Detection of Mutations by Single‐Strand Conformation Polymorphism (SSCP) Analysis and SSCP‐Hybrid Methods

William Warren1, Eivind Hovig2, Birgitte Smith‐Sørensen2, Anne‐Lise Børresen2, Frank K. Fujimura3, Qiang Liu3, Jinong Feng3, Steve S. Sommer3

1 Institute of Cancer Research, Surrey, 2 The Norwegian Radium Hospital, Oslo, 3 City of Hope National Medical Center, Duarte, California
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 7.4
DOI:  10.1002/0471142905.hg0704s15
Online Posting Date:  May, 2001
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Abstract

Single‐strand conformation polymorphism (SSCP) analysis detects mutations based on the fact that single‐nucleotide changes in DNA sequences alter the mobility of single‐stranded DNA in nondenaturing gels. Four methods for detecting mutations based on SSCP are described here. (1) Traditional SSCP analysis is technically easy and can be used for screening large numbers of samples. SSCP‐hybrid methods detect mutations based on either an SSCP effect or an altered component independent of the SSCP effect. (2) Dideoxy fingerprinting (ddF) involves PCR amplification of the target and creation of a set of dideoxy‐terminated strands with the mutation. (3) Bi‐directional dideoxy fingerprinting (Bi‐ddF) involves production of two sets of dideoxy‐terminated strands that are generated from two different primers. (4) Restriction endonuclease fingerprinting (REF) involves cleavage of the amplified target with five to six groups of restriction endonucleases.

     
 
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Table of Contents

  • Basic Protocol 1: Mutation Detection Using Single‐Strand Conformation Polymorphism Analysis
  • Basic Protocol 2: Mutation Detection Using Dideoxy Fingerprinting
  • Basic Protocol 3: Mutation Detection Using Bidirectional Dideoxy Fingerprinting
  • Basic Protocol 4: Mutation Detection Using Restriction Endonuclease Fingerprinting
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Mutation Detection Using Single‐Strand Conformation Polymorphism Analysis

  Materials
  • PCR primers A and B (1 OD 260/ml; 1 pmol/µl): forward and reverse primers designed to amplify <220 bp of DNA region of interest
  • 5 U/µl T4 polynucleotide kinase and 10× buffer ( appendix 3E)
  • 10 mCi/ml [γ‐32P]ATP (3000 Ci/mmol; Amersham)
  • 2 mM 4dNTP mix ( appendix 2D)
  • 5 U/µl Taq DNA polymerase and 10× PCR amplification buffer ( appendix 2D)
  • 25 to 250 µg/ml human genomic DNA from affected and unaffected individuals (see Critical Parameters and unit 7.1)
  • Mineral oil
  • Low gelling/melting temperature agarose (e.g., NuSieve GTG agarose, FMC Bioproducts)
  • 2% dimethyldichlorosilane (BDH Diagnostics; store at room temperature)
  • recipe50% (w/v) acrylamide stock solution (see recipe)
  • 10× TBE buffer ( appendix 2D)
  • 10% (w/v) ammonium persulfate (APS; prepare immediately before use)
  • TEMED
  • 0.1% (w/v) SDS/10 mM EDTA (pH 8.0)
  • 2× formamide loading buffer ( appendix 2D)
  • 65°C water bath
  • Thermal cycler
  • DNA sequencing gel apparatus with 31 × 38.5–cm glass plates, 0.4‐mm spacers, and sharkstooth comb
  • Waterproof tape
  • 90°C heating block
  • Whatman 3MM filter paper
  • UV‐transparent plastic wrap (e.g., Saran Wrap)
  • Additional reagents and equipment for labeling primers by T4 polynucleotide kinase ( appendix 3E), PCR amplification of sequences from affected individuals (unit 7.1), and agarose gel electrophoresis (unit 2.7)

Basic Protocol 2: Mutation Detection Using Dideoxy Fingerprinting

  Materials
  • 200 µg/ml purified sample DNAs
  • 200 µg/ml normal control DNA
  • 10× PCR amplification buffer ( appendix 2D)
  • 10 mM MgCl 2
  • 1.25 mM 4dNTP mix ( appendix 2D)
  • 2.5 µM PCR primers
  • 5 U/µl Taq DNA polymerase
  • recipe5× ddF transcription buffer (see recipe)
  • 2.5 mM 4rNTP mix
  • 100 mM DTT
  • 40 U/µl RNasin (Promega)
  • 20 U/µl T7 or SP6 RNA polymerase
  • 20 µM sequencing primer(s)
  • 10 µCi/µl [γ‐32P]ATP (6000 Ci/mmol) or 10 µCi/µl [γ‐33P]ATP (3000 Ci/mmol)
  • recipe10× ddF end‐labeling buffer (see recipe)
  • 7 to 10 U/µl T4 polynucleotide kinase
  • Annealing buffer: 250 mM KCl/ 10 mM Tris⋅Cl, pH 8.3
  • recipeddF RT buffer (see recipe)
  • 25 U/µl AMV reverse transcriptase
  • ddNTP: 0.25 mM ddCTP, 1 mM ddATP, 1 mM ddGTP, or 1 mM ddTTP
  • recipeddF stop/loading buffer (see recipe)
  • 7.5% (w/v) GeneAmp (Perkin‐Elmer) or 0.5× Mutation Detection Enhancement (MDE, J.T. Baker) gel solution
  • 10× TBE electrophoresis buffer ( appendix 2D)
  • Thermal cycler (Perkin‐Elmer 9600 or equivalent)
  • 37°, 45°, 55°, 80°C water baths
  • Sequencing gel apparatus with provision for cooling
  • 60‐ to 64‐square‐well comb
  • Gel dryer
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.7), end‐labeling primers ( appendix 3E), and preparing nondenaturing gels (see steps protocol 1, to )

Basic Protocol 3: Mutation Detection Using Bidirectional Dideoxy Fingerprinting

  Materials
  • 200 µg/ml purified sample DNAs
  • 200 µg/ml normal control DNA
  • TE buffer ( appendix 2D)
  • 20 µM sequencing primer(s)
  • recipe10× Bi‐ddF cycle‐sequencing buffer (see recipe)
  • 200 µM 4dNTP mix ( appendix 2D)
  • ddNTP: 1 mM ddCTP, ddATP, ddGTP, or ddTTP
  • 5 U/µl Taq DNA polymerase
  • recipeddF stop/loading buffer (see recipe)
  • Thermal cycler (e.g., Perkin‐Elmer 9600 or equivalent)
  • Microconcentrators (e.g., Microcon 100 or equivalent)
  • 85°C water bath
  • Sequencing gel apparatus with provision for cooling
  • Gel dryer
  • Additional reagents and equipment for amplifying target sequence by PCR (see protocol 2, steps and ), quantifying DNA ( appendix 3D), end‐labeling primers ( appendix 3E), and preparing nondenaturing sequencing gels (see protocol 2, steps and )

Basic Protocol 4: Mutation Detection Using Restriction Endonuclease Fingerprinting

  Materials
  • 200 µg/ml purified sample DNAs
  • 200 µg/ml control DNA
  • TE buffer ( appendix 2D)
  • Restriction endonucleases and appropriate 10× buffers
  • 10 U/µl calf intestine alkaline phosphatase (CIP)
  • 10 µCi/µl [γ‐32P]ATP (6000 Ci/mmol) or 10 µCi/µl [γ‐33P]ATP (3000 Ci/mmol)
  • 7 to 10 U/µl T4 polynucleotide kinase
  • recipe10× ddF end‐labeling buffer (see recipe)
  • 10 µM ATP (freshly prepared)
  • recipeddF stop/loading buffer (see recipe)
  • Microconcentrator (e.g., Microcon 100 or equivalent)
  • 80° to 85°C water bath
  • Sequencing gel apparatus with provision for cooling
  • Gel dryer
  • Additional reagents and equipment for PCR amplification of target sequences (see protocol 2, steps and ), quantifying DNA ( appendix 3D), end‐labeling DNA fragments ( appendix 3E), and preparing nondenaturing sequencing gels (see protocol 2, steps and )
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Figures

Videos

Literature Cited

Literature Cited
   Blaszyk, H., Hartmann, A., Schroeder, J.J., McGovern, R.M., Sommer, S.S., and Kovach, J.S. 1995. Rapid and efficient screening for p53 mutations by dideoxy fingerprinting. BioTechniques. 18:256‐260.
   Felmlee, T.A., Liu, Q., Whelen, A.C., Williams, D., Sommer, S.S., and Persing, D.H. 1995. Genotypic detection of Mycobacterium tuberculosis rifampicin resistance: Comparison of single‐strand conformational polymorphism and dideoxy fingerprinting. J. Clin. Microbiol. 33:1617‐1623.
   Gaidano, G., Ballerini, P., Gong, J.Z., Inghirami, G., Neri, A., Newcomb, E.W., Magrath, I.T., Knowles, D.M., and Dalla‐Favera, R. 1991. p53 mutations in human lymphoid malignancies: Association with Burkitt lymphoma and chronic lymphocytic leukemia. Proc. Natl. Acad. Sci. U.S.A. 88:5413‐5417.
   Liu, Q. and Sommer, S.S. 1994. Parameters affecting the sensitivities of dideoxy fingerprinting and SSCP. PCR Methods Appl. 4:97‐108.
   Liu, Q. and Sommer, S.S. 1995. Restriction endonuclease fingerprinting (REF): A sensitive method for screening mutations in long, contiguous segments of DNA. BioTechniques. 18:470‐477.
   Liu, Q., Feng, J., Sommer, S.S. 1996. Bi‐directional dideoxy fingerprinting (Bi‐ddF): A rapid method for quantitative detection of mutations in genomic regions of 300‐600 bp. Hum. Mol. Genet. 5:107‐114.
   Liu, Q., Feng, J., Sommer, S.S. 1997. In a blinded analysis, restriction endonuclease fingerprinting (REF) detects all the mutations in a 1.9‐kb segment. BioTechniques (In press).
   Martincic, D. and Whitlock, J.A. 1996. Improved detection of p53 point mutations by dideoxy fingerprinting (ddF). Oncogene. 13:2039‐2044.
   Mashiyama, S., Murakami, Y., Yoshimoto, T., Sekiya, T., and Hayashi, K. 1991. Detection of p53 gene mutations in human brain tumors by single‐strand conformation polymorphism analysis of polymerase chain reaction products. Oncogene 6:1313‐1318.
   Murakami, Y., Hayashi, K., Hirohashi, S., and Sekiya, T. 1991. Aberrations of the tumor suppressor p53 and retinoblastoma genes in human hepatocellular carcinomas. Cancer Res. 51:5520‐5525.
   Orita, M., Suzuki, Y., Sekiya, T., and Hayashi, K. 1989a. A rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5:874‐879.
   Orita, M., Iwahana, H., Kanazawa, H., Hayashi, K., and Sekiya, T. 1989b. Detection of polymorphisms of human DNA by gel electrophoresis as single‐strand conformation polymorphisms. Proc. Natl. Acad. Sci. U.S.A. 86:2766‐2770.
   Sarkar, G., Yoon, H‐S., and Sommer, S.S. 1992. Dideoxy fingerprinting (ddF): A rapid and efficient screen for the presence of mutations. Genomics 13:441‐443.
   Sommer, S.S. 1996. Restriction endonuclease and dideoxy fingerprinting. In Laboratory Protocols for Mutation Detection (U. Landegren, ed.) pp. 27‐32. Oxford University Press, Oxford.
   Sommer, S.S. and Vielhaber, E.L. 1994. Phage promoter–based methods for sequencing and screening for mutations. In The Polymerase Chain Reaction (K.B. Mullis, F. Ferre, and R. Gibbs, eds.) pp. 214‐221. Birkhauser, Boston.
   Spinardi, L., Mazars, R., and Theillet, C. 1991. Protocols for an improved detection of point mutations by SSCP. Nucl. Acids Res. 19:4009.
   Yandell, D.W. and Dryja, T.P. 1989. Detection of DNA sequence polymorphisms by enzymatic amplification and direct genome sequencing. Am. J. Hum. Gen. 45:547‐555.
Key References
   Blaszyk et al., 1995. See above.
  Application of ddF for blinded and prospective analysis of p53 mutations; technical tips for ddF.
   Liu et al., 1996. See above.
  Description of Bi‐ddF and comparison of ddF and SSCP.
   Liu and Sommer, 1995. See above.
  Description of REF and comparison with SSCP and ddF.
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