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1st Year sem 2: Forensic Identification > V L7: DNA isolation and quantification > Flashcards

V L7: DNA isolation and quantification Flashcards

1
Q

what are the Principle stages of DNA extraction from cells?

A
  • Cell lysis to disrupt cell and nuclear membranes including denaturing and degrading proteins which are abundant in cells and bound to DNA in chromosomes
  • Separating DNA from other cell components including cell debris, proteins, lipids, CHO and RNA
  • Isolation of the DNA into a aqueous phase.
  • Storing DNA for future analyses.
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2
Q

what does cell lysis involve?

A

heating the sample

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3
Q

why do we use ethanol precipitation?

A

because if the DNA is in water it forms a pallet of DNA

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4
Q

a detergent does what?

A

denatures

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5
Q

in what ways can we separate and isolate DNA form other cell components?

A

– Organic or phenol-chloroform method
– Ethanol precipitation
– Silica based extraction (affinity binding) and magnetic separation
– Filtration and centrifugation

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6
Q

what does SDS stand for?

A

sodium dodecyl sulphate

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7
Q

what is SDS?

A

anionic detergent that

denatures proteins.

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8
Q

what is proteinase K?

A

protease (enzyme) that degrades proteins – cleaves peptide bonds.

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9
Q

what does EDTA stand for?

A

Ethylenediamine tetra acetic acid

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10
Q

what does EDTA do?

A

chelating agent, DNase inhibition.

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11
Q

what does DTT stand for?

A

dithiotreitol

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12
Q

what is DTT?

A

reducing agent for disulphide bonds.

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13
Q

what does DNase do?

A

chew up DNA

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14
Q

for DNA storage what temperature is needed?

A

-20 degrees and lower

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15
Q

what tube material is best to store DNA in?

A

PTFE

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16
Q

To store DNA we need maintain ____ ____.

A

pH levels

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17
Q

whats an FTA card?

A

a card containing different chemicals so they can be stored at room temperature

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18
Q

what the organic method for storing DNA?

A

phenol-chloroform method

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19
Q

phenol-chloroform method is not used ______.

A

frequently

20
Q

phenol-chloroform method is used more often on a ___ sample.

A

dirty

21
Q

phenol-chloroform method involves how many steps ?

A

2

22
Q

phenol-chloroform method is like a ____ _____ extraction.

A

liquid - liquid

23
Q

phenol-chloroform method requires the constant removal of a _____ layer.

A

aquaous

24
Q

phenol-chloroform method involves what 3 steps?

A

lysis, solvent extraction and DNA seperation

25
Q

the Chelex method involves how many tubes?

A

1

26
Q

How does the Chelex method work?

A
  1. add chelex resin which inhibits DNAase
  2. add proteinase K +boil to denture and degrade
  3. centrifuge, pellet at bottom to throw away.
27
Q

what exactly does DNase break?

A

phosphodiester bonds found in DNA

28
Q

How do chelating agents inactive DNase?

A

Chelating agents bind with metal ions that are cofactors for DNase, inactivating them.

29
Q

DNA binding method

A
  1. cell lysis + denature proteins, detergent, proteinase K and chaotropic salt.
  2. DNA binds to magnetic beads everything else passess through
  3. beads collected by magnetic force
30
Q

what are FTA cards sued for?

A

to store blood and saliva samples.

31
Q

what do FTA cards contain?

A
  • Contains chemicals that causes cell lysis
  • Protects DNA from nuclease degradation
  • Prohibits microbial growth
32
Q

vaginal swabs, how to pull out male DNA?

A

differential lysis

33
Q

How is differential lysis carried out?

A

SDS EDTA proteinase K, incubate, centrifuge, remove supernatant, spermatozoa are still in tact, add SDS EDTA proteinase K + DTT, centrifuge, disulphide bonds broken, opens membranes of spermatozoa, remove supernatant (male DNA).

34
Q

whats an advantage of bones and hard tissue?

A
  • DNA is protected from degradation

- bone surface can be cleaned with abrasives and bleach or cross-linked with UV

35
Q

what are problems with bones?

A
  • need to be grinded up, hard to obtain homogenous sample

- high PCR inhibitor, high calcium (EDTA can remove it)

36
Q

what can you use to quantify DNA?

A

Ultraviolet spectroscopy

fluorescent dye

37
Q

Ultraviolet spectroscopy gives you the concentration of what?

A

DNA
protein
CHO

38
Q

whats an issue with Ultraviolet spectroscopy ?

A

it can’t differentiate between different types of DNA.

39
Q

Fluorescent dye

_____ to DNA.

A

binds

40
Q

what two dyes are mainly used?

A

– Sybergreen

– Ethidium Bromide

41
Q

whats more sensitive Interchelating Fluorescent Dyes or Ultraviolet spectroscopy?

A

Interchelating Fluorescent Dyes

42
Q

Fluorescence is recorded using what?

A

spectrofluorometer, or gels

43
Q

Interchelating Fluorescent Dyes requires a…

A

calibration graph

44
Q

what is the hybridisation method?

A
  • Sample is denatured and a small spot of sample is immobilised onto a nylon membrane
  • Addition of a 40-nucleotide labelled probe which hybridises to a primate-specific highly repetitive α- satellite D17Z1 locus in the genome – located on chromosome 17
45
Q

whats the best method for DNA quantification?

A

real-time PCR

1st Year sem 2: Forensic Identification (15 decks)

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