DNA Extraction

Question 1

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

Answer 1

375

Question 2

1989

Answer 2

the first DNA exoneration took place

Question 3

how many exonerated cases started with eyewitness misidentification?

Answer 3

69%

Question 4

reminders about DNA collection/storage

Answer 4

• dry out samples to limit contamination from mold
• breathable container

Question 5

how to collect samples (dry blood in tub)

Answer 5

• scrape dry blood into an envelope
• wet swab to get blood then let it dry

Question 6

collecting reference/known samples

Answer 6

• fast
• painless
• high DNA yield
• buccal swabs

Question 7

preferred methods of sample collections

Answer 7

buccal swabs

Question 8

sample storage

Answer 8

FTA cards

Question 9

samples inside of the lab

Answer 9

• 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

Question 10

DNA Extraction: what does the soap solution do?

Answer 10

breaks open cells by disrupting the phospholipid bilayer

Question 11

DNA Extraction: what does the saline solution do?

Answer 11

helps DNA stick together by separating macromolecules from DNA

Question 12

DNA Extraction: what does the alcohol do?

Answer 12

clumps DNA together so it can be removed

Question 13

Chelex Extraction

Answer 13

• 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

Question 14

DNA Quantification- Why?

Answer 14

• 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

Question 15

Real Time Quantification PCR (RT-qPCR)

Answer 15

• determines the amount of amplifiable DNA
• reflects both quantity and quality

Question 16

RT-qPCR analysis

Answer 16

• cycle to cycle change in fluorescence
• more DNA = more fluorescence

Question 17

steps of Polymerase Chain Reaction (PCR)

Answer 17

• denaturing
• annealing
• extending
• repeat

Question 18

PCR: denaturing

Answer 18

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

Question 19

PCR: annealing

Answer 19

lowering temperature & primers will attach through complimentary base pairing

Question 20

PCR: extending

Answer 20

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

Question 21

CT - cycle threshold

Answer 21

number of PCR cycles for fluorescence to signal threshold

Question 22

qPCR CT values inverse relationship

Answer 22

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

Question 23

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

Answer 23

2, 4

Question 24

how many DNA molecules would exist after 10 PCR cycles?

Answer 24

10^2 = 100 DNA molecules

Question 25

how many DNA molecules would exist after 30 PCR cycles?

Answer 25

30^2 = 900 DNA molecules

Question 26

PCR

Answer 26

- allowing testing of partially degraded & small samples - shortened analysis times from weeks to days - increased ability to use DNA evidence

Question 27

who developed PCR?

Answer 27

Kary Mullis

Question 28

why is PCR not the whole answer?

Answer 28

it can only duplicate smaller fragments

Question 29

short tandem regions

Answer 29

the repeat region is variable between samples while the flanking regions where PCR primers bind are constant

Question 30

homozygote

Answer 30

both alleles are the same length

Question 31

heterozygote

Answer 31

alleles differ & can be resolved from one another

Question 32

calculate: "AAGCTA" DNA fragment length: 72 bp # of repeats

Answer 32

72/6 = 12

Question 33

calculate: "TAGGG" DNA fragment length: 105 bp # of repeats

Answer 33

105/5 = 21

Question 34

where are STR's compared?

Answer 34

multiple sites