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Analysis of mutational spectra by denaturing capillary electrophoresis

Nature Protocols volume 3, pages 1153–1166 (2008)Cite this article

Abstract

The point mutational spectrum over nearly any 75- to 250-bp DNA sequence isolated from cells, tissues or large populations may be discovered using denaturing capillary electrophoresis (DCE). A modification of the standard DCE method that uses cycling temperature (e.g., ±5 °C), CyDCE, permits optimal resolution of mutant sequences using computer-defined target sequences without preliminary optimization experiments. The protocol consists of three steps: computer design of target sequence including polymerase chain reaction (PCR) primers, high-fidelity DNA amplification by PCR and mutant sequence separation by CyDCE and takes about 6 h. DCE and CyDCE have been used to define quantitative point mutational spectra relating to errors of DNA polymerases, human cells in development and carcinogenesis, common gene–disease associations and microbial populations. Detection limits are about 5 × 10−3 (mutants copies/total copies) but can be as low as 10−6 (mutants copies/total copies) when DCE is used in combination with fraction collection for mutant enrichment. No other technological approach for unknown mutant detection and enumeration offers the sensitivity, generality and efficiency of the approach described herein.

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Figure 1: Melting profile of the mitochondrial DNA sequence.
Figure 2: Noise reduction by two-stage DNA amplification method.
Figure 3: Steps in DCE protocol.
Figure 4: Theoretical DNA melting profile of complementary strands of an example target sequence.
Figure 5: Illustration of partial melting of amplified DNA.
Figure 6: Effects of applying different DNA melting algorithms.
Figure 7: Possible strand combinations after DNA amplification of heterozygote sample and heteroduplex formation.
Figure 8: Schematics of optical bench-assembled CDCE instrument.
Figure 9: Effect on allele separation with increase in denaturing temperature.
Figure 10: Visualization of Taq polymerase–induced mutants following mutant enrichment by fraction collection of the heteroduplex region.
Figure 11: Allele separation of heterozygote sample by CDCE in ABI 310 and ABI 3100 DNA sequencing instruments (Applied Biosystems).
Figure 12: Simultaneous separation of alleles in 12 different fragments using CyDCE on a 96-capillary instrument (MegaBACE 1000).
Figure 13: Reproducibility of allele separation by CyDCE.

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Authors and Affiliations

  1. Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, 02139-4307, Massachusetts, USA

    Per O Ekstrøm, Xiao-Cheng Li-Sucholeiki & William G Thilly

  2. Department of Surgical Oncology, The Norwegian Radium Hospital, Ullern chausseen 70, Oslo, 0310, Norway

    Per O Ekstrøm

  3. Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Avenue, Boston, 02215, Massachusetts, USA

    Konstantin Khrapko

  4. Department of Mechanical Engineering, BioInstrumentation Laboratory, Massachusetts Institute of Technology, Cambridge, 02139, Massachusetts, USA

    Ian W Hunter

  5. Agencourt Bioscience Corp., 500 Cummings Center, Suite 2450, Beverly, 01915, Massachusetts, USA

    Xiao-Cheng Li-Sucholeiki

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  1. Per O Ekstrøm
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  2. Konstantin Khrapko
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  3. Xiao-Cheng Li-Sucholeiki
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  4. Ian W Hunter
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  5. William G Thilly
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Correspondence to Per O Ekstrøm.

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Competing interests

The authors declare competing financial interests. Beckman-Coulter, Inc. has licensed patents for constant temperature DCE technology and related applications from MIT. Drs Khrapko, Li-Sucholeiki and Thilly as inventors receive a portion of annual royalties.

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Ekstrøm, P., Khrapko, K., Li-Sucholeiki, XC. et al. Analysis of mutational spectra by denaturing capillary electrophoresis. Nat Protoc 3, 1153–1166 (2008). https://doi.org/10.1038/nprot.2008.79

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