Characterizing mutations in samples with low-level mosaicism by collection and analysis of DHPLC fractionated heteroduplexes

Hum Mutat. 2003 Feb;21(2):112-5. doi: 10.1002/humu.10159.


Somatic mosaicism is a frequent phenomenon in mendelian disorders that exhibit a high proportion of new mutations; however, mutant alleles present at low frequency are difficult to detect and characterize. We have previously shown that denaturing high-performance liquid chromatography (DHPLC) can detect TSC1 and TSC2 mutations in tuberous sclerosis patients with low-level somatic mosaicism, even when direct sequencing cannot identify the causative lesion. Characterization of these mutations traditionally involves extensive sequencing of cloned products. To overcome this limitation, we have utilized DHPLC with an in-line fraction collector to isolate low-level heteroduplex peaks that can be directly sequenced to reveal the mutation. We have successfully applied this technique to resolve the mutations 2724-1G>C in TSC1and 1462-28del42bp, 1774del4bp, and N1643K (4947C>G) in TSC2, which were present in only 6.5-17% of the patients' alleles. We have also applied this technique to successfully resolve seven somatic APC mutations in colorectal tumor samples that were previously undetectable by direct PCR product sequencing. This method may simplify many of the currently challenging goals in mutation detection.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chromatography, High Pressure Liquid / methods*
  • DNA / blood
  • Genes, Tumor Suppressor
  • Humans
  • Leukocytes / chemistry
  • Mosaicism / genetics*
  • Mutation / genetics*
  • Nucleic Acid Denaturation / genetics*
  • Nucleic Acid Heteroduplexes / chemistry
  • Nucleic Acid Heteroduplexes / genetics*
  • Proteins / chemistry
  • Proteins / genetics
  • Repressor Proteins / chemistry
  • Repressor Proteins / genetics
  • Tuberous Sclerosis / genetics
  • Tuberous Sclerosis Complex 1 Protein
  • Tuberous Sclerosis Complex 2 Protein
  • Tumor Suppressor Proteins


  • Nucleic Acid Heteroduplexes
  • Proteins
  • Repressor Proteins
  • TSC1 protein, human
  • TSC2 protein, human
  • Tuberous Sclerosis Complex 1 Protein
  • Tuberous Sclerosis Complex 2 Protein
  • Tumor Suppressor Proteins
  • DNA