Quantification of mRNA expression by competitive PCR using non-homologous competitors containing a shifted restriction site.

Despite the recent introduction of real-time PCR methods, competitive PCR techniques continue to play an important role in nucleic acid quantification because of the significantly lower cost of equipment and consumables. Here we describe a shifted restriction-site competitive PCR (SRS-cPCR) assay based on a modified type of competitor. The competitor fragments are designed to contain a recognition site for a restriction endonuclease that is also present in the target sequence to be quantified, but in a different position. Upon completion of the PCR, the amplicons are digested in the same tube with a single restriction enzyme, without the need to purify PCR products. The generated competitor- and target-specific restriction fragments display different sizes, and can be readily separated by electrophoresis and quantified by image analysis. Suboptimal digestion affects competitor- and target-derived amplicons to the same extent, thus eliminating the problem of incorrect quantification as a result of incomplete digestion of PCR products. We have established optimized conditions for a panel of 20 common restriction endonucleases permitting efficient digestion in PCR buffer. It is possible, therefore, to find a suitable restriction site for competitive PCR in virtually any sequence of interest. The assay presented is inexpensive, widely applicable, and permits reliable and accurate quantification of nucleic acid targets.

Use of quantitative polymerase chain reaction to monitor residual disease in chronic myelogenous leukemia during treatment with interferon.

Interferon-alpha (IFN-alpha) has become a widely used treatment modality in chronic myelogenous leukemia (CML) and was shown to induce complete hematologic responses in about 70% of the patients. In a minority of cases, complete suppression of the Philadelphia (Ph)-positive clone has been observed by cytogenetic investigation or by Southern blot analysis. In most instances, however, analyses by the highly sensitive two-step polymerase chain reaction (PCR) reveal the presence of residual leukemic cells despite continuous treatment. Since PCR positivity has not been associated with an increased risk of disease recurrence, the monitoring of cells carrying the characteristic BCR/ABL rearrangement by qualitative PCR may not facilitate early identification of patients who are likely to relapse. We have therefore employed a quantitative PCR technique to monitor the BCR/ABL mRNA expression levels during the course of treatment in an attempt to assess the response to IFN-alpha at the molecular level and to provide a basis for early detection of progressive disease. Twenty CML patients who received therapy with IFN-alpha in first chronic phase of the disease were enrolled in the study. In addition, we have monitored two CML patients treated with IFN-alpha for relapse after bone marrow transplantation. Thirteen patients who displayed decreasing, constant or fluctuating levels of BCR/ABL expression during an observation period of up to 4 years (mean 25 months) have remained in hematologic remission. Two patients showed an elevation in the marker gene expression upon discontinuation of treatment, but no further increase in BCR/ABL mRNA has been observed after reinitiation of therapy with IFN, and the patients have remained in hematologic remission. In seven patients, quantitative PCR (Q-PCR) analyses revealed increasing expression of the chimeric gene during treatment with IFN-alpha. In all seven cases, the detection of elevated BCR/ABL transcripts by quantitative PCR preceded signs of hematologic or cytogenetic disease progression by up to 8 months (median 6 months). Our data show that quantitative PCR analysis facilitates the monitoring of the response to IFN-alpha therapy and provides an effective diagnostic tool for the timely detection of recurrent disease. The employment of this technique greatly enhances the diagnostic possibilities in the management of chronic myelogenous leukemia.

Determination of radiation-induced DNA strand breaks in individual cells by non-radioactive labelling of 3' OH ends.

To quantify DNA strand breaks generated by ionizing radiation in single cells with preserved morphology, the number of radiation-induced 3' OH ends of nuclear DNA was determined by enzymatic labelling. Confluent CHO-K1 cells were irradiated with doses up to 100 Gy. After fixation and permeabilization of the cell monolayer the nuclear DNA was labelled with Digoxigenin-11-dUTP using terminal transferase. The incorporated nucleotide was detected with an anti-Digoxigenin antibody conjugated with alkaline phosphatase. The phosphatase bound was quantified by a colour reaction and the integrated optical densities of the cell nuclei were measured. For doses ranging from 20 to 100 Gy a linear relationship between dose and labelling signal was obtained. Repair experiments showed a fast component of repair with a half-time of about 14 min, followed by a slower decline to background values, which were reached after 6-8 h. This method allows the measurement of radiation-induced DNA strand breaks in morphologically preserved single cells in a reproducible way, which may be of importance in the prediction of tumour response in radiotherapy.

Increase of bcr-abl chimeric mRNA expression in tumor cells of patients with chronic myeloid leukemia precedes disease progression.

The translocation t(9;22) in chronic myeloid leukemia (CML) generates a bcr-abl fusion gene that codes for an aberrant chimeric mRNA. Cell lines established from CML patients in blast crisis show higher expression of this aberrant bcr-abl transcript than cells from patients in chronic phase of the disease. This observation provided the stimulus to investigate whether increased expression of the aberrant bcr-abl fusion transcript is critical to the progression of CML from chronic phase to blast crisis. We have monitored the bcr-abl mRNA expression in 25 patients by serial quantitative polymerase chain reaction analyses during a follow-up period of 12 to 156 months after diagnosis, with a median observation time of 28 months. In all patients who have shown disease progression to accelerated phase (n = 4) or blast crisis (n = 7), an increase in bcr-abl mRNA expression was detected up to 16 months before laboratory or clinical parameters showed phenotypic transformation of the malignant clone. To investigate whether the elevated levels of bcr-abl mRNA reflected an increase in the proportion of leukemic cells in the samples analyzed or primarily enhanced transcriptional activity of the bcr-abl fusion gene, we performed quantitative analyses of the bcr-abl gene at the DNA level and of the Ph chromosome at the cytogenetic level and compared these data with steady-state bcr-abl mRNA levels. We show that increased levels of the bcr-abl transcript did not reflect increased proportions of leukemic cells but elevated steady-state levels of the chimeric mRNA in the malignant cells before disease progression. Therefore, our data strongly suggest that an increase of the chimeric mRNA expression in the leukemic cells precedes the phenotypic transformation of the malignant clone.