PCR-SSCP TechniquePCR-SSCP TechniqueProtocols and “Demonstration”Protocols and “Demonstration”

Christoph C. TebbeChristoph C. Tebbe

Institut für Agrarökologie (Dept. Agroecology)Institut für Agrarökologie (Dept. Agroecology)

Bundesforschungsanstalt für Landwirtschaft (FAL)Bundesforschungsanstalt für Landwirtschaft (FAL)

Braunschweig - GermanyBraunschweig - Germany

The SSCP-Side of Life, Part 2 The SSCP-Side of Life, Part 2

• SSCP papers of interest

• Protocols and Pictures

Basic InformationBasic Information

• Invention of SSCPOrita, 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

• First description of PCR-SSCPHayashi, K. 1991. PCR-SSCP: a simple and sensitive method for detection of mutations in the genomic DNA. PCR Methods Appl. 1:34-38

• Differentiation of pure culture bacteria by 16S rRNA targeted PCR-SSCPWidjojoatmodjo, M., A. C. Fluit, J. Verhoef. 1994. Rapid identification of bacteria by PCR-single strand conformation polymorphism. J. Clinic. Microbiol. 32:3002-3007

more Referencesmore References• Automation of PCR-SSCP product analysis for bacterial identification using the

ABI machineWidjojoatmodjo, 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.

References (community analysis)References (community analysis)

• First attempt to use SSCP for genetic profiling of microbial communitiesLee, D.-H., Y.-G. Zo, and S.-J. Kim. 1996. Non-radioactive method to study genetic profiles of bacterial communities by PCR-single-strand-conformation polymorphism. Appl. Environ. Microbiol. 62:3112-3120

• Bacterial community analysis of bioreactor studies and automated SSCP with product identification by co-migrationsZumstein, E., R. Moletta, and J. J. Gordon. 2000. Examination of two years of community dynamics in an anaerobic bioreactor using fluorescence polymerase chain reaction single-strand-conformation-polymorphism analysis. Environ. Microbiol. 2:69-78

The “Single-Stranded Community Approach”The “Single-Stranded Community Approach”

• Strategy, efficiency of the method, patterns of pure culture, mixed model communities and rhizospheresSchwieger, F. and C. C. Tebbe. 1998. A new approach to utilize PCR-single-strand-conformation polymorphism for 16S rRNA gene-based microbial community analysis. Appl. Environ. Microbiol. 64:4870-4876

• Application for monitoring compost MCs, different primers for different taxonomic groups, isolation and sequencing of single bands from profilesPeters, 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 small-subunit rRNA genes. Appl. Environ. Microbiol. 66:930-936

• Comparison of PCR-SSCP for MCs with diversity of cultivated bacterial isolatesSchwieger, F. and C. C. Tebbe. 2000. Effect of field inoculation with Sinorhizobium meliloti ...- Linking 16S rRNA gene based SSCP community profiles to the diversity of cultivated isolates. Appl.

Environ. Microbiol. 66: August issue.

Pathway of MC Analysis by PCR-SSCPPathway of MC Analysis by PCR-SSCP

• Extraction and purification of environmental DNA

• PCR and generation of single-stranded PCR products for SSCP

• Generation of SSCP profilespouring of gel - running electrophoresis - staining procedure

• Isolation and identification of products from SSCP-profilescloning and sequencing

Critical Factors for MC Analysis by Critical Factors for MC Analysis by PCR-SSCPPCR-SSCP

• Extraction and purification of nucleic acids from environmental samples

• PCR conditions, additives (e.g. T4GP32)

• Targeted gene fragments, e.g. which variable region?

• Denaturing procedure

• Concentrations and cross-linking of polyacrylamide gels, gel length, temperature

• Additives in the gel (MDE?) glycerol, formamide

• Staining procedure

• .....

Analysis and interpretation of SSCP Analysis and interpretation of SSCP profilesprofiles

• Pattern Analysisscanning of whole gels, recording on line, e.g. by ABI machine - digital image analysis, transformation of data, algorithm of pattern comparisons, e.g., Pearson, Dice, ...(GelCompar, WinCam, ...) - Cross-Gel comparisons!!!

• Identification of Single Components (Band i.d.)Isolations (crush and soak), reamplification, product check by SSCP with regenerated opposite strand, direct sequencing or cloning in E. coli etc. (non-16S primers)

PCR and generation of single-stranded PCR productsPCR and generation of single-stranded PCR products

• PCR-amplify target from environmental DNA (dilution, + T4GP321) - size 300 to 500 bp - one phosphorylated and one non-posphorylated primer

• Purify product (Qiaquick2)

• Digest with lambda exonuclease (USB, NEB) of purified PCR product; 10 U at 37°C for 1 h

• Purify by phenol-chloroform extraction or Qiaquick

• final volume should not be above 20 (l)

1, according to Tebbe and Vahjen, 1993, Appl. Environ. Microbiol. 59: 2657-2665; 2, Qiagen, Hilden, Germany

Generation of SSCP-patternsGeneration 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

Generation of SSCP patterns (cont.)Generation of SSCP patterns (cont.)

• insert gel into chamber, add running buffer (TBE) in buffer chambers, activate cooling system at, e.g., 18°C

• instead of glass plates, GelBond PAG membranes can also be used

• mix DNA samples with one vol of loading buffer (95 % formamide, 10 mM NaOH, 0.025 % bromophenol blue and xylene cyanole, resp.. Heat to 95°C for 2 min, immediately chill on ice for 3 min - load up to 8 μl into a sample well

• Addition of formamide increases the quality of the profiles by preventing non-specific annealing of DNA

• run gels: longer gels at 750 to 800 V for 18 to 24 h, shorter gels at 400 V for 14 to 16 hkeep temperature at 20°C

• remove spacers and comb, carefully detach cover glass plate

• start staining procedure ...

Generation of SSCP patterns (cont.)Generation of SSCP patterns (cont.)

• Staining: Silver staining procedure by Bassam et al.1 - stick to the protocol (very critical) use trays made of stainless steel - use shaking table

• freshly prepared:silver nitrate foraldehyde (ss)sodium bicarbonate-formaldehyde-thiosulfate (ds) at 8°C!; acetic acid (stop solution)

• wash with distilled water - control developer process and stop at the right moment (e.g., 5-10 min)

1, Anal. Biochem. 196:80-83 (1991); detection limit 15 pg ds DNA per band

Generation of SSCP patterns (cont.)Generation of SSCP patterns (cont.)

Drying under the hood

Gel on the light table

Isolation and Identification of BandsIsolation and Identification of Bands

• it even works with olde gels (> 0.5 years under the hood)

• 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

Cloning and SequencingCloning and Sequencing• purified PCR products are ligated

into pGEM-T, transformed into E. coli JM109, clones selected via blue white selection, subcultivation etc.

• selected bacterial cells are transferred into lysis solution, and PCR amplified with vector specific primers (complementary 2950-2970 for forward and 174 to 193 for reverse)

• PCR products are purified with Qiaquick and sequenced with SequiTherm EXCEL II DNA Sequencing kit (Epicentre) - analyzed on Li-Cor

AGAAAAGAAAagTGAAGGCACggAAAATAaAAGGAATTGACGGtAACCCAAAGGAATTGACGGAAACTTAAAGGAATTgACGGAAAcTCAAAGGAGTTGACGGAAACTCAAAGGAATTGACGGAAACTCAAAGGAATTGACGGAATCACTAGTGCGGCCGCCTGCAGGtCGACCATATGGGAGAGCTcMCAACGCGTTGGATGCATABTTGAGTATTCTATAGTGTCACCTAAATAGCTTGGCGTAATcATgg GGAGCTCtCCCATAtGGTCGACCKGCAGGCGGCCGCACTAGtGATTCCGTCAATTCCTTTGAGTTTCCGTCAATTCCTTTGAGTTTCCGTcAACTCCTTTGAGTTTCCGTCAATTCCTTTAAGTTTCCGTCAATTCCTTTGGGTTTCCGTCAATTCCTTTGAGTTTCCGTCAATTCCTTTGAGTTTCTGCTGCCTCcCCGCGGCCATGGCGGCgcGGGAGCATgCGACGTcGGGCCCAATTcGCCCTATAGTGAGTTCGTATTACAATTCAC GGAGCTCtCCCATAtGGTCGACCCAATTCCTTTGAGTTTCCGTCAATTCCTTTGAGTTTCCGTcAACTCCTTTGAGTTTCCGTCAATTCCTTTAAGTTTCCGTCAATTCCTTTGGGTTTCCGTCAATTCCTTTGAGTTTCCGTCAATTCCTTTGAGTTTCTGCTGCCTCCaaTcCCGCGGCCATGGCGGCgcGGGAGCATgCGACGTcGGGCCCAATTcGCCCTATAGTGAGTTCGTATTACAATTCACAGAAAAGAAAagTGAAGGCACggAAAGGAATTGACGGAAACTTAAAGGAATTgACGGAAAcTCAAAGGAGTTGACGGAAACTCAAAGGAATTGACGGAAACTCAAAGGAATTGACGGAATCACTAGTGCGGCCGCCTGCAGGtCGACCATATGGGAGAGCTcMCAACGCGTTGGATGCATABTTGAGTATTCTATAGTGTCACCTAAATAGCTTGGCGTAATcATgg GGAGCTCtCCCATAtGGTCGACCKGCAGGCGGCCGCACTAGtGATTCAATTCCTTTGAGTTTCCGTCAATTCCTTTGAGTTTCCGTcAACTCCTTTGAGTTTCCGTCAATTCCTTTAAGTTTCCGTCAATTCCTTTGGGTTTCCGTCAATTCCTTTGAGTTTCCGTCAATTCCTTTGAGTTTCTGCTGCCTCCaaTCAATTCCTTTGAGTTTCCGTCAATTCCTTTGAGTTTCCGTcAACTCCTTTGAGTTTCCGTCAATTCCTTTAAGTTTCCGTCAATTCCTTTGGGTTTCCGTCAATTCCTTTGAGTTTCCGTCAATTCCTTTGAGTTTCTGCTGCCTCCaaTcCCGCGGCCATGGCGGCgcGGGAGCATgCGACGTcGGGCCCAATTcGCCCTATAGTGAGTTCGTATTACAATTCAC GGAGCTCtCCCATAtGGTCGACCCAATTCCTTTGAGTTTCCGTCAATTCCTTTGAGTTTCCGTcAACTCCTTTGAGTTTCCGTCAATTCCTTTAAGTTTCCGTCAATTCCTTTGGGTTTCCGTCAATTCCTTTGAGTTTCCGTCAATTCCTTTGAGTTTCTGCTGCCTCCaaTcCCGCGGCCATGGCGGCgcGGGAGCATgCGACGTcGGGCCCAATTcGCCCTATAGTGAGTTCGTATTACAATTCACAGAAAAGAAAagTGAAGGCACggAAAGGAATTGACGGAAACTTAAAGGAATTgACGGAAAcTCAAAGGAGTTGACGGAAACTCAAAGGAATTGACGGAAACTCAAAGGAATTGACGGAATCACTAGTGCGGCCGCCTGCAGGtCGACCATATGGGAGAGCTcMCAACGCGTTGGATGCATABTTGAGTATTCTATAGTGTCACCTAAATAGCTTGGCGTAATcA

... and what happened to that marine community DNA from Nick Fuller and

Dave Scanlan?

V4/V5 region (Com primers)