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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

1Institute of Cancer Research, Surrey, United Kingdom
2The Norwegian Radium Hospital, Oslo, Norway
3City 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

  • Unit Introduction
  • 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
  • Bibliography
  • Figures
     
 
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Materials

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

 Materials
  • PCR primers A and B (1 OD260/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 [-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)
  • 50% (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 MgCl2
  • 1.25 mM 4dNTP mix (appendix 2D)
  • 2.5 µM PCR primers
  • 5 U/µl Taq DNA polymerase
  • 5× 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 [-32P]ATP (6000 Ci/mmol) or 10 µCi/µl [-33P]ATP (3000 Ci/mmol)
  • 10× 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
  • ddF 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
  • ddF 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 Basic 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)
  • 10× 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
  • ddF 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 Basic Protocol 2, steps and ), quantifying DNA (appendix 3D), end-labeling primers (appendix 3E), and preparing nondenaturing sequencing gels (see Basic 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 [-32P]ATP (6000 Ci/mmol) or 10 µCi/µl [-33P]ATP (3000 Ci/mmol)
  • 7 to 10 U/µl T4 polynucleotide kinase
  • 10× ddF end-labeling buffer (see recipe)
  • 10 µM ATP (freshly prepared)
  • ddF 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 Basic Protocol 2, steps and ), quantifying DNA (appendix 3D), end-labeling DNA fragments (appendix 3E), and preparing nondenaturing sequencing gels (see Basic Protocol 2, steps and )
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Figures

  • Figure 7.4.1
    SSCP analysis of exon 8 (238 bp) of the TP53 gene. DNA prepared from blood samples was PCR-amplified and analyzed on a nondenaturing (4.5%) polyacrylamide gel for 2.5 hr. The sample with aberrant mobility (arrow) carries a germline mutation in codon 273. The location of double-stranded DNA on the gel is indicated. The fragments were denatured and annealed as described in the text.

    View Image
  • Figure 7.4.2
    SSCP analysis of presumptive sequence variants of exon 6 of the TP53 gene from archival bladder-tumor DNAs. Bands of altered mobility were excised from a dried gel run and autoradiographed previously. DNAs extracted from excised bands were then reamplified by PCR to provide DNA for sequencing. This gel shows reamplified DNA analyzed on a 6% polyacrylamide gel containing 10% glycerol and run overnight at 6 W.

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  • Figure 7.4.3
    Schematic of dideoxy fingerprinting (ddF). The targeted region of DNA has a point mutation indicated by X. The bacteriophage T7 promoter sequence is present in one PCR primer. The PCR product is transcribed by T7 RNA polymerase, and the transcript is sequenced by the Sanger termination reaction using an internal primer and one ddNTP. In the example shown, the mutation results in a termination segment with the ddNTP used, resulting in a novel band after gel electrophoresis (lane 1). Lane 2 illustrates a situation where there is no new termination segment, but two segments show altered mobilities in the nondenaturing gel due to the presence of the mutation. Lane C contains normal control DNA (reprinted with permission from Liu and Sommer, 1994).

    View Image
  • Figure 7.4.4
    Analysis of a 300-bp segment of exon B of the factor IX gene. Results are shown for 20 samples analyzed by ddF. Normal control samples (C) are analyzed in every third lane so that each test sample lane (numbers) is adjacent to a control lane (only even-numbered lanes are labeled). This DNA segment contains 40% GC. Termination fragments were generated with ddCTP and the genomic amplification with transcript sequencing (GAWTS) method. Numbers along the left side represent termination products for control DNA, with 1 corresponding to the smallest segment. The nondenaturing gel contained 7.5% GeneAmp and 1× TBE electrophoresis buffer. The gel was electrophoresed at 12 W constant power for 4 hr at 7° to 8.5°C (A) or 3 hr at 22.5° to 24°C (B). Note that electrophoresis at the lower temperature results in more pronounced mobility differences for this segment with a relatively low GC content. However, electrophoresis at lower temperatures can result in band smearing for DNA segments with higher GC content.

    View Image
  • Figure 7.4.5
    (at left) Schematic of bidirectional dideoxy fingerprinting (Bi-ddF). (A) A region of genomic DNA is amplified by PCR, then a cycle-sequencing reaction is performed using two opposing nested primers, downstream (D) and upstream (U), and one ddNTP. (B) Illustration of the sensitivities of the SSCP and the dideoxy components in Bi-ddF for the downstream and upstream segments. The region shown contains two mutations (X1 and X2) lying at the center or at one end. The segments illustrated as dashed lines are <60 bases and have been electrophoresed off the gel. The mutation X2 first appears in such a segment in the downstream direction, so that any informative dideoxy component is missed. However, all the downstream fragments >60 bases also contain this mutation so that the SSCP component in the downstream direction is expected to be informative almost all the time. The segments illustrated by solid lines are within the highly informative window; those illustrated by dotted lines are >400 bases which have low resolution. The figure emphasizes how the SSCP and the dideoxy components contribute to the sensitivity of Bi-ddF. The total Bi-ddF sensitivity depends upon the sum of the SSCP and the dideoxy components in both the downstream and the upstream segments. (C) Schematic representation of an autoradiogram of Bi-ddF results using ddGTP for the two hypothetical mutations shown in (B). The directions of the arrows indicate whether the segments were derived from the downstream or the upstream primer. Although not shown in the schematic, control unidirectional ddF reactions allow the downstream and upstream components to be distinguished, thereby allowing mutations to be localized, typically within 30 nucleotides. The X represents the presence of a mutation within the fragment. A highly informative window of 60 to 400 bases is indicated. The central lane (lane C) contains a wild-type control DNA. Lane 1 contains mutation X1 and Lane 2 contains mutation X2. For mutation X1, the dideoxy component is informative in the downstream, but not the upstream, direction. The SSCP component is informative in both directions. For mutation X2, the dideoxy component cannot be detected in the downstream direction because any gained or lost fragment is <60 bases and has been run off the gels. The SSCP component is informative for X2 in the downstream direction (reprinted with permission from Liu et al., 1996).

    View Image
  • Figure 7.4.6
    Analysis of a 500-bp segment of exons B and C of the Factor IX gene by Bi-ddF. Twenty-one DNA samples were analyzed by Bi-ddF. Normal control DNA (C) was run in every third lane. The two outside lanes on either side of the gel show the pattern for the separate reactions containing only the downstream (D) or upstream (U) primer. Termination fragments were generated using ddCTP. Numbers along the sides of the gel represent termination products for control DNA from both the upstream and downstream primers. The number 1 corresponds to the smallest termination product. Note that the number of segments is much higher for Bi-ddF than for ddF (see Fig. 7.4.4). The GC content of this segment is 40%. Electrophoresis was performed using an 0.5% MDE gel and TBE electrophoresis buffer. The gel was electrophoresed at 12 W for 6 hr at 8°C.

    View Image
  • Figure 7.4.7
    Schematic of restriction endonuclease fingerprinting (REF). The targeted region of DNA has a point mutation indicated by X. The region is amplified by PCR. The PCR product is purified, then digested separately with several groups of restriction endonucleases; only three groups (A, B, and C) are illustrated. After restriction endonuclease digestion, the separate groups of fragments are combined, end-labeled, denatured, and analyzed on a nondenaturing gel. In the example shown, the mutation results in the loss of a restriction site for endonuclease group C, resulting in an informative restriction component (loss of fragments C2 and C3 in the control lane and appearance of a novel fragment, C2 + C3, in the mutant lane). Fragments A2 and B1 contain the mutation and show altered mobilities due to the SSCP effect. Bands in the figure represent single strands; complementary strands tend to migrate close to each other and are represented as doublets (reprinted with permission from Liu and Sommer, 1995).

    View Image
  • Figure 7.4.8
    Analysis of a 1941-bp segment of exons G and H of the factor IX gene by REF. Forty-two samples were analyzed by REF using the six restriction endonuclease groups indicated at the right edge of the figure. Lane C is a normal control sample. The six right-hand lanes are the patterns for each of the separate groups of restriction fragments for normal control DNA. Fragments in the control lanes (at the right) are labeled according to map position along the 1941-bp segment for each digestion group. The GC content of this DNA segment is 40%. Electrophoresis was performed using a 7.5% GeneAmp gel with 1× TBE electrophoresis buffer. The gel was electrophoresed at 12 W for 4 hr at 20°C.

    View Image

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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