620 extraction quiz

what does DNA extraction involve?

separating nucleic acids in cells away from proteins and other cellular material

what are the main methods for DNA extraction?

organic, Chelex, sold-phase

what are two commonly found PCR inhibitors?

hemoglobin + indigo dye

advantages of organic (phenol-chloroform) extraction

most effective way to obtain high molecular weight DNA

disadvantages of organic (phenol-chloroform) extraction

uses toxic chemicals

SDS full name

sodium dodecysulfate

general process for organic extraction

SDS/proK/EDTA, incubate + centrifuge, phenol chlorofrom, vortex + centrifuge, transfer aqueous phase

advantages of Chelex extraction

inexpensive, chelating-resin suspension added directly to sample, more rapid, fewer steps so less chance of contamination, removes PCR inhibitors

disadvantages of Chelex extraction

denature double-stranded DNA so can only be followed with PCR-based analysis

general Chelex extraction process

wash, remove supernatant, add chelex suspension, incubate at 100C

advantages of FTA paper extraction

consistent results without quantification, procedure may be automated

disadvantages of FTA paper extraction

dry paper punches can jump between wells

general FTA paper extraction process

apply blood to paper, take punch, wash with buffer, remove supernatant

what is solid-phase DNA extraction?

DNA selectively bound to substrate like silica beads/particles (in presence of high chaotropic salts)

types of solid-phase extractions

QIAGEN columns, DNA IQ, PrepFiler

robotic platform that automates solid-phase extraction process

QIAGEN EZ1

what is differential extraction?

separation of epithelial and sperm cells

DTT full name

dithiothreitol

general differential extraction process

add SDS/EDTA/proK, incubate + centrifuge, remove supernatant (non-sperm fraction), add SDS/EDTA/proK/DTT, sperm fraction

other methods to separate epithelial + sperm cells (if azsospermic/absence of spermatozoa)

selective cell degradtion, selective PCR (Y-STRs), selective cell capture (laser)

what is extraction?

taking biological material, adding reagents to lyse open cells, cellular components released into solution, everything else washed away

examples of degradation

UV radiation + heat

___________ inhibts DNA itself, __________ affect amplification

degradation ; inhibitors

Towson uses the ___ robot and _______________ kit

EZ1 ; DNA investigator

what is the purpose of G2 buffer?

lyse open cells

what is the purpose of proteinase K?

breaks down histones/proteins that protect the DNA

set up of the EZ1 robot

elution tubes in row 1, pipette tips + holders in row 2, nothing in row 3, sample tubes in row 4

chaotropic agent

guanidinium thiocynate

EDTA full name

ethylenediamine tetraacetic acid

robot process

adds lysis buffer, adds salt, lowers pH, DNA binds to salt bridge (decreases salt, raises pH)

DNA is eluted using an _____________, which is alkaline (raises the pH) which _________ the affinity of the DNA to the beads

elution buffer ; decreases

___ breaks down the disulfide bonds in the sperm head

dithiothretiol (DTT)

all evidence samples must be completed _______ processing any standards

prior to

pipetting QA + QC

use correct range pipette, use clean tip for each sample/reagent/master mix, never pass pipette tip over another sample, check for bubbles, pipettes are calibrated every six months

how can contamination be prevented?

wear + change gloves as needed, bleach all work areas before + after work, lay down kraft paper, clean utensils with ethanol between uses, only have one tube open at a time, only have one envelope open at a time

need _ QC per tube type

1

other QC measures

label tubes prior to cutting, tube check, add DNA stable, store extracts in refrigerator when complete

trace protcol

used on evidence only, as much DNA as possible from evidence sample

tip dance protocol

for known references