Quantitative determination of the ratio of mutated to normal ras genes in the blood of leukemia patients by allele-specific PCR

Leuk Res. 1994 Sep;18(9):693-702. doi: 10.1016/0145-2126(94)90069-8.

Abstract

By combining allele-specific PCR amplification and a PCR-based quantitation approach, a method has been developed to estimate the mutated K-ras gene content in the blood of AML patients as a percentage of total K-ras. One PCR primer set was designed not to discriminate between mutant K-ras and wild-type K-ras and thus amplified the total K-ras gene. The other PCR primer set was designed to be allele-specific for K-ras gene containing a G to C mutation at codon 12. This primer set could discriminate the mutant and wild-type genes when the proportion of the mutated sequence was 0.2% of the total K-ras gene. To test the method on biological specimens, genomic DNA samples were analyzed from the peripheral blood of a patient who had secondary AML with the same codon 12 K-ras mutation. Two samples taken from this patient 2 months apart during follow-up had myeloblast cell contents of 67 and 80%. However, the percentage of mutated K-ras was 50% in both samples, suggesting that this patient may be inherently heterozygotic in this particular mutation. This ratio of mutated to normal K-ras in the patient's cells was confirmed by RNA-SSCP analysis and RNA sequencing. This quantitation method can provide a sensitive and specific estimation of the content of mutated K-ras alleles in patient samples.

MeSH terms

  • Alleles
  • Base Sequence
  • Codon / genetics
  • DNA / blood
  • DNA Primers
  • DNA, Neoplasm / blood
  • Genes, ras*
  • Genetic Carrier Screening
  • Humans
  • Leukemia, Myeloid, Acute / blood
  • Leukemia, Myeloid, Acute / genetics*
  • Molecular Sequence Data
  • Monocytes / cytology
  • Monocytes / pathology
  • Neoplasms, Second Primary / blood
  • Neoplasms, Second Primary / genetics
  • Point Mutation*
  • Polymerase Chain Reaction / methods*
  • Reference Values

Substances

  • Codon
  • DNA Primers
  • DNA, Neoplasm
  • DNA