User:Cytosort
Chromosome Analysis (Flow Karyotype)
[edit]Chromosome analysis is a method that employs the use of flow cytometry for the classificiation and purification of chromosomes from mitotic cells. Before flow cytometric analysis, the chromosomes are treated with 2 different types of DNA fluorescent dyes; Hoechst(HO) and Chromomycin A3 (CA3). The fluorescent dyes HO binds preferentially on A-T rich sequences while CA3 binds preferentially on G-C rich sequences. The intensity of the emitted fluorescence produced from these fluorescence dyes is dependent not only on the DNA content but also on the base composition of each chromosome. It is based on this differences that distinguish the chromosomes from one another. As such the term flow karyotyping has been used for the classification of chromosomes by using flow cytometry. This technique has been proved to be very useful for quantitative detection of aberrant chromosomes due to translocations, deletions or additions.
Flow cytometric analysis of chromosome was first made possible by the development of technique for isolating intact chromosomes by Wray and Stubblefield [1] in 1970. The protocol for chromosome analysis using ethidium bromide was first described in 1975 at the Lawrence Livermore laboratory by Joe W. Gray and others [2] from University of California. This single color staining procedure was then developed into two colors [3] measurements which provided the resolution required to separate most of the human chromosomes.
Experimental Procedure
[edit]The basic features in preparing cells for flow karyotyping involves enrichment of cells number at metaphase with mitotic inhibitor, swelling of mitotic cells with hypotonic solution [4], and the release of chromosomes into polyamines [4] or magnesium ion [5] based chromosome stabilizing buffer.
Aside from HO and CA3, other fluorescent dyes combination such as DAPI and CA3, HO and olivomycin have been used previously(ref). Both DAPI and HO binds preferentially to A-T rich sequence while CA3 and oliviomycin has a binding preference for G-C rich sequence.
Doublets/Clumps discrimination
[edit]Fragmented chromosomes in the formed of DNA debris and clumped chromosomes aggregates are formed during the chromosome isolation process. Some of these debris and aggregates are discriminated by applying region gate on a plot of linear Forward scatter (FSC) versus linear pulse width (see D1), and bivariate plot of HO versus CA3 fluorescence was gated on this region (see D2). This gating led to an improvement in the resolution of the fow karyotype.
References
[edit]- ^ Wray W, Stubblefield E (1970). "A new method for the rapid isolation of chromosomes, mitotic apparatus, or nuclei from mammalian fibroblasts at near neutral pH". Exp. Cell Res. 59 (3): 469–78. PMID 5461579.
{{cite journal}}: Unknown parameter|month=ignored (help) - ^ Gray JW, Carrano AV, Moore DH; et al. (1975). "High-speed quantitative karyotyping by flow microfluorometry". Clin. Chem. 21 (9): 1258–62. PMID 1170959.
{{cite journal}}: Explicit use of et al. in:|author=(help); Unknown parameter|month=ignored (help)CS1 maint: multiple names: authors list (link) - ^ Gray JW, Langlois RG , Carrano AV, Burkhart-Schulte K, Moore Van Dilla MA; et al. (1979). "High resolution chromosome analysis: One and two Parameter flow cytometry". Chromosoma. 73: 9–27.
{{cite journal}}: Explicit use of et al. in:|author=(help); Unknown parameter|month=ignored (help)CS1 maint: multiple names: authors list (link) - ^ a b Ng BL, Carter NP (2006). "Factors affecting flow karyotype resolution". Cytometry A. 69 (9): 1028–36. doi:10.1002/cyto.a.20330. PMID 16969800.
{{cite journal}}: Unknown parameter|month=ignored (help) - ^ van den Engh G, Trask B, Cram S, Bartholdi M (1984). "Preparation of chromosome suspensions for flow cytometry". Cytometry. 5 (2): 108–17. doi:10.1002/cyto.990050203. PMID 6201326.
{{cite journal}}: Unknown parameter|month=ignored (help)CS1 maint: multiple names: authors list (link)