Amplification‐Refractory Mutation System (ARMS) Analysis of Point Mutations

Stephen Little1

1 Zeneca Diagnostics, Cheshire, null
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 9.8
DOI:  10.1002/0471142905.hg0908s07
Online Posting Date:  May, 2001
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Abstract

The amplification‐refractory mutation system (ARMS) is a simple method for detecting any mutation involving single base changes or small deletions. ARMS is based on the use of sequence‐specific PCR primers that allow amplification of test DNA only when the target allele is contained within the sample. Following an ARMS reaction the presence or absence of a PCR product is diagnostic for the presence or absence of the target allele. The protocols detailed here outline methods that can be used to analyze human genomic DNA for one or more mutations. The describes the development and application of an ARMS test for a single mutation; the extends this to multiplex ARMS for the analysis of two or more mutations. The describes a rapid DNA extraction method from blood or mouthwash samples that yields DNA compatible with the type of tests described.The amplification‐refractory mutation system (ARMS) is a simple method for detecting any mutation involving single base change The amplification‐refractory mutation system (ARMS) is a simple method for detecting any mutation involving single base change

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Analysis of Single Mutations by ARMS Tests
  • Alternate Protocol 1: Analysis of Multiple Mutations by Multiplex ARMS
  • Support Protocol 1: Rapid DNA Extraction from Mouthwash and Blood Samples
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Analysis of Single Mutations by ARMS Tests

  MaterialsFor recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.
  • 50 µM normal ARMS primer
  • 50 µM mutant ARMS primer
  • 50 µM common primer
  • 50 µM each control primer A and B (or other appropriate pair; see recipe)
  • 1 mM 4dNTP mix ( appendix 2A)
  • 10× PCR amplification buffer containing 12 mM MgCl 2 ( appendix 2A)
  • Control DNA samples of known genotype (10 to 50 ng/µl in H 2O)
  • Light mineral oil
  • 5 U/µl Taq DNA polymerase (Perkin‐Elmer Cetus AmpliTaq or equivalent)
  • Nusieve 3:1 agarose (FMC Bioproducts)
  • 1× TBE buffer ( appendix 2A) containing 0.5 µg/ml ethidium bromide
  • recipeLoading buffer (see recipe)
  • DNA molecular size markers
  • Test DNA samples (10 to 50 ng/µl in H 2O; support protocol)
  • Perkin‐Elmer Cetus thermal cycler (480 or TC) and suitable reaction tubes
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.7)

Alternate Protocol 1: Analysis of Multiple Mutations by Multiplex ARMS

  MaterialsFor common stock solutions, see appendix 2D.
  • 170 mM ammonium chloride (prepare fresh)
  • Blood sample, fresh or frozen
  • 10 mM NaCl/10 mM EDTA
  • 4% (w/v) sucrose in H 2O
  • 50 mM sodium hydroxide
  • 1 M Tris⋅Cl, pH 7.5 ( appendix 2A)
  • 1.5‐ml screw‐cap microcentrifuge tube
  • Rotator
  • 25‐ml plastic sample tube, sterile
  • Low‐speed centrifuge
  • Boiling water bath
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Figures

  •   FigureFigure 9.8.1 ARMS primer sequences for a single ARMS test. Sequences of the ARMS primers and target DNA sequences around the R117H mutation of the CFTR gene (G→A at position 482; Dean et al., ). The base that is altered is indicated in the normal and mutant DNA sequences by a box. The presence of an arrow indicates that primer/target combinations can be extended by Taq DNA polymerase; an “X” indicates extension does not occur. Bases in the ARMS primers that are not complementary to the target are shown displaced from the target sequence. A single mismatch (in this case a C/C) at the penultimate base is not sufficient to prevent extension whereas a primer with two adjacent mismatches, at the terminal and the penultimate base, is not extended.
  •   FigureFigure 9.8.2 Single and multiplex ARMS tests. The upper panel shows the results from four individuals tested for the R117H mutation of the CFTR gene. Each ARMS test consists of two ARMS reactions specific for the normal (N) or mutant (M) sequences. The 3′ sequences of the ARMS primers are given in Figure . All sample lanes contain the 360‐bp PCR control product amplified using control primer pair A and B. Samples 1 and 2 are from an individual heterozygous for the R117H mutation, whereas samples 3 and 4 are from normal individuals. The lower panel shows the results of seven individuals analyzed using a multiplex ARMS test for four common mutations of the CFTR gene (ΔF508, G542X, G551D and 621+1G→T). There are two lanes for each sample. The first lane contains products of the normal ARMS primers for 621+1G→T and ΔF508 and the mutant ARMS primers for G551D and G542X. The second lane contains the corresponding products for the 621+1G→T and 508 mutant primers and the G551D and G542X normal primers. The location of the products of each primer set is indicated at the side of the figure. The genotypes of the seven samples are: 1, normal; 2, ΔF508 heterozygote; 3, G551D heterozygote; 4, G542X heterozygote; 5, 621+1G→T heterozygote; 6, G542X, ΔF508 heterozygote; 7, G551D, ΔF508 heterozygote.

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Literature Cited

Literature Cited
   Dean, M., White, M.B., Amos, J., Gerrard, B., Stewart, C., Khaw, K.‐T., and Leppert, M. 1990. Multiple mutations in highly conserved residues are found in mildly affected cystic fibrosis patients. Cell 61:863‐70.
   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.
   Fortina, P., Conant, R., Monokian, G., Dotti, G., Parrella, T., Hitchcock, W., Kant, J., Scanlin, T., Rappaport, E., Schwartz, E., and Surrey, S. 1992. Nonradioactive detection of the most common mutations in the cystic fibrosis transmembrane conductance regulator gene by multiplex allele specific polymerase chain reaction. Hum. Genet. 90:375‐378.
   Mullis, K.B. 1991. The polymerase chain reaction in an anemic mode: How to avoid cold oligodeoxyribonuclear fusion. PCR Meth. Appl. 1:1‐4.
   Newton, C.R., Graham, A., Heptinstall, L.E., Powell, S.J., Summers, C., Kalsheker, N., Smith, J., and Markham, A.F. 1989. Analysis of any point mutation in DNA: The amplification refractory mutation system (ARMS). Nucl. Acid Res. 17:2503‐2516.
   Okayama, H., Curiel, D.T., Brantly, M.L., Holmes, M.D., and Crystal, R.G. 1989. Rapid nonradioactive detection of mutations in the human genome by allele specific amplification. J. Lab. Clin. Med. 114:105‐113.
   Sarkar, G., Cassady, J., Bottema, C.D.K., and Sommer, S.S. 1990. Characterization of polymerase chain reaction amplification of specific alleles. Anal. Biochem. 186:64‐68.
   Sidransky, D., Tokino, T., Hamilton, S.R., Kinzler, K.W., Levin, B., Frost, P., and Vogelstein, B. 1992. Identification of ras oncogene mutations in the stool of patients with curable colorectal tumors. Science 256:102‐105.
   Sommer, S.S., Groszbach, A.R., and Bottema, C.D.K. 1992. PCR amplification of specific alleles (PASA) is a general technique for rapidly detecting known single base changes. BioTechniques 12:82‐86.
   Wu, D.Y., Ugozzoli, L., Pal, B.K., and Wallace, R.B. 1989. Allele specific enzymatic amplification of β‐globin genomic DNA for the diagnosis of sickle cell anemia. Proc. Natl. Acad. Sci. U.S.A. 86:2757‐2760
Key Reference
   Ferrie et al., 1992. See above.
  Provides background information on ARMS and gives clinical data that support the reliability claims for the method.
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