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 containing nitrogen bases similar to those of DNA (Kapoor and Telford, 2004). The backbone of PNA consists of N-(2-aminoethyl)-glycine units linked by peptide bonds unlike DNA backbone. Nitrogen bases are linked to the backbone by methylene carbonyl bonds instead of DNA N-glycoside bonds. Melting temperature of the PNA-DNA hybrid is higher than of DNA-DNA hybrid (Egholm et al., 1993), allowing hybridisation at high temperatures. The probe is labelled with dyes which are excited in the ‘blue’ visible light range (440-490 nm) since flow cytometers have a blue laser. Most often, fluoresceine isothiocyanate (FITC) is used as a label. If flow cytometer is equipped with a red laser, fluorescent labels with excitation peaks in the red visible range are used to label the PNA probe. To control cell cycle phase, the DNA is stained with dyes capable of binding to the DNA in a quantitative fashion such as propidium iodide (PI) or 4,6-diamidino-phenylindole (DAPI) (Nielsen et at.,1991). DAPI is easier to use, just like PI, it will bind to RNA, but the emissi...
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...ge, inhibitors accumulate, or even inactivation of the taq polymerase has started which may affect the efficiency of the PCR reaction. Fluorescence readings at early amplification cycles will gauge the amplified template quantity where the reaction is much more reproducible from sample to sample than at the endpoint. Use of a standard curve is a direct and accurate approach for analyzing the quantity of DNA .Standard curve is usually prepared from a dilution series of template whose concentration is known. The standard curve approach is used when it is important to the experimental design and objective of the project to measure the exact level of template in the samples. A variety of sources can be used as standard template such as plasmid which has a cloned gene of interest, genomic DNA, copy DNA, synthetic oligos, in vitro transcripts and total RNA besides others.