| 1. |
- Heiskanen, M, et al.
(författare)
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A novel method to quantitate methylation of specific genomic regions
- 1994
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Ingår i: PCR methods and applications. - 1054-9803. ; 4:1, s. 26-30
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Tidskriftsartikel (refereegranskat)abstract
- A new solid-phase primer extension method has been developed for the quantitation of methylation differences and is described here. The method is less cumbersome than Southern blot analysis, expresses the results in a numerical format, can be adapted to a microtitration well format, and thus allows the analysis of a large series of samples. The model gene analyzed here is the calcitonin gene, but the method can be adapted to the analysis of methylation alterations in any area of the genome. The primer extension method clearly differentiated hypermethylated samples from normally methylated samples and a range for normal values could be determined. In quantitation experiments the method showed linearity in a range from 2% to 100% malignant blasts diluted with normal leukocytes.
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| 2. |
- Ihalainen, J, et al.
(författare)
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Towards automatic detection of point mutations : use of scintillating microplates in solid-phase minisequencing
- 1994
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Ingår i: BioTechniques. - 0736-6205 .- 1940-9818. ; 16:5, s. 938-943
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Tidskriftsartikel (refereegranskat)abstract
- Simplification of molecular genetic techniques is one of the main features of large-scale clinical applications of mutation analysis. The solid-phase minisequencing method, which is based on single-nucleotide primer extension by a DNA polymerase on a solid support, is an easy way of detecting point mutations of previously known locations. Here the procedure was further simplified by the use of microplates made of scintillating plastics, a microplate format scintillation counter and an automatic microplate washer. DNA samples from patients with either a hereditary aspartylglucosaminidase (AGA) gene point mutation or an acquired N-ras gene mutation were analyzed by three different minisequencing detection procedures utilizing tritiated nucleotides. The new counting method with scintillating plates was compared to traditional liquid scintillation counting in scintillation vials or to another microplate format procedure, which requires addition of scintillation liquid. In all three methods, normal individuals, heterozygous carriers of the AGA mutation and homozygous patients could be unequivocally discriminated. The N-ras mutation in leukemic blasts could also be detected with high resolution. The coefficients of variation and reproducibility of the scintillating microplate method were almost identical to those of the traditional liquid scintillation assay, which was used as a reference method. The technical innovations adopted here for performing minisequencing assays reduce significantly the labor required without affecting the quality of the results.
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| 3. |
- Palotie, A, et al.
(författare)
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Development of molecular genetic methods for monitoring myeloid malignancies
- 1993
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Ingår i: Scandinavian journal of clinical & laboratory investigation. Supplementum. - 0085-591X. ; 213, s. 29-38
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Tidskriftsartikel (refereegranskat)abstract
- The malignant diagnosis of a haematological disorder can in most cases be made by clinical signs and routine microscopic examination. However, it has become necessary to characterize the malignant clone with various markers, which give either knowledge of the prognosis of the disease or give tools for the laboratory follow up of the patient. In lymphatic diseases there are excellent markers of clonality. On the contrary in myeloid malignancies the few well characterized markers are mostly helpful in the clinical management of rare myeloid subgroups. The aim of our project has been to develop methods for laboratory monitoring of myeloid diseases by two major approaches 1) detection of methylation alterations in the short arm of chromosome 11 and 2) novel approaches for sensitive point mutation detection. The short arm of chromosome 11 has areas where the DNA becomes hypermethylated in acute leukemias and lymphomas. In this chromosomal area the calcitonin gene serves as a good marker for methylation alterations due to several CpG sites in the 5'area of the gene. Even if the gene is normally methylated in most cases of chronic myeloid leukemia (CML), we have found that the hypermethylation of the calcitonin gene marks progression of CML and precedes any other signs of acceleration with several months. The point mutations of certain proto-oncogenes, such as the N-ras gene, are attractive markers for detecting residual diseases after chemotherapy of high malignant haematological disorders. However, conventional methods for detecting point mutations have been both insensitive and cumbersome, and thus unsuitable for clinical routine laboratories. With the solid-phase minisequencing we can technically easily and accurately detect small quantities of mutated cells.
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| 4. |
- Syvänen, Ann-Christine, et al.
(författare)
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N-ras gene mutations in acute myeloid leukemia : accurate detection by solid-phase minisequencing
- 1992
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Ingår i: International Journal of Cancer. - 0020-7136 .- 1097-0215. ; 50:5, s. 713-718
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Tidskriftsartikel (refereegranskat)abstract
- Mutations in the N-ras gene are found in one-third of patients with acute myeloid leukemia. The N-ras mutations could serve as markers for residual cells, if a highly sensitive method for detecting the mutations was available. We applied a new method, solid-phase minisequencing, to analyze bone-marrow cells from 16 patients with acute myeloid leukemia for mutations in codon 12, 13 and 61 of the N-ras gene. In the solid-phase minisequencing technique the mutations are identified by a primer extension reaction, in which a single labelled nucleoside triphosphate is incorporated into an immobilized DNA fragment previously amplified by the polymerase chain reaction. We identified N-ras mutations in 5 of the patients (30%). In one patient, we observed 2 mutations that were shown to be located in different alleles. With the solid-phase minisequencing method, we were able to determine the proportion of mutated cells in the samples. We found that in 4 of the samples only a fraction (7-64%) of the blasts carried an N-ras mutation, and in one sample practically all blast cells were mutated. The method was highly sensitive, allowing us to identify N-ras mutations even when the sample consisted of 99.7% normal cells and only 0.3% mutated blasts.
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