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#PCR detection

2 public questions tagged with this topic.

PCR

Which is best method for checking mycoplasma contamination in a mammalian cell line?

Mycoplasma contamination in mammalian cell cultures is difficult to detect by microscopy or culture because organisms lack a cell wall and grow slowly. PCR targeting conserved 16S rRNA or 16S-23S spacer regions offers highly sensitive, specific, and rapid detection of few genome copies in culture supernatant. Southern hybridization requires larger DNA amounts and is less sensitive. ELISA and Western hybridization detect proteins and lack broad Mycoplasma genus coverage. Hence PCR is the gold standard for routine screening mandated by cell banks, preventing altered growth, metabolism, and exper

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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PCR Modifications

Which step in Real-Time PCR with Molecular Beacons leads to fluorescence emission?

Molecular beacons are hairpin-shaped probes bearing a fluorophore at one end and a quencher at the other. In free solution, the stem keeps them close, enabling FRET quenching with negligible emission. Hybridization occurs specifically during the annealing phase, when temperature permits complementary base pairing to target amplicons. Binding forces stem separation, physically distancing fluorophore from quencher and restoring fluorescence. Fluorescence intensity therefore reflects target-specific duplex formation at annealing, not denaturation or extension. This reversible, hybridization-depen

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

View Details