620 oral boards

what is extraction in general sense?

taking biological material, adding reagents to lyse cells, cellular components released

what is the objective of extraction?

remove DNA from stain/substrate, isolate DNA from other cellular materials and inhibitors. chemicals are used to break open cells to release DNA and unwind DNA from bound proteins. solution chemically washed to remove unwanted cellular materials. final product is a liquid containing only DNA

what are two examples of inhibitors?

indigo dye and hemoglobin

what are two examples of degradation?

UV radiation and heat

what robot is used for extraction?

EZ1 or EZ1 advanced XL with DNA investigator kit

what is the purpose of G2 buffer?

lyse open cells

what is the purpose of proteinase K?

breaks down histones/proteins that protect DNA

what is the purpose of incubation steps in extraction?

complete lyses and activate enzymes

how many QCs do you need on each instrument?

one

the _____ protocol is used to get as much DNA as possible from evidence

trace

the ________ protocol is used for known references

tip dance

what is the robot extraction process in a general sense?

adds lysis buffer, adds salt, lower pH, DNA binds to salt bridge

what is the robot extraction process?

lysis buffer (to increase affinity to beads), sample moves to well with resin that contains paramagnetic beads , DNA binds to beads with low pH, wash steps (to remove cellular debris and proteins that can degrade DNA, elution buffer decreases affinity of DNA to beads, DNA purified and transferred

what does the lysis buffer contain to increase affinity?

chaotropic agents (guanidinium thiocynate)

what is EDTA and what does it do?

ethylenedimine tetraacetic acid inhibits nuclease activity, chelates Mg2+ and Ca2+

what does paramagnetic mean?

not attracted to each other, but when magent applied attracted to each other (holds DNA in place)

DNA binds to beads with ___ pH

low

what type of extraction separates sperm from non-sperm cells?

differential

what is the purpose of dithiothreitol (DTT)?

breaks down disulfide bonds in sperm heads (because G2 is not strong enough)

T/F: all evidence samples should be processed before processing known references

true

T/F: for differential extractions, a QC is needed for both the non-sperm and sperm fractions

true

what are examples of ways to help prevent contamination?

bleach work areas, use kraft paper, clean supplies with ethanol, only open one tube or one envelope at a time, wear appropriate PPE (gloves, lab coat, goggles), clearly label tubes

what is the purpose of quantitation?

determine the amount of amplifiable DNA

T/F: the FBI QAs require that all evidence samples be quanted

true

what happens if too much or too little DNA is amplified?

too much causes overblown electropherograms, too little causes possible loss of alleles

what are the three steps of polymerase chain reaction (PCR)?

denaturing, annealing, extension

what happens in the denaturing step of PCR?

high temperatures (95 C) break hydrogen bonds in double stranded DNA to make single stranded DNA

what happens in the annealing step of PCR?

primers use Brownian motion to find complementary primer binding sites (occurs at 60 C)

what happens in the extension step of PCR?

DNA polymerase attaches to the end of primers to bring in complementary dNTPs to make double stranded DNA

how many cycles does PCR repeat for?

40

what is quantitative PCR (qPCR)?

PCR has the ability to detect/read fluorescence at the end of each cycle

what are the three phases of qPCR?

exponential, linear, plateau

what happens in the exponential phase of qPCR?

DNA is doubling after each cycle, all master mix components are abundantly available

during what phase of qPCR is DNA concentration measured?

exponential

what is the cycle threshold (CT) and what is it used for?

an arbitrary fluorescence value determined by the kit manufactuer that is used to differentiate between background noise and PCR product

what happens in the linear phase of qPCR?

less reagents are available for PCR, DNA is not doubling each cycle, one or more components start to fall below critical concentration, dNTPs and primers are used at differential rates

what happens in the plataeu phase of qPCR?

reagents are almost used up, PCR process comes to an end, multiple components have reached end of effectiveness, increase in fluorescence levels out

what is the standard curve used for?

determining unknown quants (it is a plot of CT versus log DNA concentration)

in the best fit line of standard curve equation, y=mx+b, what does each value represent?

y is CT (cycle in which level of fluorescence crosses threshold), m is slope (always negative, corresponds to PCR efficiency), x is log DNA concentration, b is y-intercept

what is R2, in relation to the standard curve?

measures the fit of data points in the curve in relation to the best fit line

what kit do we use for quantitation?

investigator quantiplex pro RGQ kit

what does the quant kit detect?

human autosomal DNA and male DNA

what does the quant kit estimate?

degradation of autosomal DNA and male DNA

T/F: the quant kit contains an internal control (IC)

true

what is the purpose of the IC?

ensure PCR is proceeding appropriately

a degradation ratio greater than 2:1 indicates ____ degradation

more

fluorescence is detected using ______ probes

TaqMan

the taqman probe is labled with which dyes that emit different wavelengths?

reporter dye on the 5’ end and quencher dye on the 3’ end

what is the taqman assay process?

probe hybridizes to the DNA target region located within the locus being amplified, when the reporter and quencher dyes are close almost no fluorescenece is emitted, quantinova DNA polymerase cleaves the fluorophore from the quencher, when reporter is no longer close to quencher there is detectable fluorescence

what is the general procedure for non-differential extraction?

add G2 and proK, vortex, incubate at 56C for 30 minutes, centrifuge, substrate transfer to basket, remove substrate from buffer and place in spin basket, centrifuge at max, discard basket and retain liquid in flat-bottom tube

what is the general procedure for differential extraction?

add G2 and proK, vortex, incubate at 56C for 30 minutes, centrifuge, substrate transfer to basket, remove substrate from buffer and place in spin basket, centrifuge at max, discard basket and transfer supernatant (non-sperm) to new tube. wash pellet with G2, centrifuge at max, discard supernatant, add G2/DTT/proK, vortex, incubate at 70C for 15 minutes, centrifuge, transfer liquid (sperm) to clean tube

what parameters are used in the extraction instrument set-up?

trace protocol, TE elution, 50 uL elution volume

what goes in each of the four rows in the extraction set-up?

1 has the elution tube, 2 has the pipette tip and holder, 3 is blank, 4 has the lysed sample

what instrument is used for quantitation?

real-time thermal cycler (rotor-gene Q)

what is the general quantitation procedure?

dilution of control DNA to create standard curve, make mastermix, vortex, add reagents to 100uL tubes, place tubes on instrument (make sure to balance), update computer sample layout and keep log, open Q-rex software, start run