LESSON 4: NUCLEIC ACID EXTRACTION METHODS Flashcards

1
Q
  • Release of nucleic acid from the cell
  • Should be free of contaminants (e.g. proteins,
    carbohydrates, lipids)
A

NUCLEIC ACID EXTRACTION

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2
Q

___of extraction and cost effectivity

A

Efficiency

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3
Q

____of the quantity extracted for downstream
applications

A

Sufficiency

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4
Q

____ of final nucleic acid extract

A

Purity

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5
Q

USES OF NUCLEIC ACID EXTRACTION
what are those 3?

A

Scientific Research
Medical
Forensic

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6
Q

DNA EXTRACTION - 5 STEPS

A
  1. Pre-treatment and Washing
  2. Cell Lysis
  3. Removal of Contaminants
  4. DNA Precipitation
  5. DNA RESUSPENSION
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7
Q

Cell Lysis - physical steps

A
  1. Grinding
  2. Shearing
  3. Bead beating
  4. Freeze-thaw
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8
Q

Solution-Based

  • ____- Alkali (NaOH), Detergents (SDS, CTAB), Chaotropic agents (EDTA)
  • ____- Proteinase K, Lysozyme, Lipase
A

Chemical
Enzymatic

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9
Q

Removal of Contaminants

+ proteases/ detergents

A

PROTEINS

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10
Q

Removal of Contaminants

broad-spectrum serine protease that can also
degrade nucleases

A

PROTEINASE K

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11
Q

Removal of Contaminants

makes proteins anionic

A

SDS (Sodium dodecyl sulfate)

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12
Q

cluster of solvent molecules
surrounding and attaching to solute molecules in
solution

A

Solvation shell

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13
Q

Also called “salting out”

A

INORGANIC - under DNA Precipitation

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14
Q

RNA EXTRACTION
How is it different?

A
  • RNA is not as stable as DNA
  • RNAses are ubiquitously present in the environment and hardy
  • While dissociating tissue, sample must be frozen in liquid nitrogen or immersed in buffer to inactivate intracellular RNAses
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15
Q

ORGANIC RNA EXTRACTION
ADVANTAGES and DISADVATAGES

A

AD
* Rapid elimination of nucleases
* Stabilization of RNA
* Can be used for smaller or larger samples
* Protocols are wellestablished and more routinely used
DISAD
* Time-consuming
* Laborious
* Makes use of hazardous reagents

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16
Q

SOLID PHASE EXTRACTION
ADVANTAGES and DISADVATAGES

A

AD
* Simple and straightforward
* Used in kits, making them highly convenient
* Usable in largescale extractions and automated methods
DISAD
* Large amounts of sample cannot be used in one go as it can clog the membranes
* Expensive

17
Q

QUANTITY AND QUALITY
WHY ASSESS?

A
  • To check the concentration and purity of yield
  • Determines success of isolation
  • Calculations used for downstream applications
18
Q

Makes use of Beer-Lambert’s Law to determine
concentration of DNA

A

SPECTROPHOTOMETRY

19
Q
  • Nucleic acids have conjugated double bonds in their
    purine and pyrimidine rings
  • Maximum absorbance at ___ nm
A

260

20
Q

NANODROP SPECTROPHOTOMETRY
Requires only ___ of sample instead of needing
50-75 uL

A

1-2 uL

21
Q

Separation method based on size under the
influence of an electric current (max: 15 V)

A

AGAROSE GEL ELECTROPHORESIS

22
Q

PURITY OF NUCLEIC ACID
Determined most commonly by ____

A

UV spectrophotometry

23
Q

____ ratio often used to assess purity

A

A260/A280

24
Q

Absorbance at ___ nm for nucleic acids
Absorbance at ___ nm for proteins

A

260 nm
280 nm

25
Q

Additional ___ nm for other contaminants
and ___ nm for phenols

A

A230
270