Magnetic particles for the Separation and purification of Nucleic Acids

1. Nucleic Acid Extraction
By: Neeraj Kumar Fauzdar
M.Sc. Biotechnology
Botany Department, UOR,
Jaipur.

2. Contents
• What is Nucleic Acid?
• Why we isolate Nucleic acid?
• What is gene?
• What is enzyme?
• Isolation methods for nucleic acid
• Precautions and best practices for nucleic acid
extraction.

3. Nucleic Acid
Definition A linear sequence of nucleotides linked together by
phosphodiester bond.
History Friedrich Miescher in 1869 isolated what he called
nuclein from the nuclei of pus cells Nuclein was shown
to have acidic properties, hence it became called
nucleic acid.
Types Two types:
• Deoxyribonucleic acid (DNA)
• Ribonucleic acid (RNA)
Occurrence All cells and viruses

4. Nucleic Acid

6. Nucleoside
Phosphate
Nucleotide Polynucleotide
RNA
Nucleic
Acid
DNA
Sugar
+
Nitrogenous
Base
SUGAR
a. Ribose or
b. Deoxyribose
Basic overview of Structure of
Nucleic Acid

7. 3. PURINE
a.Adenine (A)
b.Guanine(G)
2. PYRIMIDINE
a.Cytosine (C)
b.Thymine (T)
c.Uracil (U)
Nitrogenous Bases

8. DNA v/s RNA
Spot the difference
DNA RNA
Acid Name Deoxyribonucleic Ribonucleic
Stability Very stable Less stable
Found in Nucleus Nucleus &
Cytosol (throughout the cell)
Mitochondria (Most eukaryotes)
Plastids (plant cell)
Function Static, Data storage Dynamic, many varied functions
Copier enzyme DNA polymerase RNA polymerase
Structure Long nucleotide chain Short nucleotide chain
Two complementary strands One strand
A-, B-, C-form helix A-form helix only
Backbone Inorganic phosphate
Deoxyribose (D in DNA) Ribose (R in RNA)
Nulceobases Thymine, Cytosine, Adenine, Guanine Uracil replaced by Thymine
EM Radiations Somewhat UV sensitive Relatively UV resistant

10. Gene
Gene is a small segment of DNA which codes for
RNA. So, gene is a transcription unit.
Transcription unit must consist of Promoter Coding
sequence Terminator sequence.

11. Gene Hypothesis
• One gene, One enzyme- This hypothesis states
that all proteins are enzymes (which is not
true).
• One gene, One protein
• One gene, One RNA

12. Enzyme
Enzymes are biological catalysts (also known as biocatalysts) that
speed up biochemical reactions in living organisms.
All known enzymes are proteins. They are high molecular weight
compounds made up principally of chains of amino acids linked
together by peptide bonds.
Examples : DNAse, RNAse, Cellulase, Pectinase DNA Pol., RNA Polymerage

14. General Workflow
Extract
Sample(Cell or
tissue
Lysis of
Buffer (Mild
or Harsh)
Removal of cell
Debris
Removal of other
Bimolecules
Precipitation of DNA
Washing and
Purification of DNA
Isolation method of Nucleic Acid

15. Isolation method of Nucleic Acid
• The purification of DNA and RNA molecules is required to avoid contaminations with
other intracellular components, such as proteins and metabolites.
• Some commonly used methods for DNA and RNA purification are precipitation with
phenol-chloroform or isopropanol or by spin columns with silica gel.
• DNA and RNA can also be isolated from the same biological sample by extracting a
total nucleic acid fraction and dividing it into two parts:
– One of which will be treated with Dnase 1.
– While the other part will be treated with Rnase A to recover RNA and DNA,
respectively.

16. Methods of RNA Isolation
There are three major techniques extensively used for RNA extraction:
1.Organic extraction, such as phenol-Guanidine Isothiocyanate (GITC)-based
solutions,
2.Silica-membrane based spin column technology, and
3.Paramagnetic particle technology.
One of the most commonly used methods is the phenol-GITC-based organic
extraction.
However, RNA samples isolated by this method are frequently contaminated
with proteins and other cellular materials, organic solvents such as phenol-
chloroform, salts and ethanol.

17. 2.Silica-membrane based spin column
technology
• There are different types of spin-column purification material :
Glass fiber, derivatized silica, or ion exchange membrane in column
• Samples are lysed and passed through the membrane using
centrifugal or vacuum force. Wash and elution solutions are
subsequently passed through the membrane, and the sample is
collected into a tube by centrifugation or vacuum.
• Solid phase extraction such as using a spin-column based extraction
method takes advantage of the fact that DNA binds to silica.
• The sample containing DNA is added to a column containing a silica
gel or silica beads and chaotropic salts.
• The chaotropic salts disrupt the hydrogen bonding between
strands and facilitate of the DNA to silica by causing the nucleic
acids to become hydrophobic.
•

18. • This exposes the phosphate residues so they are available for adsorption.
•The DNA binds to the silica, while the rest of the solution is washed out
using ethanol to remove chaotropic salts and other unnecessary
constituents.
•The DNA can then be rehydrated with aqueous low salt solutions allowing
for elution of the DNA from the beads.

19. • Benefits of spin basket formats
• Ability to automate
• Ability to manufacture membranes of various
dimensions
• Drawbacks of spin basket formats
• Propensity to clog with particulate material
• Retention of large nucleic acids such as gDNA
• Fixed binding capacity within a manufactured
format

20. Magnetic particles for the Separation and
purification of Nucleic Acids
• Magnetic particle or beads are the first option to eliminate centrifuge-
dependent steps in the extraction process.
• Magnetic beads make use of different ligands such as antibodies,
antigens, oligonucleotides, , which bind specifically to its target in sample.
• The first magnetic particle used for extraction consisted of an iron-oxide
core covered by functional carboxylic group, which then binds DNA or
RNA.

21. Material Required
• Binding Solution
• Guanidine thiocyanate is a denaturing agent and is used routinely in RNA isolation. It is used
as a storage buffer for whole blood samples. Guanidine thiocyanate inactivates nucleases and is
ideal for storing and freezing fecal samples for DNA studies.
• Binding Beads
• Magnetic particle methods utilize small (0.5–1 µm) particles particles that contain a
paramagnetic core and surrounding shell modified to bind to entities of interest. Paramagnetic
particles migrate when exposed to a magnetic field, but retain minimal magnetic memory once
the field is removed.
• This allows the particles to interact with molecules of interest based on their surface
modifications, be collected rapidly using an external magnetic field, and then be resuspended
easily once the field is removed.
• Samples are lysed in a solution containing RNase inhibitors and allowed to bind to magnetic
particles. The magnetic particles and associated cargo are collected by applying a magnetic field.
After several rounds of release, resuspension in wash solutions, and recapture, the RNA is
released into an elution solution and the particles are removed.

22. PK ( Proteinase K)
Proteins K is used during DNA extraction to digest many contaminating
proteins present. It also degrades nucleases that may be present in DNA
extraction and protects the nucleic acids from nuclease attack.
Procedure

23. Benefits & Drawbacks
• Benefits of magnetic particle–mediated purification
• Paramagnetic particle based RNA isolation systems do not require the use of toxic organic
solvents, are relatively simple, efficient, low cost, and yield total intact RNA with low levels of
contamination from proteins and other cellular materials. However, these methods can often
result in significant levels of genomic DNA contamination.
• No risk of filter clogging
• Solution-based binding kinetics increase the efficiency of target capture
• The magnetic format allows for rapid collection/concentration of sample Ability to automate
• Wide availability of surface chemistries
• Drawbacks of magnetic particles
• Potential carry-through of magnetic particles into eluted samples
• Slow migration of magnetic particles in viscous solutions
• Capture/release of particles can be laborious (Time and Efforts) when performed manually.
• These methods can often result in significant levels of genomic DNA contamination

24. Thank you