdings etwas länger.
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Literatur
Hutchison CA (2007) DNA sequencing: bench to bedside and beyond. Nucl.Acids Res. 35, 6227–6237
Maxam AM, Gilbert W (1977) A new method for sequencing DNA. Proc. Nat. Acad. Sci. USA 74, 560–564
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc. Nat. Acad. Sci. USA 74, 5463–5467
Literatur
Venter JC et al. (1998) Shotgun sequencing of the human genome. Science 280, 1540–1542
Adams MD et al. (1991) Complementary DNA Sequencing: Expressed Sequence Tags and Human Genome Project. Science 252, 1651–1656
Literatur
Hardin SH, Jones LB, Homayouni R, McCollum JC (1996) Octamer-primed cycle sequencing: design of an optimized primer library. Genome Res. 6, 545–550.
Jones LB, Hardin SH (1998) Octamer-primed cycle sequencing using dye-terminator chemistry. Nucl. Acids Res. 26, 2824–2826
Ball S, Reeve MA, Robinson PS, Hill F, Brown DM, Loakes D (1998) The use of tailed octamer primers for cycle sequencing. Nucl. Acids Res. 26, 5225–5227
Literatur
Sokolov BP (1990) Primer extension technique for the detection of single nucleotide in genomic DNA. Nucl. Acids Res. 18, 3671
Pastinen T et al. (1997) Minisequencing: a specific tool for DNA analysis and diagnosis on oligonucleotide arrays. Genome Res. 7, 606–614
Syvänen A-C (1999) From gels to chips:“Minisequencing”primer extension for analysis of point mutations and single nucleotide polymorphisms. Hum. Mutat. 13, 1–10
Literatur
Nyren P, Pettersson B, Uhlen M (1993) Solid phase DNA minisequencing by an enzymatic luminometric inorganic pyrophosphate detection assay. Anal. Biochem. 208, 171–175
Ronaghi M et al. (1996) Real-time DNA sequencing using detection of pyrophosphate release. Anal. Biochem. 242, 84–89
Ronaghi M, Uhlen M, Nyren P (1998) A sequencing method based on real-time pyrophosphate. Science 281, 363–365
Literatur
Biémont C., Vieira C. (2006) Genetics: junk DNA as an evolutionary force. Nature 443, 521–524
Green RE et al. (2006) Analysis of one million base pairs of Neanderthal DNA. Nature 444, 330–336
Lander ES et al. (2001) Initial sequencing and analysis of the human genome. Nature, 409, 860–921
Levy S, Sutton G et al. (2007) The Diploid Genome Sequence of an Individual Human. PLoS Biol. 5, e254. doi:10.1371/journal.pbio.0050254
Margulies M et al. (2005) Genome sequencing in microfabricated high-density picolitre reactors. Nature 437, 376–380
Venter JC et al. (2001) The sequence of the human genome. Science, 291, 1304–1351
Wheeler DA et al. (2008) The complete genome of an individual by massively parallel DNA sequencing. Nature 452, 872–876
Literatur
Chan EY (2005) Advances in sequencing technology. Mutat.Res. 573, 13–40
Deamer DW, Akeson M (2000) Nanopores and nucleic acids: prospects for ultrarapid sequencing. Trends Biotechol. 18, 147–151
Jett JH et al. (1989) High-speed DNA sequencing: an approach based upon fluorescence detection of single molecules. J.Biomol.Struct.Dyn. 7, 301–309
Ju J et al. (2006) Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators. Proc.Natl.Acad.Sci. USA 103, 19635–19640
Shendure J et al. (2005) Accurate multiplex polony sequencing of an evolved bacterial genome. Science 309, 1728–1732
Literatur
Sogin ML et al. (2006) Microbial diversity in the deep sea and the underexplored“rare biosphere”. Proc.Natl. Acad.Sci. USA 103, 12115–12120
Venter JC et al. (2004) Environmental Genome Shotgun Sequencing of the Sargasso Sea. Science 304, 66–74
Sultan M et al. (2008) A Global View of Gene Activity and Alternative Splicing by Deep Sequencing of the Human Transcriptome. Science (published online, 3. Juli 2008) DOI: 10.1126/science.1160342
Literatur
Orita M et al. (1989) Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc. Natl. Adac. Sci USA 86, 2766–2770
Orita M et al. (1989) Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5, 874–879
Yap EPH, McGee JOD (1992) Nonisotopic SSCP detection in PCR products by ethidium bromide staining. Trends Genet. 8, 49
Hayashi K, Yandell DW (1993) How sensitive is PCR-SSCP? Hum. Mutat. 2, 338–346
Suzuki Y, Sekiya T, Hayashi K (1991) Allele-specific polymerase chain reaction: a method for amplification and sequence determination of a single component among a mixture of sequence variants. Anal. Biochem. 192, 82–84.
Literatur
Fischer SG, Lerman LS (1983) DNA fragments differing by single base-pair substitutions are separated in denaturing gradient gels: Correspondence with melting theory. Proc. Natl. Acad. Sci. USA 80, 1579–1583
Borresen A-L et al. (1991) Constant denaturant gel electrophoresis as a rapid screening technique for p53 mutations. Proc. Natl. Acad. Sci. USA 88, 8405–8409
Literatur
Riesner D et al. (1989) Temperature-gradient gel electrophoresis of nucleic acids: analysis of conformational transitions, sequence variations, and protein-nucleic acid interactions. Electrophoresis 10, 377–389
Wiese U et al. (1995) Scanning for mutations in the human prion protein open reading frame by temporal temperature gradient gel electrophoresis. Electrophoresis 16, 1851–1860
Literatur
Zimmermann PA, Carrington MN, Nutman TB (1993) Exploiting structural differences among heteroduplex molecules to simplify genotyping the DQA1 and DQB1 alleles in human lymphocyte typing. Nucl. Acids Res. 21, 4541–4547
D’Amato M, Sorrentino R (1995) Short insertions in the partner strands greatly enhance the discriminating power of DNA heteroduplex analysis: resolution of HLA-DQB1 polymorphisms. Nucl. Acids Res. 23, 2078–2079.
Literatur
Babon J, Youil R, Cotton RGH (1995) Improved strategy for mutation detection — a modification to the enzyme mismatch cleavage method. Nucl. Acids Res. 23, 5082–5084
Literatur
Roest PA et al. (1993) Protein truncation test (PTT) for rapid detection of translation-terminating mutations. Hum. Mol. Genet. 2, 1719–1721
Hogervorst FBL (1997) Promega Notes 62, 7
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(2009). DNA-Analyse. In: Der Experimentator: Molekularbiologie/ Genomics. Experimentator. Spektrum Akademischer Verlag. https://doi.org/10.1007/978-3-8274-2158-6_8
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