Modeling, CRISPR, RT-PCR, Sequencing

what kid of mutations does CRIPSR - Cas9 create

Point mutations - site-directed mutations, transgene inserts, knockouts

how can we use cas9 guide RNA to create site-directed mutations, transgene inserts or knockouts

gRNA directs Cas 9 to a specific DNA sequence (PAM), Cas9 cuts DNA at target site and makes blunt ends,

knockout - cas 9 cut in 2 spots

what role does the “spacer” on gRNA play in this process

20 base sequence complementary to DNA target, binds to matching DNA sequence, ensures Cas 9 cuts at right location

what is the PAM (protospacer adjacent motif) and where is if fount?

found upstream of target DNA sequence (not on gRNA) ca9 wont cut unless PAM is nearby, downstream of what you want to change

what binds to PAM

Cas( protein, binds and unwinds nearby DNA, spacer on gRNA binds to target DNA

what is reverse transcription PCR

converting RNA into complementary DNA, then amplified using PCR

What does Revers transcription PCR allow us to detect, and how does it work (what kind of protein do we use)

RNA based info by converting it to cDNA use reverse transcriptase (RNA dependent, DNA polymerase)

What is real time PCR or quantitative PCR

allows you to quantify amount of DNA or RNA in real time during amplification process

explain the fluorophore/quencher system on DNA probes and how is it used to quantity the amount of PCR done

Probe - short single-strand complementary DNA sequence, forms hairpin and has fluorophore and quencher on each end. When next to each other, fluorophore doesn’t glow, but when attached to DNA, it spreads out and is expressed, amount of fluorescence emitted = amount of DNA being amplified

how can you use Poly A polymerase and dTTP terminal transferase to create sticky ends

one sequence has a bunch of A’s the other a bunch of T’s and then they can come together