RESUMEN
SUMMARY: The detection of mutant tumor genes holds great promise for an early diagnosis of primary tumors and residual malignant disease. When few tumor cells are present with an excess of nonmalignant cells of the same lineage, the excess of wild-type alleles over mutant tumor alleles presents an analytical problem. The subtractive iterative PCR (siPCR) assay presents a new approach to solving this problem. To achieve an enrichment of mutant alleles, wild-type alleles are removed by differential hybridization to complementary oligonucleotides spanning the region of the gene in which point mutations are expected. The nonbound fraction is reamplified by PCR. By iterating this process, mutant alleles can be detected in the presence of an excess of wild-type alleles with high sensitivity. To prove the feasibility of siPCR, pancreatic juice samples were analyzed for KRAS mutations. Pancreatic juice obtained from patients with pancreatic carcinoma or chronic pancreatitis during endoscopic retrograde cholangiopancreatography was analyzed for point mutations in codons 12 and 13 of the KRAS gene. In each of six samples from tumor patients, mutations in codon 12 were detected. One of nine samples from patients with chronic pancreatitis scored positive.
Asunto(s)
Alelos , Genes ras/genética , Mutación , Jugo Pancreático/fisiología , Reacción en Cadena de la Polimerasa/métodos , Carcinoma/genética , Enfermedad Crónica , Codón/genética , Estudios de Factibilidad , Humanos , Neoplasias Pancreáticas/genética , Pancreatitis/genética , Mutación PuntualRESUMEN
The detection of cardiac troponins in peripheral blood as protein markers of myocardial infarction is a new diagnostic tool in the diagnosis of cardiac disease. In order to increase the sensitivity and specificity of this diagnostic approach, a reverse transcription polymerase chain reaction assay has been developed to detect the mRNA encoding cardiac troponin I from myocardial cells hypothetically released from damaged cardiac tissue. The detection is specific for cardiac troponin I mRNA, with no amplification of homologous sequences of other troponin I isoforms, i.e., troponin I from skeletal muscle cells. However, a strong amplification signal for cardiac troponin I mRNA was detected in samples of peripheral blood from healthy human volunteers. In patients with acute myocardial infarction or angina pectoris, the cardiac troponin I mRNA levels were not increased over background levels. In conclusion, a reverse transcription polymerase chain reaction approach based on the amplification of cardiac troponin I mRNA is not feasible in the diagnosis of cardiac diseases.
Asunto(s)
Angina de Pecho/sangre , Infarto del Miocardio/sangre , Miocardio/metabolismo , Isoformas de Proteínas/genética , ARN Mensajero/sangre , Troponina I/genética , Angina de Pecho/diagnóstico , Angina Inestable/sangre , Angina Inestable/diagnóstico , Secuencia de Bases , Biomarcadores , Estudios de Factibilidad , Humanos , Datos de Secuencia Molecular , Infarto del Miocardio/diagnóstico , Reacción en Cadena de la Polimerasa , Valor Predictivo de las Pruebas , Alineación de Secuencia , Homología de Secuencia de Ácido NucleicoRESUMEN
In human carcinomas, mutations that alter tumour genes such as the KRAS, P53, or APC genes, are mostly point mutations. The detection of mutant alleles of tumour genes in specimens such as urine, pancreatic juice, sputum, and stool holds great promise for an early diagnosis of cancer. In addition, the detection of mutant tumour genes in tissue samples, such as lymph nodes or resection margins, may allow a sensitive diagnosis of residual malignant disease. However, the reliable detection of mutant alleles in excess of wild type alleles remains an unresolved analytical problem when the mutations are not known a priori. In the present communication, a new approach is described which makes possible the detection of unknown point mutations in tumour genes at excess of wild type alleles. The method is based on the removal of wild type alleles by hybridisation to immobilised complementary oligonucleotides. Using this approach, an enrichment of mutant KRAS, P53 and APC alleles of one mutant in up to 10(3) normal alleles has been achieved. Parallel miniaturised separation units with oligonucleotides complementary to defined sequences of a wild type allele should allow the detection of unknown point mutations as well as small insertions or deletions which occur in the sequence range covered by the oligonucleotides.
Asunto(s)
Alelos , Cromatografía/métodos , ADN de Neoplasias/química , Mutación Puntual , Humanos , Polimorfismo de Longitud del Fragmento de Restricción , Análisis de Secuencia de ADN , Células Tumorales CultivadasRESUMEN
Processed pseudogenes of residual contaminating genomic DNA interfere with a sensitive detection of cytokeratin 18 (CK18) mRNA by reverse transcription and polymerase chain reaction (RT-PCR). This may cause false-positive results when CK18 mRNA is used as a marker for ectopic tumor cells in specimens from cancer patients. To establish a sensitive CK18 RT-PCR by excluding the amplification of processed pseudogenes, the following strategy was chosen: (a) CK18 pseudogene sequences were cloned from genomic DNA by PCR; (b) cDNA-specific primers were designed on the basis of mismatches between pseudogenes and cDNA; (c) PCR conditions were adjusted to reach maximum sensitivity and specificity. Epithelial cells (1-10) could be detected in 1 mL of blood. Among the numerous CK18 genes homologous to the transcribed gene, at least two different processed pseudogenes exist that are highly homologous to each other and to the exons of the transcribed CK18 gene.
Asunto(s)
Biomarcadores de Tumor/genética , ADN/genética , Queratinas/genética , Reacción en Cadena de la Polimerasa/métodos , Seudogenes/genética , ARN Mensajero/análisis , ARN Neoplásico/análisis , Médula Ósea/patología , Cardias , Cartilla de ADN/genética , ADN Complementario/genética , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Datos de Secuencia Molecular , Sensibilidad y Especificidad , Homología de Secuencia de Ácido Nucleico , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologíaRESUMEN
The N-terminal domains of two different highly homologous isoforms of pregnancy-specific beta 1 glycoproteins (PSGs) were expressed in bacteria. The N-terminal domain of PSG1 (PSG1-N) and PSG3 (PSG3-N) were chosen since PSG3-N, but not PSG1-N, contains an RGD sequence. Immunosorbents were prepared using bacterially expressed fusion proteins with the respective N domains. Antibodies from a polyclonal antiserum against native PSG were eluted from PSG1-N and were subsequently absorbed against PSG3-N. Using this procedure, antibodies were generated that were able to bind to native PSG and PSG1-N, but not to PSG3-N. These results show that the antiserum against native PSG crossreacts with PSG isoforms of two subgroups. From the PSG antiserum, antibodies can be isolated that differentially bind to V-like PSG domains which differ by eight non-conservative amino acid substitutions, three of which are clustered in a position corresponding to the CDR III of immunoglobulin V region domains. Purification of antibody populations by this technique should make it possible to distinguish rapidly between highly homologous PSG isoforms in tissues and body fluids.