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Träfflista för sökning "WFRF:(Sitbon M) srt2:(1995-1999)"

Search: WFRF:(Sitbon M) > (1995-1999)

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  • Sitbon, G, et al. (author)
  • A colorimetric minisequencing assay for the mutation in codon 506 of the coagulation factor V gene
  • 1997
  • In: Thrombosis and Haemostasis. - 0340-6245 .- 2567-689X. ; 77:4, s. 701-703
  • Journal article (peer-reviewed)abstract
    • We describe a colorimetric screening method for the mutation in codon 506 of the coagulation factor V gene. The nucleotide at the site of the FV:Q506 mutation is identified in an immobilized amplified DNA template by extension of a primer with a hapten-labelled dNTP using a DNA polymerase. The incorporated hapten is detected by an antibody-alkaline phosphatase conjugate that catalyses the formation of a coloured end product. The assay is carried out in a microtiter plate format, and the procedure is identical to that of enzyme immunoassays. It unequivocally identifies the FV:Q506 mutation in heterozygous and homozygous form. The colorimetric minisequencing method gave the same result as a 3H-based minisequencing assay and restriction site analysis with Mn11 used as reference methods. Because of its simple format and numeric result, the novel colorimetric minisequencing method should be an attractive alternative for screening for the FV:Q506 mutation in clinical laboratories.
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  • Williams, Cecilia, 1969-, et al. (author)
  • A high frequency of sequence alterations is due to formalin fixation of archival specimens.
  • 1999
  • In: American Journal of Pathology. - 0002-9440 .- 1525-2191. ; 155:5
  • Journal article (peer-reviewed)abstract
    • Genomic analysis of archival tissues fixed in formalin is of fundamental importance in biomedical research, and numerous studies have used such material. Although the possibility of polymerase chain reaction (PCR)-introduced artifacts is known, the use of direct sequencing has been thought to overcome such problems. Here we report the results from a controlled study, performed in parallel on frozen and formalin-fixed material, where a high frequency of nonreproducible sequence alterations was detected with the use of formalin-fixed tissues. Defined numbers of well-characterized tumor cells were amplified and analyzed by direct DNA sequencing. No nonreproducible sequence alterations were found in frozen tissues. In formalin-fixed material up to one mutation artifact per 500 bases was recorded. The chance of such artificial mutations in formalin-fixed material was inversely correlated with the number of cells used in the PCR-the fewer cells, the more artifacts. A total of 28 artificial mutations were recorded, of which 27 were C-T or G-A transitions. Through confirmational sequencing of independent amplification products artifacts can be distinguished from true mutations. However, because this problem was not acknowledged earlier, the presence of artifacts may have profoundly influenced previously reported mutations in formalin-fixed material, including those inserted into mutation databases.
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