High Throughput Detection of Microsatellite Instability by Denaturing High-Performance Liquid Chromatography

Kai Feng Pan, Wanguo Liu, You Yong Lu, Lian Zhang, Zhen Pu Li, Wan Li Lu, Stephen N Thibodeau, Wei Cheng You

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

Microsatellite instability (MSI) is a hallmark of the DNA replication error phenotype, due to the inactivation of mismatch repair genes. MSI has been implicated in colon and many other gastrointestinal cancers. MSI usually can be analyzed by PCR amplification of microsatellite markers followed by electrophoresis and detected using autoradiography or fluorescence techniques. We report here a novel method for high-throughput detection of MSI using denaturing high performance liquid chromatography (DHPLC). Amplification of two mononucleotide markers (BAT25 and BAT26) by polymerase chain reaction (PCR) is followed by DHPLC analysis to display alteration in the length of repetitive sequences. These two markers were tested in 84 colorectal cancer samples confirmed to be 44 MSI-H and 40 MSI-L or MSS, previously defined by multiple microsatellite markers and/or by immunohistochemical analyses of MLH1 and MSH2 proteins. Among 44 MSI-H samples, sequence variations in BAT26 and BAT25 were detected in 44 (100%) and 43 (98%), respectively, while no sequence variation in the two markers was detected in 40 MSI-L or MSS samples. A total of 96 gastric cancers and their matched normal tissues were then analyzed for MSI-H using this method. Sequence variations in BAT26 and BAT25 were detected in nine (9.4%) samples. Seven of the nine cases were shown unstable at both BAT26 and BAT25; one each was unstable at BAT26 or BAT25. These results were confirmed by autoradiography analyses. Together, our results demonstrate high sensitivity and specificity of DHPLC in the analysis of sequence variations in BAT25 and BAT26 to determine MSI status. This simple, efficient, and high-throughput approach will facilitate analysis of MSI in large sample sets of any cancers.

Original languageEnglish (US)
Pages (from-to)388-394
Number of pages7
JournalHuman Mutation
Volume22
Issue number5
DOIs
StatePublished - 2003

Fingerprint

Microsatellite Instability
High Pressure Liquid Chromatography
Autoradiography
Microsatellite Repeats
Polymerase Chain Reaction
DNA Mismatch Repair
Gastrointestinal Neoplasms
Nucleic Acid Repetitive Sequences
DNA Replication
Stomach Neoplasms
Sequence Analysis
Electrophoresis
Colorectal Neoplasms
Colon
Fluorescence

Keywords

  • BAT25
  • BAT26
  • Cancer
  • DHPLC
  • Gastric cancer
  • Microsatellite instability
  • MLH1
  • MLH2

ASJC Scopus subject areas

  • Genetics
  • Genetics(clinical)

Cite this

Pan, K. F., Liu, W., Lu, Y. Y., Zhang, L., Li, Z. P., Lu, W. L., ... You, W. C. (2003). High Throughput Detection of Microsatellite Instability by Denaturing High-Performance Liquid Chromatography. Human Mutation, 22(5), 388-394. https://doi.org/10.1002/humu.10271

High Throughput Detection of Microsatellite Instability by Denaturing High-Performance Liquid Chromatography. / Pan, Kai Feng; Liu, Wanguo; Lu, You Yong; Zhang, Lian; Li, Zhen Pu; Lu, Wan Li; Thibodeau, Stephen N; You, Wei Cheng.

In: Human Mutation, Vol. 22, No. 5, 2003, p. 388-394.

Research output: Contribution to journalArticle

Pan, Kai Feng ; Liu, Wanguo ; Lu, You Yong ; Zhang, Lian ; Li, Zhen Pu ; Lu, Wan Li ; Thibodeau, Stephen N ; You, Wei Cheng. / High Throughput Detection of Microsatellite Instability by Denaturing High-Performance Liquid Chromatography. In: Human Mutation. 2003 ; Vol. 22, No. 5. pp. 388-394.
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