Lecture 7 Flashcards
What range do the fluorescent dyes fluoresce
Visible range
Commercial STR kits use what dye approach?
dye primer approach (only one labeled primer/locus) and the primer +dye combination is patented
- dye is added to 5’ end
How does fluorescence work in PCR? (like chem process)
excitation by laser causes a fluorescent dye molecule to absorb a photon and then emit a photon (with lower energy / longer wavelength than the initial, so detector can tell them apart)
What is a stokes shift
The photon emitted is a longer wavelength than one absorbed
missing a slide here i think
What color is 5-TAMRA
yellow :3
How does the quencher method of qPCR work?
uses Taq polymerases function of 5’ exonuclease activity will cause it to destroy the probe as it comes across it, releasing it and emitting the signal, while if Taq pol does not amplify the sequence containing the probe, then the quencher will annul the flourencence
What is the issue with using many dyes in a multiplex rxn?
There is a bit of overlap in emission spectra. Cant use dyes that are close in wavelength
How did they used to multiplex mult STR loci of a sample?
Using a big PAGE with two different dyes, software can call out alleles for you as well
How does detetector look kfor each possible color?
The detector systems cycles through (used to be real) virtual optical filters
The instrument needs to be calibrated to accurately distinguish dyes. (QC)
Massive signal way downstream to left is…
Primers that never annealed
What is a pull up?
When the instrument is not correctly calibrated and the detector miscalls an underlying peak as an actual allele
artifactual peak
can also happen if there is a really big peak
Can the allelic ladder be co-electrophoresis?
What about the internal size standard?
- allelic ladder no
- size standard yes, so you need a diff color for dye
