Detection of Mutations by RNase Cleavage

Hugh C. Watkins1, Marianna Goldrick2

1 University of Oxford, Oxford, 2 Ambion, Inc., Austin, Texas
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 7.2
DOI:  10.1002/0471142905.hg0702s14
Online Posting Date:  May, 2001
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Abstract

Ribonucleases can specifically recognize and cleave RNA at the site of sequence mismatches in RNA‐DNA or RNA‐RNA hybrids. The cleavage products are then characterized by gel electrophoresis. In this unit, a procedure is presented for RNase cleavage of 32P‐labeled riboprobes (transcribed from a cloned copy of the normal sequence) that have been annealed to amplified sequences of a candidate gene or cDNA obtained from affected individuals. A explains how to prepare riboprobes from a genomic or cDNA template obtained from a nonmutant individual. An alternate protocol describes cleavage of RNARNA hybrids using a nonisotopic RNase cleavage mutation assay. Sequential PCR and in vitro transcription steps generate sufficient quantities of duplex RNA targets so that the cleavage products can be detected on a gel by ethidium bromide staining. The unit also discusses the use of alternative ribonucleases for cleaving singlebase mismatches.

     
 
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Table of Contents

  • Basic Protocol 1: Cleavage of Radiolabeled RNA‐DNA Hybrids at Mutation Sites Using RNase A
  • Support Protocol 1: Preparation of 32P‐Labeled Riboprobes
  • Alternate Protocol 1: Cleavage of RNA‐RNA Hybrids at Mutation Sites Using the Nonisotopic RNase Cleavage Assay (NIRCA)
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Cleavage of Radiolabeled RNA‐DNA Hybrids at Mutation Sites Using RNase A

  Materials
  • 0.05 to 0.1 µg/µl PCR‐amplified DNA from an affected individual (unit 7.1)
  • recipeHybridization buffer for isotopic mismatch detection (see recipe)
  • Formamide
  • 0.5 × 106 cpm/µl [α‐32 P]UTP sense and antisense riboprobes (see protocol 2)
  • recipeRNase digestion buffer for isotopic mismatch detection (see recipe)
  • 2 mg/ml DNase‐free RNase A stock solution ( appendix 2D), prepared in DEPC‐treated H 2O (unit 7.1)
  • 20% (w/v) SDS ( appendix 2D)
  • 10 mg/ml proteinase K (store aliquots ≤6 months at −20°C)
  • 2× formamide loading buffer ( appendix 2D)
  • 1.5‐mm‐thick denaturing polyacrylamide gel containing 6% (w/w) 29:1 acrylamide/bisacrylamide/7 M urea and with large wells (e.g., 20 mm deep × 5 mm wide; appendix 3F)
  • RNase‐free microcentrifuge tubes
  • 45°, 90°, and 95°C water baths or heating blocks
  • UV‐transparent plastic wrap (e.g., Saran Wrap)
  • X‐ray film
  • Intensifying screen
  • Additional reagents and equipment for denaturing polyacrylamide gel electrophoresis ( appendix 3F)

Support Protocol 1: Preparation of 32P‐Labeled Riboprobes

  Materials
  • Plasmid DNA: DNA from plasmid vector containing T7 and T3 promoters (e.g., pBluescript II SK, Stratagene) with gene of interest inserted (e.g., CPMB UNIT )
  • Restriction enzyme that cuts once in polylinker downstream of insert DNA in plasmid
  • 1:1 (v/v) phenol/chloroform (made with buffered phenol; appendix 3B)
  • 100% ethanol
  • TE buffer, pH 7.6 ( appendix 2D)
  • DEPC‐treated H 2O (unit 7.1)
  • recipe5× transcription buffer for isotopic mismatch detection (see recipe)
  • 200 mM DTT (store in aliquots at −20°C)
  • 40,000 U/ml placental ribonuclease inhibitor (e.g., RNasin, Promega)
  • 10 mM 3NTP mix: 10 mM each ATP, GTP, CTP (store in single‐use aliquots at −20°C)
  • 10 mM UTP
  • 10 mCi/ml [α‐32P]UTP (800 Ci/mmol)
  • 50 U/µl T7 and T3 bacteriophage RNA polymerases
  • 5000 U/ml (2.5 mg/ml) RNase‐free DNase I (e.g., Worthington DPRF grade)
  • RNase‐free microcentrifuge tubes
Additional reagents and equipment for agarose gel electrophoresis (unit 2.7), phenol extraction and ethanol precipitation of DNA ( appendix 3C), handling RNA (CPMB UNIT ), and measuring radioactivity in RNA by TCA precipitation ( appendix 3E)

Alternate Protocol 1: Cleavage of RNA‐RNA Hybrids at Mutation Sites Using the Nonisotopic RNase Cleavage Assay (NIRCA)

  Materials
  • ∼12.5 to 50 ng/µl DNA or cDNA from wild‐type control and affected individuals (unit 7.1), PCR‐amplified with forward and reverse primers containing T7 and SP6 promoters, respectively, on their 5′ ends (see )
  • 10× transcription buffer for nonisotopic mismatch detection (see recipe)
  • 4NTP mix: 2.5 mM each of ATP, CTP, UTP, and GTP in 10 mM HEPES, pH 7.5
  • 20 U/µl T7 and SP6 RNA polymerases
  • RNase‐free H 2O (unit 7.1)
  • Hybridization buffer for nonisotopic mismatch detection (see recipe)
  • 100× RNase A stock solution (see recipe)
  • RNase digestion buffer for nonisotopic mismatch detection (see recipe)
  • Gel loading solution (see recipe)
  • 1× TBE buffer ( appendix 2D)
  • 0.5‐ml microcentrifuge tubes, RNase‐free
  • Heating block
  • 96‐well round‐bottom microtiter plates (optional)
Additional reagents and equipment for agarose gel electrophoresis (unit 2.7)
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Figures

Videos

Literature Cited

Literature Cited
   De Souza, A.T., Hankins, G.R., Washington, M.K., Orton, T.C., and Jirtle, R.L. 1995. M6P/IGF2R gene is mutated in human hepatocellular carcinomas with loss of heterozygosity. Nature Genet. 11:447‐449.
   Ekenberg, S. and Hudson, G. 1994. RNase protection assay system: a versatile technique for the analysis of RNA. Promega Notes 46:14‐16. Promega Corporation (see SUPPLIERS APPENDIX).
   Forrester, K., Almoguera, C., Han, K., Grizzle, W.E., and Perucho, M. 1987. Detection of high incidence of K‐ras oncogenes during human colon tumorigenesis. Nature 327:298‐303.
   Gibbs, R.A. and Caskey, C.T. 1987. Identification and localization of mutations at the Lesch‐Nyhan locus by Ribonuclease A cleavage. Science 236:303‐305.
   Goldrick, M.M., Kimball, G.R., Liu, Q., Martin, L.A., Sommer, S.S., and Tseng, J.Y‐H. 1996. NIRCA: A rapid robust method for screening for unknown point mutations. BioTechniques 21:106‐112.
   Ingles, S.A., Diep, A., Xue, S‐Y., Shattuck‐Eidens, D., Sparkes, R., and Haile, R. 1995. BRCA1 mutation testing: Results from 68 early‐onset bilateral breast cancer families. Am. J. Hum. Genet. 57:A67 (abstract 354).
   Johnson, M. 1996. Use of RNase I for the efficient elimination of RNA from DNA preparations and mismatch detection. Epicentre Forum. 3:7‐8. Epicentre Technologies (see SUPPLIERS APPENDIX).
   Kaufman, D.L., Ramesh, V., McClatchey, A.I., Menkes, J.H., and Tobin, A.J. 1990. Detection of point mutations associated with genetic diseases by an exon scanning technique. Genomics 8:656‐663.
   MacRae, C.A., Watkins, H., Jarcho, J.A., Thierfelder, L., Rosenzweig, A., Hwang, D.S., McKenna, W.J., Seidman, J.G., and Seidman, C.E. 1994. An evaluation of RNase protection for the detection of beta cardiac myosin heavy chain gene mutations. Circulation 89:33‐35.
   Maslen, C., Babcock, D., Raghunath, M., and Steinmann, B. 1997. A rare branch‐point mutation is associated with missplicing of fibrillin‐Z in a large family with congenital contractural arachnodactyly. Am. J. Hum. Genet. 60:1389‐1398.
   Meador, J. and Kennell, D. 1990. Cloning and sequencing the gene encoding Escherichia coli ribonuclease I: Exact physical mapping using the genome library. Gene 95:1‐7.
   Murthy, K.K., Shen, S.H., and Banville, D. 1995. A sensitive method for detection of mutations—A PCR‐based RNase protection assay. DNA Cell Biol. 14:87‐94.
   Myers, R.M., Larin, Z., and Maniatis, T. 1985. Detection of single base substitutions by ribonuclease cleavage at mismatches in RNA:DNA duplexes. Science 230:1242‐1246.
   Nash, K.A. and Inderlied, C.B. 1996. Rapid detection of mutations associated with macrolide resistance in Mycobacterium avium complex. Antimicrob. Agents Chemother. 40:1748‐1750.
   Peral, B., Gamble, V., Strong, C., Ong, A.C.M., Sloane‐Stanley, J., Zerres, K., Winearls, C.G., and Harris, P.C. 1997. Identification of mutations in the duplicated region of the polycystic kidney disease 1 gene (PKD1) by a novel approach. Am. J. Hum. Genet. 60:1399‐1410.
   Rosenzweig, A., Watkins, H., Hwang, D.‐S., Miri, M., McKenna, W.J., Traill, T., Seidman, J.G., and Seidman, C.E. 1991. Preclinical diagnosis of familial hypertrophic cardiomyopathy by genetic analysis of blood lymphocytes. New Engl. J. Med. 325:1753‐1760.
   Stoflet, E.S., Koeberl, D.D., Sarkar, G., and Sommer, S.S. 1988. Genomic amplification with transcript sequencing. Science 239:491‐494.
   Watkins, H., Rosenzweig, A., Hwang, D.‐S., Levi, T., McKenna, W.J., Seidman, C.E., and Seidman, J.G. 1992. Distribution and prognostic significance of myosin missense mutations in familial hypertrophic cardiomyopathy. New Engl. J. Med. 326:1108‐1114.
   Winter, E., Yamamoto, F., Almoguera, C., and Perucho, M. 1985. A method to detect and characterize point mutations in transcribed genes: Amplification and overexpression of the mutant c‐Ki‐ras allele in human tumor cells. Proc. Natl. Acad. Sci. U.S.A. 82:7575‐7579.
Key References
   Goldrick et al., 1996. See above.
  Describes use of PCR to generate duplex RNA targets and use of multiple RNases to cleave mismatches.
   Myers et al., 1985. See above.
  Initial evaluation of use of RNase A to detect single‐base substitutions in DNA and most definitive assessment of cleavage at specific RNA‐DNA mismatches.
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