Mutation Detection by Cycle Sequencing

Ludwig Thierfelder1

1 Max‐Delbrück‐Centrum für Molekulare Medizin, Berlin
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 7.7
DOI:  10.1002/0471142905.hg0707s16
Online Posting Date:  May, 2001
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Abstract

Candidate genes are screened for mutations by a DNA sequencing procedure known as cycle sequencing. First, a segment of the candidate gene is PCR amplified from the genomic DNA of an affected individual. The PCR product is then subjected to multiple rounds of further amplification in a thermal cycler using a heat‐stable DNA polymerase in the presence of different dideoxynucleotides and a radiolabeled primer. The resulting 32P‐labeled sequence reaction products are fractionated on a denaturing polyacrylamide gel and visualized by autoradiography. DNA segments on the order of 200 bp from 10 to 30 individuals can be screened on each gel. Cycle sequencing eliminates the need to subclone genomic fragments or PCR products, which makes it a much simpler method than conventional sequencing for identifying mutations.

     
 
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Table of Contents

  • Basic Protocol 1: Cycle Sequencing with a 5′‐End‐Labeled Primer
  • Reagents and Solutions
  • Commentary
  • Tables
     
 
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Materials

Basic Protocol 1: Cycle Sequencing with a 5′‐End‐Labeled Primer

  Materials
  • Blood or other DNA source from individual(s) to be tested
  • 5 M sodium acetate, pH 4.8
  • Isopropanol (pure)
  • TE buffer, pH 7.4 ( appendix 2D)
  • Molecular weight markers: e.g., ϕX‐174 plasmid digested with HaeIII (Hoefer Pharmacia Biotech or Life Technologies)
  • 12.5 pmol oligonucleotide sequencing primer: PCR primer used in step , or nested primer
  • recipe10× T4 polynucleotide kinase reaction buffer (see recipe)
  • 10 µCi/µl [γ‐32P]ATP (3000 Ci/mmol), 10 µCi/µl [γ‐33P]ATP (3000 Ci/mmol), or 10 µCi/µl [γ‐35S]ATP (1000 Ci/mmol)
  • 5 U/µl T4 polynucleotide kinase
  • Dideoxynucleotides: 100 µM ddGTP (or 7‐deaza‐dGTP for GC‐rich templates), 600 µM ddATP, 1000 µM ddTTP, and 600 µM ddCTP
  • 2 U/µl Taq DNA polymerase
  • recipe10× cycle sequencing reaction buffer (see recipe)
  • Mineral oil (if needed, depending on thermal cycler)
  • recipeStop solution (see recipe)
  • Thermal cycler and thermal cycler tubes
  • Additional reagents and equipment for isolating and purifying human genomic DNA ( appendix 3B or units 9.8, 10.5 & 14.4), agarose gel electrophoresis (unit 2.7), nucleic acid quantification ( 3.NaN), PCR amplification of genomic DNA (unit 7.1), optimization of PCR conditions (CPMB UNIT 15.1), and preparation, electrophoresis, and drying of polyacrylamide gels ( 3.NaN)
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Figures

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Literature Cited

Literature Cited
   Innis, M.A., Myambo, K.B., Gelfand, D.H., and Brow, A.D. 1988. DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction amplified DNA. Proc. Natl. Acad. Sci. U.S.A. 85:9436‐9440.
   Mizusawa, S., Nishimura, S., and Seela, F. 1986. Improvement of the dideoxy chain termination method of DNA sequencing by use of deoxy‐7‐deazaguanosine triphosphate in place of dGTP. Nucl. Acids Res. 14:1319‐1324.
   Murray, V. 1989. Improved double‐stranded DNA sequencing using the linear polymerase chain reaction. Nucl. Acids Res. 17:8889.
   Wong, C., Dowling, C.E., Saiki, R.K., Higuchi, R.G., Erlich, H.A., and Kazazian, H. 1987. Characterization of β thalassaemia mutations using direct genomic sequencing of amplified single copy DNA. Nature 330:384‐386.
   Wrischnik, L.A., Higuchi, R.G., Stoneking, M., Erlich, H.A., Arnheim, N., and Wilson, A.C. 1987. Length mutations in human mitochondrial DNA: Direct sequencing of enzymatically amplified DNA. Nucl. Acids Res. 15:529‐542.
Key Reference
   Murray, 1989. See above.
  Original paper describing cycle sequencing.
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