*2.2.2 Polymerase chain reaction-based techniques (qPCR, MMqPCR, and aTL qPCR)*

This method was created in order to address the issue of requiring a significant amount of DNA in order to evaluate telomere lengths [50]. Quantitative PCR (qPCR), monochromatic multiplex quantitative PCR (MMqPCR), and absolute telomere length quantification are employed to carry out these processes. The DNA sequence is amplified by PCR using specially designed primers over the course of 20–40 cycles, with each run doubling the amount of the PCR product (the amplicon typically), a fluorophore is employed in qPCR to quantify the quantity of the target DNA sequence. After each turn, the amount of emitted fluorescence is evaluated [75]. Seven years later, the same study group improved this technique by completing the amplification of both the telomeric and single-copy DNA regions from the same tube. Monochrome multiplex quantitative PCR (MMqPCR) is the name of this novel method [76, 77]. The underlying qPCR-based method was further modified by the other research team [78]. This group referred to it as an absolute telomere length (aTL) qPCR method. Briefly, the procedure for using this approach is the same as for using the initial qPCR experiment.

Limitations of this method

