extraction Flashcards
DNA workflow
DNA extraction
DNA quantitation
Amplification
Electrophoresis
Data Analysis/Interpretation
purpose of extraction
Step 1
lyse the cells
also protect and unwind the DNA by denaturing protiens
Step 2
separate it from other cell componenets
Step 3
purify and concentrate the dna
another goal of extraction
remove inhibitors from the sample
substances that interfere with he per reaction may interact with tap DNA polymerase itself, or bind to target sequences of primers
examples of inhibitors
heme from blood
melanin from hair and skin
bile salts, polysaccharides, fecal matter
humid compounds, soil/dirt
urea from urine
textile dyes, denim blue dye
industrial oils/chemicals from guns, fluids on bottles, residue on tools
2 diff types of extraction
cellular extraction (non-sperm) or blood
any type epithelial / blood cells
differential extraction
separate sperm cells from non-sperm (epithelial) cells
two step extraction
preferentially lyse the epithelial cells, and then you lyse the sperm cells
get two different fractions for dna analysis
sperm and epithelial
types of cell and tissue disruption
part of or before step 1 (lysis)
enzymatic digestions, such as proteinase K (proK)
boiling
alkali treatment pH>7 basic
those there are most common
materials such as bones and teeth can be frozen n liquid nitrogen and then ground into a fine powder
step 1: lysis of membranes and organelles
performed during or immediately after tissue disruption
release dna from nuclei or mitochondria
use of a lysis buffer which contains all or a combination of
detergent
buffer system
high salt conc
reducing agent
chelating agent
detergent
anionic compounds, sarkosyl, and sodium dodecyl sulfate (SDS) to destroy cell membranes, denature proteins, and dissociate proteins from DNA
laundry detergent
fatty lipid bilayer and proteins
buffer system
Tris-HCl to maintain the pH in a range that avoids the activity of degrading enzymes
nucleases will activate when pH changes
high salt concentration
dissociate nuclear proteins such as histones from DNA (+ charge helps shield phosphate backbone once unwound)
backbone is -
reducing agent
mercaptoethanol or dithiothreitol (DTT) (breaks sperm heads disulfide bonds) to inhibit oxidation processes that can damage DNA
chelating agent
ethylenediaminetetraacedit acid EDTA or Chelex to capture divalent metal ions that are cofactors of endogenous nucleases that degrade DNA
LASD cell lysis
stain extraction buffer SEB contains:
TRIS-buffer (7-9 pH)
SDS detergent
EDTA chelating agnet
NaCl salt
DTT cleaves cystine bonds (disulfide bonds, sperm heads)
not added immediately for differential extraction
proteinase K (proK) cleaves peptide bonds and digests proteins
56C digest, proK most efficient temp
hour long enzymatic digestion
SDS opens up cells
SDS and proK work together, SDS opens proteins up to allow the proK to cleave them
step 2
clean or purify dna
separate dna from all other cellular components and inhibitors
techniques
phenol - chloroform
chelex
silica-based extraction - EZ1
organic: phenol-chloroform (PCl)
goal is to remove proteins and other stuff, leaving only DNA
goal for all extractions
use phenol:chloroform:isoamyl alcohol solution (25:24:1)
phenol is used to extract proteins from aqueous sol
chloroform is used to help distinguish organic layer from acquits layer due to its high density
dissolves with phenol, like dissolves like
chloroform heavier than water
phenol/water is less different than chloroform and water
isoamyl alcohol is added to reduce foaming
DNA is solubilized in the aqueous phase (top)
lipids solubilized in the organic phase
proteins are located at the interface between the 2 phases
wash aqueous DNA with PCI multiple times to really clean the DNA, get out tract cellular componenets
results in dsDNA > can be used for RFLP or PCR based analysis
cons: time consuming, involves use of hazardous reagents and requires transferring samples amount tubes, more chances to switch samples
concentration after PCI
step 3
result from step 2 is dilute, 3-4 mL
2 common methods
ethanol precipitation
ultrafiltration - microcon and centricon
utilizes centrifuge
microcon starts at bottom, add sample to reservoir at top, spin and discard filtrate
dna trapped in filter
flip microcon cartridge and spin again to release concentrated DNA