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Documentos de Profesional
Documentos de Cultura
Christoph C. Tebbe Institut fr Agrarkologie (Dept. Agroecology) Bundesforschungsanstalt fr Landwirtschaft (FAL) Braunschweig - Germany
Basic Information
Invention of SSCP
Orita, M., H. Iwahana, H. Kanazawa, K. Hayashi, and T. Sekyia. 1989. Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc. Nat. Acad. Sci. USA 86:2766-2770
more References
Automation of PCR-SSCP product analysis for bacterial identification using the ABI machine
Widjojoatmodjo, M. C., A. C. Fluit, and J. Verhoef. 1995. Molecular identification of bacteria by fluorescence-based PCR-single-strand-conformation polymorphism of the 16S rRNA gene. J. Clinic. Microbiol. 33:2601-2606
Use of PCR-SSCP for ITS analysis for identification of fungi (pure cultures) and suggested for community analysis
Simon, L., R. C. Levesque, and M. Lalonde. 1993. Identification of endomycorrhizal fungi colonizing roots by fluorescent single-strand conformation polymorphism-PCR. Appl. Environ. Microbiol. 59:4211-4215 Kumeda, Y., and T. Asao. 1996. Single-strand conformation polymorphism analysis of PCR-amplified ribosomal DNA internal transcribed spacers to differentiate species of Aspergillus section flavi. Appl. Environ. Microbiol. 62:2947-2952 ... Clapp, J. P. 1999. The identification of root-associated fungi by PCR-SSCP, p. 1-18. In A. D. Akkermans, J. D. van Elsas, and F. J. de Bruijn, Molecular Microbial Ecology Manual, 3.4.7. Kluwer Academic Publ., Dordrecht, The Netherlands.
Bacterial community analysis of bioreactor studies and automated SSCP with product identification by co-migrations
Zumstein, E., R. Moletta, and J. J. Gordon. 2000. Examination of two years of community dynamics in an anaerobic bioreactor using fluorescence polymerase chain reaction singlestrand-conformation-polymorphism analysis. Environ. Microbiol. 2:69-78
Application for monitoring compost MCs, different primers for different taxonomic groups, isolation and sequencing of single bands from profiles
Peters, S., S. Koschinsky, F. Schwieger, and C. C. Tebbe. 2000. Succession of microbial communities during hot composting as detected by PCR-SSCP-based genetic profiles of smallsubunit rRNA genes. Appl. Environ. Microbiol. 66:930-936
, according to Tebbe and Vahjen, 1993, Appl. Environ. Microbiol. 59: 2657-2665; 2, Qiagen, Hilden, Germany
Generation of SSCP-patterns
Macrophor System (Pharmacia) MDE (0.6-fold; corresp. to approx. 7 % polyacryl amide; FMC BioWhittaker), no glycerol etc. cast on thermostatic plate treated with repel silane, cover plate with bind silane - spacer 0.4 mm, fixed with clamps MDE in 1 x TBE, filtrate and degas add TEMED and APS for polymerisation cast gel, add comb 2 h at room temperature
dried gels are rehydrated with A. dest. for 2 to 5 min cut out selected bands with a razor blade - transer to a microfuge tube - crush and soak (Sambrook et al., 1989) reamplify with same primers by PCR, purify with Qiaquick, check on SSCP next to a community profile. Products which match with the intended bands can then be sequenced
AGAAAAGAAAagTGAAGGCACggAAAATAaAAGGAATTGACGGtAACCCA AAGGAATTGACGGAAACTTAAAGGAATTgACGGAAAcTCAAAGGAGTTGA CGGAAACTCAAAGGAATTGACGGAAACTCAAAGGAATTGACGGAATCACT AGTGCGGCCGCCTGCAGGtCGACCATATGGGAGAGCTcMCAACGCGTTGG ATGCATABTTGAGTATTCTATAGTGTCACCTAAATAGCTTGGCGTAATcA Tgg GGAGCTCtCCCATAtGGTCGACCKGCAGGCGGCCGCACTAGtGATT CCGTCAATTCCTTTGAGTTTCCGTCAATTCCTTTGAGTTTCCGTcAACTC CTTTGAGTTTCCGTCAATTCCTTTAAGTTTCCGTCAATTCCTTTGGGTTT CCGTCAATTCCTTTGAGTTTCCGTCAATTCCTTTGAGTTTCTGCTGCCTC cCCGCGGCCATGGCGGCgcGGGAGCATgCGACGTcGGGCCCAATTcGCCC TATAGTGAGTTCGTATTACAATTCAC GGAGCTCtCCCATAtGGTCGACC CAATTCCTTTGAGTTTCCGTCAATTCCTTTGAGTTTCCGTcAACTCCTTT GAGTTTCCGTCAATTCCTTTAAGTTTCCGTCAATTCCTTTGGGTTTCCGT CAATTCCTTTGAGTTTCCGTCAATTCCTTTGAGTTTCTGCTGCCTCCaaT cCCGCGGCCATGGCGGCgcGGGAGCATgCGACGTcGGGCCCAATTcGCCC TATAGTGAGTTCGTATTACAATTCACAGAAAAGAAAagTGAAGGCACggA AAGGAATTGACGGAAACTTAAAGGAATTgACGGAAAcTCAAAGGAGTTGA CGGAAACTCAAAGGAATTGACGGAAACTCAAAGGAATTGACGGAATCACT AGTGCGGCCGCCTGCAGGtCGACCATATGGGAGAGCTcMCAACGCGTTGG ATGCATABTTGAGTATTCTATAGTGTCACCTAAATAGCTTGGCGTAATcA Tgg GGAGCTCtCCCATAtGGTCGACCKGCAGGCGGCCGCACTAGtGATT CAATTCCTTTGAGTTTCCGTCAATTCCTTTGAGTTTCCGTcAACTCCTTT GAGTTTCCGTCAATTCCTTTAAGTTTCCGTCAATTCCTTTGGGTTTCCGT CAATTCCTTTGAGTTTCCGTCAATTCCTTTGAGTTTCTGCTGCCTCCaaT CAATTCCTTTGAGTTTCCGTCAATTCCTTTGAGTTTCCGTcAACTCCTTT GAGTTTCCGTCAATTCCTTTAAGTTTCCGTCAATTCCTTTGGGTTTCCGT CAATTCCTTTGAGTTTCCGTCAATTCCTTTGAGTTTCTGCTGCCTCCaaT cCCGCGGCCATGGCGGCgcGGGAGCATgCGACGTcGGGCCCAATTcGCCC TATAGTGAGTTCGTATTACAATTCAC GGAGCTCtCCCATAtGGTCGACC CAATTCCTTTGAGTTTCCGTCAATTCCTTTGAGTTTCCGTcAACTCCTTT GAGTTTCCGTCAATTCCTTTAAGTTTCCGTCAATTCCTTTGGGTTTCCGT CAATTCCTTTGAGTTTCCGTCAATTCCTTTGAGTTTCTGCTGCCTCCaaT cCCGCGGCCATGGCGGCgcGGGAGCATgCGACGTcGGGCCCAATTcGCCC TATAGTGAGTTCGTATTACAATTCACAGAAAAGAAAagTGAAGGCACggA AAGGAATTGACGGAAACTTAAAGGAATTgACGGAAAcTCAAAGGAGTTGA CGGAAACTCAAAGGAATTGACGGAAACTCAAAGGAATTGACGGAATCACT AGTGCGGCCGCCTGCAGGtCGACCATATGGGAGAGCTcMCAACGCGTTGG ATGCATABTTGAGTATTCTATAGTGTCACCTAAATAGCTTGGCGTAATcA
... and what happened to that marine community DNA from Nick Fuller and Dave Scanlan?
V4/V5 region (Com primers)