Diagnostics

DNA Helicase location

double-stranded DNA

DNA Helicase process

unwinds and separates the dsDNA into singeele strands

RNase H location

binds to a DNA/RNA duplex

RNase H

cleaves the phosphate backbone of the RNA strand, “chewing up” the RNA while leaving the DNA intact

cas12 location

binds to a specific target dsDNA sequence but its collateral activitiy targets nearby ssDNA reporters

cas12 process

performs indiscriminate “collateral” cutting of the ssDNA reporter molecules to separate a fluorophore from a quencher, generating a signal

reverse transcriptase locatioin

binds to a ssRNA template

reverse transcriptase process

acts as an RNA-dependent DNA polymerase, extending a primer to synthesize a complementary DNA (cDNA) strand

major inhibiotors you would expect to find in a blood sample

heme, lactoferrin, host DNA

Chaotropic salts

chemically lyse cells/viruses, denature nucleases, and create the necessary high-salt environment that drives nucleic acids to bind to the silica membrane.

ethanol wash

Ethanol washes away salts and impurities while keeping the DNA/RNA precipitated and bound to the silica

inhibitors from sample

proteins, salt

inhibitors from sample prep

alcohol, chaotropic salt

gram-positive

thick peptidoglycan layer

gram-negative

less thick peptidoglycan layer, typically easier to lyse

enveloped virus

easier to disrupt than non-enveloped viruses, have a lipid membrane

non-enveloped virus

tolerate heat, acid, drying

DNase

breaks down DNA

RNAase

breaks down RNA, broad range of activity and can be functional with EDTA, SDS, and chaotropic agents

polar components

DNA, salts, sugars, metal ions, detergents

nonpolar components

proteins, lipids

260nm

nucleic acids

280nm

mostly protein

230nm

salts, phenol, chaotropes, other organics

A260/A280

mainly a protein-contaminator check

A260/A230

mainly a carryover/inhibitor check

Sensitivity

how well the test finds true positives.

Specificity

how well it excludes true negatives.

Low sensitivity

more false negatives.

Low specificity

more false positives.

Low PPV

many positives are wrong.

Low NPV

many negatives are wrong.