LESSON 4: NUCLEIC ACID EXTRACTION METHODS Flashcards
- Release of nucleic acid from the cell
- Should be free of contaminants (e.g. proteins,
carbohydrates, lipids)
NUCLEIC ACID EXTRACTION
___of extraction and cost effectivity
Efficiency
____of the quantity extracted for downstream
applications
Sufficiency
____ of final nucleic acid extract
Purity
USES OF NUCLEIC ACID EXTRACTION
what are those 3?
Scientific Research
Medical
Forensic
DNA EXTRACTION - 5 STEPS
- Pre-treatment and Washing
- Cell Lysis
- Removal of Contaminants
- DNA Precipitation
- DNA RESUSPENSION
Cell Lysis - physical steps
- Grinding
- Shearing
- Bead beating
- Freeze-thaw
Solution-Based
- ____- Alkali (NaOH), Detergents (SDS, CTAB), Chaotropic agents (EDTA)
- ____- Proteinase K, Lysozyme, Lipase
Chemical
Enzymatic
Removal of Contaminants
+ proteases/ detergents
PROTEINS
Removal of Contaminants
broad-spectrum serine protease that can also
degrade nucleases
PROTEINASE K
Removal of Contaminants
makes proteins anionic
SDS (Sodium dodecyl sulfate)
cluster of solvent molecules
surrounding and attaching to solute molecules in
solution
Solvation shell
Also called “salting out”
INORGANIC - under DNA Precipitation
RNA EXTRACTION
How is it different?
- 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
ORGANIC RNA EXTRACTION
ADVANTAGES and DISADVATAGES
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