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Application of some molecular biology techniques in diagnosis of human genetic diseases

100%
Slomski R., Kwiatkowska J., Chlebowska H.
Biotechnologia
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1993
|
issue 4
125-131
EN
Following the initial reports of the cloning of a DNA fragment, modern molecular genetics found immediate practical application in molecular analysis and diagnosis of human diseases.Genomic DNA, RNA, nucleic acids from archival specimen or cloned DNA may be starting materials for gene analysis.In extrame cases complete analysis can be performed on the DNA from a single cell or a few microdissected chromosome fragments, or on RNA from only few cells. Many variations of the basic analytical procedures have nov been described and applied to a range of medical disciplines. These include, the polymerase chain reaction (PCR), which rapid detection of fast or slow growing microorganisms and viruses, such as mycobacteria and HIV, the detection of minimal residual diseases in leukaemia and in HLA typing. The analysis of archival and forensic material has applications in forensic pathology and evolutionary biology. PCR technique has also established a central role in the human genome project.
2

A modified procedure for quantitative analysis of mtDNA, detecting mtDNA depletion

88%
Weglewska A., Jakobkiewicz-Banecka J., Wegrzyn G.
|
EN
Quantitative analysis of mitochondrial DNA (mtDNA) is crucial for proper diagnosis of diseases that are caused by or associated with mtDNA depletion. However, such a quantitative characterization of mtDNA is not a simple procedure and requires several laboratory steps at which potential errors can accumulate. Here, we describe a modified procedure for quantitative human mtDNA analysis. The procedure is based on using two PCR-amplified, fluorescein-labeled DNA probes, complementary to mtDNA (detection probe) and chromosomal 18S rDNA (reference probe), both of similar length. Thus, equal amounts of these probes can be used and, contrary to previously published procedures, no mtDNA purification (apart from total DNA isolation) or 18S rDNA cloning is necessary for probe preparation. Two separate hybridizations (each with one probe) are suggested instead of one hybridization with both probes; this decreases background signals and enables adjustment of the strength of specific signals from both probes, which is useful in the subsequent densitometric analysis after superimposing of both pictures. Using different DNA amounts for reactions, we have proved that the procedure is quantitative in a broad range of sample DNA concentrations. Moreover, we were able to detect mtDNA depletion unambiguously in tissue samples from patients suffering from diseases caused by dysfunction of mtDNA.
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