Simultaneous Detection of Multiple Point Mutations Using Allele‐Specific Oligonucleotides

Nichole M. Napolitano1, Elizabeth M. Rohlfs1, Ruth A. Heim1

1 Genzyme Genetics, Westborough, Massachusetts
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 9.4
DOI:  10.1002/0471142905.hg0904s41
Online Posting Date:  September, 2004
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Abstract

This unit describes high‐throughput mutation analysis using hybridization with pooled allele‐specific oligonucleotide (ASO) probes. The approach can be used to screen one gene for many allelic mutations or to screen several loci for several allelic mutations each. Because tetramethyl ammonium chloride (TMAC) is added to the hybridization solution, the melting temperature of each oligonucleotide is independent of G–C content and oligonucleotides of the same length can be hybridized simultaneously. The pooled probes will give a positive hybridization signal from any PCR‐amplified DNA sample containing a sequence complementary to any of the ASOs in the pool of oligonucleotide sequences. If many PCR‐amplified samples are spotted onto a single membrane, multiple individuals can then be screened simultaneously for many mutant sequences. This multiple ASO hybridization technique is appropriate only for circumstances when hybridization with any one of the pooled probes is expected to be uncommon.

     
 
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Table of Contents

  • Basic Protocol 1: Screening PCR‐Amplified DNA with Multiple Pooled ASOs
  • Support Protocol 1: Stripping Old Probes and Rehybridization
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Screening PCR‐Amplified DNA with Multiple Pooled ASOs

  Materials
  • 10× T4 polynucleotide kinase buffer (see recipe or use buffer supplied by manufacturer), stored at −20°C
  • 2 µM allele‐specific oligonucleotide (ASO) working solutions in 1× TE buffer ( appendix 2D), for all normal and mutant sequences, prepared by diluting 100 µM stocks, stored at −80°C or pooled lyophilized ASOs in 1× TE buffer, stored at −80°C (see Critical Parameters)
  • 10 mCi/ml [γ‐32P]dATP (∼3000 Ci/mmol), stored at −20°C
  • 10 U/µl T4 polynucleotide kinase, stored at −20°C
  • 2× SSC ( appendix 2D)
  • Denaturing solution (see recipe), freshly prepared
  • PCR‐amplified DNA from genes of interest (see Critical Parameters)
  • TMAC hybridization solution (see recipe), freshly prepared
  • TMAC wash solution (see recipe), freshly prepared, at room temperature and 52°C
  • 37°C heat block
  • Dot‐blot apparatus (volumes provided are for 6‐mm dots)
  • Nylon membrane: e.g., Biotrans+ (ICN Biomedicals) or Biodyne (Pall)
  • Whatman 3MM filter paper
  • 80°C vacuum oven
  • Sealable bag, Tupperware container, or hybridization tube
  • 52°C shaking water bath or hybridization oven
  • Kodak Biomax MS film
  • X‐ray film cassettes and intensifying screens
  • Additional reagents and equipment for PCR (units 7.1 & 9.3; CPMB UNIT 15.1) and preparing dot blots, (CPMB UNIT 2.9)
CAUTION:32P and TMAC are hazardous; see appendix 2A for guidelines on handling, storage, and disposal.

Support Protocol 1: Stripping Old Probes and Rehybridization

  • Hybridized membrane (see protocol 1)
  • TMAC wash solution (see recipe), freshly prepared and prewarmed to 15°C above previous hybridization temperature
  • Agitating water bath, 15°C above previous hybridization temperature
CAUTION: Radiolabeled hybridized membranes and TMAC are hazardous; see appendix 2A for guidelines on handling, storage, and disposal.
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Figures

Videos

Literature Cited

   Ferrie, R.M., Schwarz, M.J., Robertson, N.H., Vaudin, S., Super, M., Malone, G., and Little, S. 1992. Development, multiplexing, and application of ARMS tests for common mutations in the CFTR gene. Am. J. Hum. Genet. 51:251‐262.
   Marky, L.A., Blumenfeld, K.S., and Breslauer, K.J. 1988. Differential effect of tetramethylammonium chloride and sodium chloride on duplex melting temperature of deoxyoligonucleotides: Resolution of a salt effect into specific and nonspecific components. Can. J. Chem. 66:836‐838.
   Melchior, W.B. and von Hippel, P.H. 1973. Alteration of the relative stability of dA‐dT and dG‐dC base pairs in DNA. Proc. Nat. Acad. Sci. U.S.A. 70:298‐302.
   Shuber, A.P., Skoletsky, J., Stern, R., and Handelin, B.L. 1992. Efficient 12‐mutation testing in the CFTR gene: A general model for complex mutation analysis. Hum. Molec. Genet. 2:159‐163.
   Shuber, A.P., Michalowsky, L.A., Nass, G.S., Skoletsky, J., Hire, L.M., Kotsopoulos, S.K., Phipps, M.F., Barberio, D.M., and Klinger, K.W. 1997. High throughput parallel analysis of hundreds of patient samples for more than 100 mutations in multiple disease genes. Hum. Mol. Genet. 6:337‐47.
Key Reference
   Shuber et al., 1992. See above.
  Original description and validation of the method.
   Wood, W.I., Gitschier, J., Lasky, L.A., and Lawn, R.M. 1985. Base composition‐independent hybridization in tetramethylammonium chloride: A method for oligonucleotide screening of highly complex gene libraries. Proc. Natl. Acad. Sci. U.S.A. 82:1585‐1588.
  Establishes the empirical conditions for use of TMAC in hybridizing degenerate oligonucleotide pools to cDNA library clones. This is an important application of TMAC conditions and is the closest to the application described here.
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