Detection of Mutations by Fluorescence‐Assisted Mismatch Analysis (FAMA)

Mario Tosi1, Elisabeth Verpy1, Tommaso Meo1

1 Institut Pasteur, Paris, null
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 7.8
DOI:  10.1002/0471142905.hg0708s12
Online Posting Date:  May, 2001
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Abstract

Fluorescence-assisted mismatch analysis (FAMA) methodology uses bifluorescent double-stranded DNA substrates to maximize the reliability and sensitivity of mutation-scanning procedures that rely on cleavage of mismatches using chemical. This unit presents a nested PCR procedure to fluorescently label each DNA strand, followed by chemical cleavage to detect the point mutations. Fluorescent end labeling with strand-specific fluorophores, electrophoretic separation of cleavage products in an automated Perkin-Elmer ABI 373 or 377 DNA sequencer and analysis using the Perkin-Elmer GENESCAN software expands sensitivity by highlighting weak signals through superimposition of strand-specific fluorescence electropherograms for different samples.

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol: Chemical Cleavage of Bifluorescent DNA Heteroduplexes
  • Reagents and Solutions
  • Commentary
  • Bibliography
  • Figures
     
 
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Materials

Basic Protocol: Chemical Cleavage of Bifluorescent DNA Heteroduplexes

 Materials
  • Genomic DNA or reverse-transcribed RNA suitable for PCR amplification (see unit 7.1)
  • Oligonucleotide primers (annealing temperature ~55°C), unlabeled (see Background Information)
  • Fluorescent primers (see recipe)
  • 3 M sodium acetate, pH 5.2 (appendix 2D)
  • TE buffer, pH 7.5 (appendix 2D)
  • 10× hybridization buffer A (see recipe)
  • 10× hydrization buffer B (optional; see recipe)
  • Paraffin oil
  • 20 mg/ml glycogen (from mussels; Boehringer Mannheim)
  • 70% and 100% ethanol (prechilled to –20°C)
  • 5 M hydroxylamine working solution (see recipe)
  • Osmium tetroxide mix (see recipe)
  • 1% osmium tetroxide working solution (see recipe)
  • Control DNA comprising two or more mismatches (see Critical Parameters)
  • Stop solution: 0.3 M sodium acetate, pH 5.2/0.1 mM Na2 EDTA (ice-cold)
  • 1 M piperidine (Aldrich) in distilled H2O (store in aliquots at –80°C)
  • 0.6 M sodium acetate, pH 5.2
  • Formamide/EDTA loading buffer: mix 5 vol formamide and 1 vol 50 mM EDTA, pH 8.0; store up to 1 year at –20°C
  • 37°, 65°, and 90°C water baths
  • 1.5-ml microcentrifuge tubes with O-rings and screw-caps (e.g., Sarstedt), silanized (cpmb appendix 3) and unsilanized
  • Eppendorf shaker
  • Centrifuge or microcentrifuge prechilled to 2°C
  • GENESCAN-2,500 ROX-labeled size standards (Applied Biosystems)
  • Perkin-Elmer ABI Model 373 or 377 DNA analysis system (Applied Biosystems) including:
  •     Electrophoresis and gel-casting apparatus
  •     GENESCAN data collection and analysis system
  •     Computer
  • Additional reagents and equipment for PCR amplification of genomic DNA (unit 7.1), agarose gel electrophoresis (unit 2.7), ethidium bromide staining (appendix 2D), and DNA precipitation (appendix 3C)

CAUTION: See appendix 2A for guidelines on handling, storage and disposal of osmium tetroxide, piperidine, and formamide.
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Figures

  •  FigureFigure 7.8.1 FAMA labeling of target amplicons. Note that PCR amplification and fluorescent end labeling are performed directly on patient's DNA when mutations are expected to be heterozygous. For detection of homozygous or hemizygous mutations, an equal amount of reference genomic DNA should be added prior to step of the Basic Protocol. When scanning for hemizygous mutations at the mRNA level, it is suggested that a reference RT-PCR product be added before fluorescent labeling (see Basic Protocol, step ).
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  •  FigureFigure 7.8.2 Illustration of analysis of fluorescence profiles using the GENESCAN software (Applied Biosystems). (A) Schematic depiction of the T G transversion analyzed. (B) Electropherogram of the hydroxylamine modification of the heteroduplex for this transversion. In this panel and panel C, the tall peaks above 800 bp represent the uncleaved sense and antisense strand, which do not always display the same electrophoretic mobility. (C) Electropherogram of the osmium tetroxide modification of the same heteroduplex. Note that this T G transversion can be predicted without sequencing, because of the precise positional information provided by this analysis, the knowledge of the strand undergoing cleavage, and the specificity of the chemical modification reactions. In the electropherograms, the horizontal scale (in nucleotide positions) is generated automatically from the internal size markers in each lane. The inset in panel C illustrates the superimposition of strand-specific fluorescence profiles from different lanes (in this case those of upper strands upon osmium tetroxide modifications), to highlight weak signals.
    View Image
  •  FigureFigure 7.8.3 Simultaneous fluorescence labeling of all DNA strands (i.e., those from the mutant DNA and those from the reference DNA), with a different fluorophore for the upper strands (squares) and for the lower strands (circles) ensures detection of two cleavage products for each point mutation. Note however that all kinds of microdeletions or microinsertions are also detected (see, for example, Verpy et al., 1996).
    View Image

Videos

Literature Cited

 Literature Cited
    Biasotto, M., Meo, T., Tosi, M., and Verpy, E. 1996. FAMA: Fluorescence-assisted mismatch analysis by chemical cleavage. In Laboratory Protocols for Mutation Detection (U. Landegren, ed.) pp. 54-60. Oxford University Press, New York.
    Cecchi, C., Biasotto, M., Tosi, M., and Avner, P. 1997. The mottled mouse as a model for human Menkes disease: Identification of mutations in the Atp7a gene. Hum. Mol. Genet. In press.
    Cotton, R. 1993. Current methods of mutation detection. Mutat. Res. 285:125-144.
    Cotton, R.G. and Campbell, R.D. 1989. Chemical reactivity of matched cytosine and thymine bases near mismatched and unmatched bases in heteroduplex between DNA strands with multiple differences. Nucl. Acids Res. 17:4223-4233.
    Cotton, R.G., Rodrigues, N.R., and Campbell, R.D. 1988. Reactivity of cytosine and thymine in single-base-pair mismatches with hydroxylamine and osmium tetroxide and its application to the study of mutations. Proc. Natl. Acad. Sci. U.S.A. 85:4397-4401.
    Germain, D., Biasotto, M., Tosi, M., Meo, T., Kahn, A., and Poenaru, L. 1996. Fluorescence-assisted mismatch analysis (FAMA) for exhaustive screening of the -galactosidase A gene and detection of carriers in Fabry disease. Hum. Genet. 98:719-726.
    Griffais, R., Andre, P.M., and Thibon, M. 1991. K-tuple frequency in the human genome and polymerase chain reaction. Nucl. Acids Res. 19:3887-3891.
    Grompe, M., Muzny, D.M., and Caskey, C.T. 1989. Scanning detection of mutations in human ornithine transcarbamoylase by chemical mismatch cleavage. Proc. Natl. Acad. Sci. U.S.A. 86:5888-5892.
    Hansen, L.-L., Justesen, J., and Kruse, T.A. 1996. Sensitive and fast mutation detection by solid phase chemical cleavage. Hum. Mutat. 7:256-263.
    Haris, I.I., Green, P.M., Bentley, D.R., and Giannelli, F. 1994. Mutation detection by fluorescent chemical cleavage: Application to hemophilia B. PCR Methods Appl. 3:268-271.
    Inazuka, M., Tahira, T., and Hayashi, K. 1996. One-tube post-PCR fluorescent labeling of DNA fragments. Genome Res. 6:551-557.
    Lener, M., Poirier, C., Tosi, M., Guenet, J.L., and Meo, T. 1996. Direct and ready readout of nucleotide diversity by FAMA: Mapping of C1 INH on mouse chromosome 2 in absence of RFLPs. Abstr. HUGO's Human Genome Meeting, Heidelberg, Germany.
    Lu, A.-L. and Hsu, I.-L. 1991. Detection of single DNA base mutations with mismatch repair enzymes. Genomics 14:249-255.
    Mashal, R.D., Koontz, J., and Sklar, J. 1995. Detection of mutations by cleavage of DNA duplexes with bacteriophage resolvases. Nature Genet. 9:177-183.
    Perkin-Elmer 1995. Fluorescence-assisted mismatch analysis for mutation detection. User Bulletin 28. Perkin-Elmer, Foster City, Calif.
    Rowley, G., Saad, S., Giannelli, F., and Green, P.M. 1995. Ultrarapid mutation detection by multiplex, solid-phase chemical cleavage. Genomics 30:574-582.
    Rychlick, W. and Rhoads, R.E. 1989. A computer program for choosing optimal oligonucleotides for filter hybridization, sequencing and in vitro amplification of DNA. Nucl. Acids Res. 17:8543-8555.
    Smith, J. and Modrich, P. 1996. Mutation detection with MutH, MutL, and MutS mismatch repair proteins. Proc. Natl. Acad. Sci. U.S.A. 93:4374-4379.
    Veitia, R., Ion, A., Barbaux, S., Jobling, M.A., Soleyreau, N., Ennis, K., Oster, H., Tosi, M., Chibani, J., Fellous, M., and McElreavey, K. 1997. Mutations and sequence variants in the testis determining region of the Y chromosome in individuals with a 46,XY female genotype. Hum. Genet. In press
    Verpy, E., Biasotto, M., Meo, T., and Tosi, M. 1994. Efficient detection of point mutations on color-coded strands of target DNA. Proc. Natl. Acad. Sci. U.S.A. 91: 1873-1877.
    Verpy, E., Biasotto, M., Brai, M., Misiano, G., Meo, T., and Tosi, M. 1996. Exhaustive mutation scanning by fluorescence assisted mismatch analysis discloses new genotype-phenotype correlations in angioedema. Am. J. Hum. Genet. 59:308-319.
    Youil, R., Kemper, B.W., and Cotton, R.G.H. 1995. Screening for mutations by enzyme mismatch cleavage with T4 endonuclease VII. Proc. Natl. Acad. Sci. U.S.A. 92:87-91.
 Key References
    Verpy et al., 1994. See above.

Original description of the FAMA method. Illustrates the precision of positional information and the sensitivity upon dilution of the mutant DNA.

    Verpy et al., 1996, see above.

Demonstrates the efficacy of detection of unknown mutations in a diagnostic context and on a large series of patients.

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