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Wildlife Forensic Science > DNA/RNA extractions > Flashcards

DNA/RNA extractions Flashcards

1
Q

What tissue is the easiest to work with

A

muscle

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2
Q

what will be needed to analyze plant cells?

A

lysis buffers for membranes and mixer mills to get rid of the cell walls

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3
Q

what is in cells?

A

RNA, DNA, proteins, small ions, sugars, fats, nucleic acids

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4
Q

when extracting DNA what is a bad sign?

A

colour

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5
Q

what is an inhibitor that we must always be mindful of?

A

calcium ions

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6
Q

in bones, teeth and tusks what are inhibitors and how do we get DNA?

A

Ca2+, melt down the bone until it becomes floppy

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7
Q

where do we sample on a feather/what types of feathers?

A

bottom we want either growing quills or blood quills

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8
Q

why is blood not a good sample?

A

because of all the +2 ions in the sample

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9
Q

what is the best place to extract DNA on a shell?

A

the inner membrane will give species ID

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10
Q

what do fecal samples tell you?

A

diet and sometimes mucosal coat telling you what/who deposited it

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11
Q

why is urine not very good?

A

most cells are absorbed into the ground/not deposited at all

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12
Q

how do you deal with bark, leaves, and seeds?

A

grind them up in mixer mills with metal beads

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13
Q

how many cells are needed for a full mtDNA profile?

A

1

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14
Q

how many cells are needed for a full nDNA profile?

A

167

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15
Q

how many pg of DNA are needed for a full profile?

A

500pg

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16
Q

What are good preservation buffers?

A

RNA later or lysis buffer

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17
Q

what is the best way to preserve blood?

A

FTA cards

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18
Q

what are sample preservation methods in the field?

A
  • freezing
  • buffers (RNA later, or Lysis buffer)
  • EtOH
  • adding tissue to desiccant
  • FTA cards
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19
Q

what is the worst tissue choice in degraded tissues?

A

organs: too many enzymes and quick autolysis

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20
Q

where is the best DNA on dried/desiccated tissues?

A

ear tips, edges of pelts

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21
Q

if the tissue is clearly rotten what will be in higher amounts? where should you take DNA from?

A

bacterial DNA. plucked hairs, feathers, drilled out pulp of teeth or marrow

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22
Q

what is the correct concentration of DNA for PCR?

A

250pg/uL

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23
Q

Why is DNA negatively charged?

A

because of the phosphate sugar backbone tightly complexed with positively charged histones

24
Q

what is the first step in DNA extraction?

A

dissolve tissue in buffer: breaks cell/nuclear membranes, disrupts DNA -protein complexes, binds multivalent ions

25
Q

During step one of DNA extraction what solutions are needed?

A
  • Urea
  • n-lauroyl sarcosine (or SDS)
  • CDTA (or EDTA)
26
Q

what does n-lauroyl sarcosine do?

A

binds proteins and unfolds them while long hydrophobic aliphatic chains bind to hydrophobic backbones of DNA the charged group makes complex soluble in aqueous buffers

27
Q

what does CDTA do?

A

Chelating agent binding multivalent ions (Mg and Fe)

28
Q

what is step 2 of the DNA extraction?

A

add proteinase-K to digest proteins

29
Q

What happens to cells lysed with DTT?

A

breaks protein disulfide bridges prevalent in hair, nails, horns, claws, antlers, and other structures high in keratin content

30
Q

what is an extra step for materials like bone, teeth and ivory? and why?

A

soaking in EDTA to decalcify prior to extraction

31
Q

what is step 3 in the DNA extraction?

A

separating DNA from digested proteins

32
Q

how is DNA separated from digested proteins

A
  • organic solvents
  • silica to bind DNA
  • magnetic beads bind to DNA
33
Q

In step 3 what was the traditional method of DNA separation?

A

adding organic solvents such as phenol and chloroform to precipitate and denature the proteins

34
Q

When cell lysate is treated with buffer saturated phenol and chloroform what happens?

A

DNA remains in the aqueous phase while cellular proteins extracted into organic phase

35
Q

When do nucleid acids bind to silica?

A

in the presence of chaotropic salts

36
Q

Iron from RBC is a PCR inhibitor how is this fixed?

A

FTA; ACK, and centrifugation

37
Q

dilution is a solution for what inhibitor(s)?

A

melanin (hair), and proteins (not successfully removed in extraction)

38
Q

ethanol is a PCR inhibitor how is this fixed?

A

heat elution with lid off

39
Q

minerals from bone is a PCR inhibitor how is this fixed?

A

decalcification prior to extraction

40
Q

what is swabbed on a fecal sample?

A

mucosal coat because there are fewer inhibitors

41
Q

what are the best fecal samples to collect?

A

fresh or on snow

42
Q

why is species-specific quantification important?

A
  1. estimate efficieny of extraction method; various methods yield different amounts and quality of DNA
  2. it is especially important to dilute samples to known concentration for use in PCR, conserves DNA, and reduces the possibility of introducing inhibitors which may be present in the extracted solution
43
Q

What is phenol?

A

an organic solvent that removes proteins it is a benzene ring with an OH on it

44
Q

what does the ethanol step do during phenol-chloroform extraction?

A

dehydrates the DNA and makes it precipitate

45
Q

how do you rehydrate the pellet?

A

add in 70% ethanol

46
Q

why do you need to get rid of all the ethanol during a phenol-chloroform extraction?

A

because it is a PCR inhibitor

47
Q

T/F: ethanol prevents DNA from dissociating

A

False, you will always lose some DNA during this protocol.

48
Q

why do you add a weak solution of EDTA in the last step?

A

because it is a preservative

49
Q

why might an aqueous phase be slightly red?

A

caused by some iron in the solution.

50
Q

what is a chaotropic salt?

A
51
Q

what do the magnetic beads do?

A

prequantify how much DNA you might get

52
Q

why do you have a negative and positive control?

A

negative says if reagents are contaminated (at the start), positive evaluates efficiency of extraction (at the end)

53
Q

in negative if there is any DNA amplification, is this bad?

A

not the case FSC will accept 1:10 ratio since its kinda impossible to get 100% clean

54
Q

why is melanin hair dilution kinda bad?

A

because its not super concentrated so it might just dilute out the small amount of DNA in the hair

55
Q

T/F:

A

Wildlife Forensic Science (3 decks)

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