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5530 midterm 2 > extraction > Flashcards

extraction Flashcards

1
Q

DNA workflow

A

DNA extraction
DNA quantitation
Amplification
Electrophoresis
Data Analysis/Interpretation

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2
Q

purpose of extraction

A

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

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3
Q

another goal of extraction

A

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

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4
Q

2 diff types of extraction

A

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

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5
Q

types of cell and tissue disruption

A

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

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6
Q

step 1: lysis of membranes and organelles

A

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

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7
Q

detergent

A

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

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8
Q

buffer system

A

Tris-HCl to maintain the pH in a range that avoids the activity of degrading enzymes
nucleases will activate when pH changes

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9
Q

high salt concentration

A

dissociate nuclear proteins such as histones from DNA (+ charge helps shield phosphate backbone once unwound)
backbone is -

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10
Q

reducing agent

A

mercaptoethanol or dithiothreitol (DTT) (breaks sperm heads disulfide bonds) to inhibit oxidation processes that can damage DNA

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11
Q

chelating agent

A

ethylenediaminetetraacedit acid EDTA or Chelex to capture divalent metal ions that are cofactors of endogenous nucleases that degrade DNA

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12
Q

LASD cell lysis

A

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

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13
Q

step 2
clean or purify dna

A

separate dna from all other cellular components and inhibitors
techniques
phenol - chloroform
chelex
silica-based extraction - EZ1

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14
Q

organic: phenol-chloroform (PCl)

A

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

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15
Q

concentration after PCI
step 3

A

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

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16
Q

extraction by boiling lysis and chelation

A

boiling for cellular disruption
results in ssDNA, can be a con b/c some methods req dsDNA
chelex extraction
introduced n early 1990s
includes steps:
washing - removes contaminants and inhibitors (heme)
boiling (step 1)
cells are suspended in solution, then lysed by boiling
chelating resin. (chelex 100) is employed, binds to polyvalent metal ions such as Mg2+
centrifugation is performed to pull the chelex 100 resin and cellular debris to the bottom of the tube (step 2), pellet
supernatant is used for dna analysis
you would still need to concentrate the ssDNA (step 3)
method is simple, rapid, and only uses one tube, reducing sample switch
cons: quick and dirty, good if you have lots of dna, not good for trace amounts, low % recovery

17
Q

silica-based extraction

A
  • backbone bound to counter ions bound to silica
    dna is reversibly adsorbed to silica in the presence of high concentrations of chaotropic salts
    chaotropes destabilize all IMF ( H bonds, VDW, and hydrophobic interactions (London dispersion))
    proteins are destabilized, included nucleases and the association of nucleic acids with water (H bonds) is disrupted
    setting up the conditions for the transfer to silica
    DNA (Hydrated) + silica (hydrated) + counter ions <-> neutral DNA-silica complex + water
    decreasing the conc of free water in solution through addition of perchlorate (a chaotrope) drives the reaction to the right, makes DNA and silica less hydrated
    three effects make the dominant contributions to the overall driving of adsorption
    shielded intermolecular electrostatic forces, chaotropes are the driving force for this
    dehydration of the dna and silica surfaces, remove water, chaotropes also drive this
    intermolecular hydrogen bond formatin in the DNA-silixa contact layer, counter ions facilitate this
    once you add water, dna is free
    step 1: cell lysis and protein digestion is carried out by extraction and specifically proK
    step 2
    dna adsorption onto silica, employs silica as the stationary phase in a column in the presence of chaotropic salts, dna is double stranded
    washing
    removes chaotropic agents and other contaminants
    the adsorbed DNA cannot be eluted from the silica matrix by certain solvents
    step 3
    elution of dna
    rehydration with acquits low-salt solution
    original device used vacuum filtration (centricon) to wash the DNA, followed by centrifugation to elute the purified DNA
    results in dsDNA
    yields high-quality DNA
    removes inhibitors as well
    adapts well to automation
    % recovery is really good, good for trace
18
Q

silica based extraction with magnet

A

employs silica-coated paramagnetic particles (magnetic beads) that adsorb DNA in the solution
a magnet is employed for particle capture instead of centrifugation or vacuum filtration
the sol containing contaminants and cellular debris is discarded
adsorbed DNA is washed several times using the magnetic provess
DNA is eluted after washing
concentration is partially determined by user selecting fluent volume
select element, water or Tris TE
can be chosen even through automation
also adapted to automation and high volume
example is EZ1/2

19
Q

when using automated magnetic bead separation such as EX1/2

A

last step in EZ1 is to elute DNA
35uL sample, 2uL for qPCR, 33 uL remains
you have a choice to elute with water or buffer, you can also pick your volume (concentrate)
50 uL or 200uL
always eluted in H2), dry down volume to dryness and rehydrate with Tris-EDTA buffer, total vol 35 u: DNA + TE
cannot be done if elute into Tris-EDTA (TE)
not good for PCR, inhibition
important: if you plan on drying down sample, use water as concentrated salts and EDTA in buffer can cause inhibition
if you screw up, rehydrate with the opposite

20
Q

EZ1

A

clean with ethanol, not bleach because it is reactive with chemicals and can produce hazardous gas
UV to sterilize

21
Q

FTATM paper
liquid blood samples

A

absorbent cellulose paper
used for reference samples
cells lyse on contact, DNA immobilized
paper contains 4 chemicals that prevent nuclease degradation and bacteria growth
DNA stable at RT for years
consistent results without quantification, small punch is placed in buffer and amplified directly

22
Q

differential extraction

A

can be cleaned by chelex (ssDNA) or EZ1 (dsDNA)
separate sperm cells from non-sperm cells
useful for extraction of DNA from biological evidence derived from sexual assault cases
selectively lyses the non-sperm and sperm cells in separate steps based on the differences in cell membrane properties of spermatozoa and other cells
add the SEb (stain extraction buffer, minus the DTT) to lyse all non-sperm cells
digest for 1-2 hours at 56C (optimal temp for proK)
remove supernatant from sample, place in labeled tube- this is the epithelial fraction
sperm cells, along with other cellular debris, will be in the pellet
to the original tube, add SEB + proK + DTT
re-extract
DTT breaks disulfide bonds that make sperm head so durable
sperm fraction
should always be male unless pellet isn’t washed well enough, causes low level F
digest 2hr to overnight
can be cleaned using PCI or EZ1

23
Q

setting up for extraction

A

multiple samples from same case, and multiple cases can be extracted together in one batch
label each sample with unique ID
always include reagent blank, will indicate if you have any contamination in reagents
tubes, DTT, buffers
this is your negative control for extraction
this will follow the workflow through dna anlaysis
if differential extraction is performed
you will have two fractions of the same sample, epithelial and sperm, Lebel accordingly
you will also have two RBs, an epithelial fraction and a sperm fraction (DTT included)

5530 midterm 2 (4 decks)

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