Non-radioactive detection of Mycobacterium tuberculosis LCR products in a microtitre plate format.
As part of the development of the ligase chain reaction (LCR) into a tool which can be used by a wide variety of researchers, we have investigated several analytical detection systems for the products of this amplification reaction. While early work with this technology has used gel electrophoresis to separate the LCR probes from the ligated product, solid phase capture techniques are also applicable, particularly when one of the probes is modified with a 'hook' such as biotin, and the adjoining probe modified with a detectable label. In this study we report a comparison of eight different non-radioactive detection techniques and discuss the analytical sensitivity of each. Detection with laser scanning fluorescent gel electrophoresis remains the most sensitive, with the assay described herein capable of detecting 100 molecules of the Mycobacterium tuberculosis insertion element IS6110 in a background of 4 micrograms of unrelated DNA. This method was followed closely by solid-phase capture and chemiluminescence detection which gave a sensitivity of 1000 molecules of IS6110. Fluorescence detection was approximately 10-fold less sensitive than chemiluminescence detection, and absorbance detection was a further 10-fold less sensitive than fluorescence detection. However, absorbance detection even at this level can still be useful for systems where visual interpretation is desired.
Winn-Deen
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Applied Biosystems, Division of Perkin-Elmer Corp., Foster City, CA 94404.
Batt
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CA
Wiedmann
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D000469:Alkaline Phosphatase / Q000378:metabolism; D001483:Base Sequence; D001710:Biotin; D011088:DNA Ligases / Q000032:analysis*; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics*; D004586:Electrophoresis / Q000379:methods; D005456:Fluorescent Dyes; D005784:Gene Amplification; D006923:Hymecromone / Q000031:analogs & derivatives / Q000378:metabolism; D007211:Indoles / Q000378:metabolism; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D009580:Nitroblue Tetrazolium / Q000378:metabolism; D009596:Nitrophenols / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D014376:Tuberculosis / Q000175:diagnosis
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D005456:Fluorescent Dyes; D007211:Indoles; D009596:Nitrophenols; D009943:Organophosphorus Compounds; C075487:AttoPhos; D009580:Nitroblue Tetrazolium; C008644:nitrophenylphosphate; C005359:4-methylumbelliferyl phosphate; C035455:5-bromo-4-chloro-3-indoxyl phosphate; D006923:Hymecromone; D009249:NADP; D001710:Biotin; D000469:Alkaline Phosphatase; C076545:Ampligase; D011088:DNA Ligases