Quantitative determination of the ratio of mutated to normal ras genes in the blood of leukemia patients by allele-specific PCR.
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.
Horikoshi
T
T
Kenneth Norris Jr Comprehensive Cancer Center, University of Southern California School of Medicine, Los Angeles 90033.
Lenz
H J
HJ
Danenberg
K
K
Koch
O M
OM
Bertino
J R
JR
Danenberg
P V
PV
D000483:Alleles; D001483:Base Sequence; D003062:Codon / Q000235:genetics; D004247:DNA / Q000097:blood; D017931:DNA Primers; D004273:DNA, Neoplasm / Q000097:blood; D011905:Genes, ras; D006580:Genetic Carrier Screening; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000097:blood / Q000235:genetics*; D008969:Molecular Sequence Data; D009000:Monocytes / Q000166:cytology / Q000473:pathology; D016609:Neoplasms, Second Primary / Q000097:blood / Q000235:genetics; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012016:Reference Values
D003062:Codon; D017931:DNA Primers; D004273:DNA, Neoplasm; D004247:DNA