DNA Extraction Flashcards

1
Q

As of 2020, how many people have the Innocence Project exonerated using DNA?

A

375

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

1989

A

the first DNA exoneration took place

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

how many exonerated cases started with eyewitness misidentification?

A

69%

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

reminders about DNA collection/storage

A
  • dry out samples to limit contamination from mold
  • breathable container
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

how to collect samples (dry blood in tub)

A
  • scrape dry blood into an envelope
  • wet swab to get blood then let it dry
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

collecting reference/known samples

A
  • fast
  • painless
  • high DNA yield
  • buccal swabs
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

preferred methods of sample collections

A

buccal swabs

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

sample storage

A

FTA cards

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

samples inside of the lab

A
  • 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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

DNA Extraction: what does the soap solution do?

A

breaks open cells by disrupting the phospholipid bilayer

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

DNA Extraction: what does the saline solution do?

A

helps DNA stick together by separating macromolecules from DNA

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

DNA Extraction: what does the alcohol do?

A

clumps DNA together so it can be removed

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

Chelex Extraction

A
  • 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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

DNA Quantification- Why?

A
  • 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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

Real Time Quantification PCR (RT-qPCR)

A
  • determines the amount of amplifiable DNA
  • reflects both quantity and quality
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

RT-qPCR analysis

A
  • cycle to cycle change in fluorescence
  • more DNA = more fluorescence
17
Q

steps of Polymerase Chain Reaction (PCR)

A
  • denaturing
  • annealing
  • extending
  • repeat
18
Q

PCR: denaturing

A

expose DNA to raised temperature & the H2 bonds between base pairs break & the 2 strands of the DNA molecules separate

19
Q

PCR: annealing

A

lowering temperature & primers will attach through complimentary base pairing

20
Q

PCR: extending

A
  • raise the temperature
  • DNA polymerase extends the new strand of DNA from the primers using dNTPs
21
Q

CT - cycle threshold

A

number of PCR cycles for fluorescence to signal threshold

22
Q

qPCR CT values inverse relationship

A

inverse relationship between CT values & amount of DNA in the sample

23
Q

after 1 round of PCR, 1 molecule of DNA consisting of 2 complementary strands yields ___ molecules of DNA, for a total of ___ strands

24
Q

how many DNA molecules would exist after 10 PCR cycles?

A

10^2 = 100 DNA molecules

25
how many DNA molecules would exist after 30 PCR cycles?
30^2 = 900 DNA molecules
26
PCR
- allowing testing of partially degraded & small samples - shortened analysis times from weeks to days - increased ability to use DNA evidence
27
who developed PCR?
Kary Mullis
28
why is PCR not the whole answer?
it can only duplicate smaller fragments
29
short tandem regions
the repeat region is variable between samples while the flanking regions where PCR primers bind are constant
30
homozygote
both alleles are the same length
31
heterozygote
alleles differ & can be resolved from one another
32
calculate: "AAGCTA" DNA fragment length: 72 bp # of repeats
72/6 = 12
33
calculate: "TAGGG" DNA fragment length: 105 bp # of repeats
105/5 = 21
34
where are STR's compared?
multiple sites