DNA Extraction Flashcards
As of 2020, how many people have the Innocence Project exonerated using DNA?
375
1989
the first DNA exoneration took place
how many exonerated cases started with eyewitness misidentification?
69%
reminders about DNA collection/storage
- dry out samples to limit contamination from mold
- breathable container
how to collect samples (dry blood in tub)
- scrape dry blood into an envelope
- wet swab to get blood then let it dry
collecting reference/known samples
- fast
- painless
- high DNA yield
- buccal swabs
preferred methods of sample collections
buccal swabs
sample storage
FTA cards
samples inside of the lab
- DNA samples are extracted & either stored in a refrigerator at 4°C or a freezer at -20°C
- for long term storage, extracted DNA samples may be stored at -70°C
DNA Extraction: what does the soap solution do?
breaks open cells by disrupting the phospholipid bilayer
DNA Extraction: what does the saline solution do?
helps DNA stick together by separating macromolecules from DNA
DNA Extraction: what does the alcohol do?
clumps DNA together so it can be removed
Chelex Extraction
- add Chelex 100 suspension to sample & mix
- heat sample at 95°C for 2-10 minutes
- separate Chelex 100 beads from sample by centrifugation, setting, or filtration
- use supernatant in downstream step such as PCR, RT-qPCR, RT-ddPCR, or RT-LAMP
DNA Quantification- Why?
- cost effective to screen for DNA first (could be none present)
- need to determine before PCR because a narrow range of DNA is optimal for most STR typing
Real Time Quantification PCR (RT-qPCR)
- determines the amount of amplifiable DNA
- reflects both quantity and quality
RT-qPCR analysis
- cycle to cycle change in fluorescence
- more DNA = more fluorescence
steps of Polymerase Chain Reaction (PCR)
- denaturing
- annealing
- extending
- repeat
PCR: denaturing
expose DNA to raised temperature & the H2 bonds between base pairs break & the 2 strands of the DNA molecules separate
PCR: annealing
lowering temperature & primers will attach through complimentary base pairing
PCR: extending
- raise the temperature
- DNA polymerase extends the new strand of DNA from the primers using dNTPs
CT - cycle threshold
number of PCR cycles for fluorescence to signal threshold
qPCR CT values inverse relationship
inverse relationship between CT values & amount of DNA in the sample
after 1 round of PCR, 1 molecule of DNA consisting of 2 complementary strands yields ___ molecules of DNA, for a total of ___ strands
2, 4
how many DNA molecules would exist after 10 PCR cycles?
10^2 = 100 DNA molecules
how many DNA molecules would exist after 30 PCR cycles?
30^2 = 900 DNA molecules
PCR
- allowing testing of partially degraded & small samples
- shortened analysis times from weeks to days
- increased ability to use DNA evidence
who developed PCR?
Kary Mullis
why is PCR not the whole answer?
it can only duplicate smaller fragments
short tandem regions
the repeat region is variable between samples while the flanking regions where PCR primers bind are constant
homozygote
both alleles are the same length
heterozygote
alleles differ & can be resolved from one another
calculate: “AAGCTA” DNA fragment length: 72 bp # of repeats
72/6 = 12
calculate: “TAGGG” DNA fragment length: 105 bp # of repeats
105/5 = 21
where are STR’s compared?
multiple sites
