LESSON 4: NUCLEIC ACID EXTRACTION METHODS

Question 1

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

Answer 1

NUCLEIC ACID EXTRACTION

Question 2

___of extraction and cost effectivity

Answer 2

Efficiency

Question 3

____of the quantity extracted for downstream
applications

Answer 3

Sufficiency

Question 4

____ of final nucleic acid extract

Answer 4

Purity

Question 5

USES OF NUCLEIC ACID EXTRACTION
what are those 3?

Answer 5

Scientific Research
Medical
Forensic

Question 6

DNA EXTRACTION - 5 STEPS

Answer 6

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

Question 7

Cell Lysis - physical steps

Answer 7

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

Question 8

Solution-Based

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

Answer 8

Chemical
Enzymatic

Question 9

Removal of Contaminants

+ proteases/ detergents

Answer 9

PROTEINS

Question 10

Removal of Contaminants

broad-spectrum serine protease that can also
degrade nucleases

Answer 10

PROTEINASE K

Question 11

Removal of Contaminants

makes proteins anionic

Answer 11

SDS (Sodium dodecyl sulfate)

Question 12

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

Answer 12

Solvation shell

Question 13

Also called “salting out”

Answer 13

INORGANIC - under DNA Precipitation

Question 14

RNA EXTRACTION
How is it different?

Answer 14

• 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

Question 15

ORGANIC RNA EXTRACTION
ADVANTAGES and DISADVATAGES

Answer 15

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

Question 16

SOLID PHASE EXTRACTION
ADVANTAGES and DISADVATAGES

Answer 16

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

Question 17

QUANTITY AND QUALITY
WHY ASSESS?

Answer 17

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

Question 18

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

Answer 18

SPECTROPHOTOMETRY

Question 19

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

Answer 19

260

Question 20

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

Answer 20

1-2 uL

Question 21

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

Answer 21

AGAROSE GEL ELECTROPHORESIS

Question 22

PURITY OF NUCLEIC ACID
Determined most commonly by ____

Answer 22

UV spectrophotometry

Question 23

____ ratio often used to assess purity

Answer 23

A260/A280

Question 24

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

Answer 24

260 nm
280 nm

Question 25

Additional ___ nm for other contaminants
and ___ nm for phenols

Answer 25

A230
270