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Biology: Determining The Lengths Of Telomeres Using Flow Fish Method

1366 words - 6 pages

Flow FISH is a method for determining the lengths of telomeres which is common nowadays due to its versatility, its more rapid and its able to process samples which contain few cells (Lansdorp,1996). The major limitation of flow-FISH is the requirement of the equipment to do the analysis which is expensive, the configuration of the equipment (flow cytometer) and the nature of the probe used for hybridization is also expensive and requires a well trained personnel. Flow-FISH involves hybridisation of telomeric DNA of fixed sample cells with a fluorescently labeled peptide-nucleotide probe with sequence which is complementary to the telomere repeat DNA sequences. PNA is a synthetic polymer ...view middle of the document...

A disadvantage of DAPI staining is that it is excited by UV light, therefore calling for availability of an UV laser and the corresponding detector array as well. DNA staining allows for distinguishing the cell populations that are in the G0/G1 cell cycle phase to measure telomere length only in cells which would have a fixed telomere DNA copies/genome equivalent ratio and for the calculation of DNA index, which is an estimate of the proportion of cells in the sample that are in G0/G1 phase compared to control cells (Kapoor and Telford, 2004).
Briefly, G0/G1 cell populations are separated and the intensity of the fluorescent signal is measured in relationship to the number of genomic proportions in the sample used. The more the intensity the fluorescent signal is, the more the hybridization of the fluorescent probe implying that there is a high copy number of the telomere TTAGGG repeat sequences in the tested sample. The fluorescence of the tested sample is compared to the fluorescence of a control sample with known telomere length while background fluorescence is accounted for by measuring the fluorescence of an unstained sample of the sample cells and the control cells.
1.6.4. Quantitative PCR
Quantitative PCR (qPCR) monitors the increase in the amount of a double-stranded amplification product during a PCR reaction. It involves primers labeled with a fluorescent dye and a quencher, designed such in a way that does not allow the primer to engaged in a reaction leading to formation of Double stranded DNA product .Generation of PCR product causes significant net increase in fluorescence which could be quantified. One of the most common techniques for measuring telomere length by means of qPCR is Cawthon’s method (Telomere/Single Copy Gene ratio also known as T/S method) (Cawthon, 2002). This method measures the how the ratio of the TTAGGG telomere repeat copy number to single-gene copy number varies between the sample tested and a control sample. Cawthon method relies on the evaluation of the kinetics of the amplification of a telomere fragment and a single-copy gene fragment so as to come up with a telomere/single-copy gene ratio. Sample tested is then compared to the standard control sample whose length is known. The number of telomere repeats in the sample tested is calculated using the level of dilution of the control DNA that would make the number of cycles of PCR required to produce a given amount of telomere PCR product from the sample tested comparable to the number of cycles needed to come up with the same amount of telomere product from the control sample. Single-copy gene used to standardize the assay is normally a housekeeping cellular gene, amplified in the same run with the telomere fragment. Amplification of the telomere fragment and the single-copy gene fragment can be done in a single tube and even by use of the same intercalating dye basing on the differential reaction kinetics for the two amplicons. Other variations of the...

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