DNA ISOLATION AND PURIFICATION

DNA Isolation / Extraction

One of the most critical part in molecular techniques because downstream procedures and analysis relies on the quality of the DNA used for the assay

DNA Isolation / Extraction

It refers to the process of separating DNA from other cellular materials such as proteins and membranes.

• Plasmid DNA

• Viral Nucleic Acids

• Genomic DNA from Blood and Biological Fluids

• Genomic DNA from Tissue and Cells

• Genomic DNA from Forensic Samples

• Genomic DNA from Plant and Fungi

• Genomic DNA from Food and Feed

• Ancient DNA

Sources of DNA

DNA isolation

It requires the removal various cellular materials to free up the DNA material inside the cell.

Inhibit DNA analysis

DNA needs to be separated from other cellular materials because?

• Cell lysis / disruption

• To expose DNA

• It refers to the breaking open of cells.

• It is done because?

Detergent

How are membrane lipids removed?

Protease

How are proteins removed?

Ice-cold ethanol or isopropanol

How is DNA precipitated?

• Slightly alkaline buffer

• Ultra-pure water

Where is DNA resolubilized?

Chelating agents

It binds divalent cations to stop DNAse activities.

• Protease

• Precipitation by sodium/ ammonium acetate

• Extraction by phenol-chloroform mixture

How are cellular and histone proteins bound to DNA removed?

• Lysis of cells to free the DNA material,

• Separation of DNA from other cell components

• Isolation of the DNA

3 basic steps of DNA extraction

• Separate WBCs from RBCs, if necessary

• Lyse WBCs or other nucleated cells

• Denature/digest proteins

• Separate contaminants (e.g., proteins, heme) from DNA

• Precipitate DNA if necessary

• Resuspend DNA in final buffer

Basic steps in isolating DNA

Chemical lysis

It is the simplest and cheapest method that can be employed.

• Chaotropic agents

• Detergents

• Salts

• Strong bases

Chemical lysis include what agents / reagents?

Enzymes

Examples include proteinase K and lysozymes which target proteins in the cell or organism to induce lysis.

• Lysosomes

• Gram positive bacteria (esp. peptidoglycan)

• Lysozymes are secreted by?

• It targets?

Mortar and Pestle

The most common method of mechanical lysing of cells.

• Bacterial spores

• Fungal cells

• Protozoan oocysts

What organisms are more resistant to lysis?

• Histones

• Other accessory proteins

The isolation of DNA is prone to protein contamination

due to the presence of?

• Sodium dodecyl sulfate

• Triton X-100

What detergents can be used to remove proteins from DNA through solubilization?

Chaotropic acids

• Guanidine hydrochloride

• Guanidinium thiocyanate

What chemicals can be used to denature proteins and employed to lyse bacteria and yeasts?

Serine protease Proteinase K

This is a common protease, originally extracted from the fungus Engyodontium album that breaks down proteins into smaller molecules by cleaving peptide bonds and is thus useful in reducing the protein background as well as in the lysis of cells.

Engyodontium album

Serine protease proteinase K originally extracted from this fungus.

Protease digestion

This removes unwanted proteins.

True

Modified True or False.

Isolation of the DNA

After lysis, the DNA must be isolated from other samples or cellular materials.

• Liquid-liquid extraction

• Solid-phase extraction

Two separation methods that can be employed in isolation of the DNA.

Liquid-liquid Extraction

This is an organic extraction, which involves liquid phase separation and precipitation

False.

The use of liquid phase extraction is based on the differential solubility of DNA from other molecules in immiscible liquids.

Modified True or False.

The use of liquid phase extraction is based on the differential solubility of DNA from other molecules in miscible liquids.

Phenol

Mixed with Chloroform and Isoamyl alcohol

What are the primary organic solvents used in the Liquid-liquid extraction technique? and is/are usually mixed with?

25:24:1 (Respectively)

Ratio of Phenol, Chloroform, and Isoamyl alcohol (Respectively) in Liquid-liquid Extraction?

Process of Conventional Chemical Liquid/Liquid Extraction?

• False.

  • The addition of Chloroform or Isoamyl alcohol aids in the partitioning of the different phases and prevents foaming

• False

  • DNA is then precipitated from the aqueous phase using alcohol

• True

Modified True or False.

Liquid-liquid Extraction

• The addition of Phenol aids in the partitioning of the different phases and prevents foaming

• DNA is then aspirated from the aqueous phase using alcohol

• Although it is an effective method for DNA separation, it is manual laborious. It poses risks due to the hazards posed by the chemicals used and wastes generated by the procedure.

Precipitation of DNA

This is a liquid phase method that yields a relatively pure product and concentrates the DNA

• Ethanol

• Isopropanol

This alcohol is usually used to precipitate DNA

• High Concentration of Salt (0.1-0.5M)

  • Sodium chloride

  • Ammonium chloride

  • Ammonium acetate

Other mixture/chemical that can also be used to precipitate DNA

Buffer or Ultrapure Water

After precipitation, the sample is centrifuged to concentrate and separate the DNA into pellet and is then dried and resuspended in?

Solid-phase Extraction

This process involves DNA separation either by size or affinity.

Is commonly performed because it is less hazardous, easier to perform, adaptable for automation and suitable for high-volume sample separation.

• Less hazardous

• Easier to perform

• Adaptable for automation

• Suitable for high-volume of sample separation

Why is Solid-phase Extraction preferred over Liquid-liquid Extraction?

1. Lysis

2. Binding

3. Washing

4. Eluting

The basic steps in a solid phase extraction lysis

• Gel Filtration

• Ion-exchange Chromatography

• Affinity Chromatography

Techniques used in Solid-phase extraction

Gel Filtration

This technique separates the components of a mixture on the basis of molecular size, and is the simplest form of chromatography for oligonucleotide purification

Sephadex

What common gel matrix is used in Gel filtration?

1. Equilibration

2. Binding

3. Washing

4. Elution

   ^searched ko lang to^ but refer to the image na lang po

Process of Gel filtration

1. Larger molecule

2. DNA

3. Small-molecule impurities

In Gel Filtration, What is/are

1. Elutes more quickly through the column

2. Elutes quickly after the previous asked ^

3. retained on the column

To the right

What is the flow of mobile phase in Gel filtration?

Ion-exchange Chromatography

This technique is based on the selective binding of negatively charged DNA to surfaces with charged groups.

Charged DNA

This exchange places with ions and bind to the surface by charge and unbound substances are washed away.

Side or walls of the tube

Where does DNA in common anion exchange resin binds/attaches?

Diethylaminoethyl cellulose (DEAE-C)

What is a common anion exchange resin used in Ion-exchange Chromatography?

Salt

What separates DNA molecule in Ion-exchange particles?

Protein+RNA

What is/are discarded in DNA purification by ion-exchange chromatography?

Affinity Chromatography

This technique uses reversible adsorption of DNA to surfaces such as silica and is the most common method for DNA extraction/isolation

DNA binds to Silica Surfaces

In Affinity Chromatography, when specific binding conditions are met, especially upon addition of chaotropic salts, where does DNA binds to?

True

Modified True or False.

Affinity Chromatography, Is used for many DNA preparation procedures and is commonly used in automated methods.

because of complex hydrogen bond formation between the silica and DNA surfaces in the presence of chaotropic salts or alcohols at high concentration and low pH (below pH 7) resulting to binding

In Affinity Chromatography, Why does Linear DNA adsorbs lengthwise to silica surfaces?

False.

Silica and DNA surfaces are both negatively charged, thus, the binding is due to adsorption in high ionic strength conditions and hydrogen bonding when water is removed from the surfaces

Modified True or False.

Silica and DNA surfaces are both negatively and positively charged, thus, the binding is due to adsorption in high ionic strength conditions and hydrogen bonding when water is added from the surfaces

DNA

What is released when salt or alcohol is removed and the surfaces are hydrated?

Silica-Gel-Membrane Technology

This is based on a simple bind-wash-elute procedure.

Polysaccharides and Proteins

In Silica-Gel-Membrane Technology, what are not adsorb and are removed instead?

Pure Nucleic Acids

In Silica-Gel-Membrane Technology, after wash step, these are eluted under low- or no-salt conditions in small volumes, ready for immediate use without further concentration.

• Nucleic acids

• Presence of Chaotropic Salts

In Silica-Gel-Membrane Technology, these are adsorbed to the silica-gel membrane in the presence of? which remove water from hydrated molecules in solution.

• Fast

• Convenient

• Economical

• No time-consuming phenol—chloroform extractions or alcohol precipitations

Advantages of Silica-Gel-Membrane Technology

Bacteria, Prokaryotes

DNA extraction from different sources:

The simplest cells are composed of a lipid bilayer outer membrane and a cytoplasm containing circular chromosomes, proteins, inorganic salts, metal ions, CHO, and other cellular components.

• lipid bilayer outer membrane

• cytoplasm containing:

  • circular chromosomes

  • proteins

  • inorganic salts

  • metal ions

  • CHO

  • other cellular components.

DNA extraction from different sources:

Bacteria, prokaryotes, are composed of?

Prokaryotic Cells

Lysis of these releases chromosomal material where DNA can be extracted.

Isolation of genomic DNA from bacteria

This is traditionally achieved using organic extraction of the soluble DNA while the insoluble debris is removed

1. True

2. False

• Many plant species have a high content of polysaccharides and polyphenols which are not removed by phenol extraction

  3. False

• It is much easier to lyse and extract genomic DNA from human and animal cells because of the absence of cell walls and chloroplasts

Modified True or False.

1. The cell walls of plants for example complicate the DNA extraction process by presenting a barrier that makes it more difficult to lyse the cells

2. Many plant species have a low content of polysaccharides and polyphenols which are removed by phenol extraction

3. It is much easier to lyse and extract genomic DNA from human and animal cells because of the presence of cell walls and chloroplasts

• True.

• False

  • Another major difference from prokaryotes lies largely on the presence of membrane-enclosed organelles in the cytoplasm

Modified True or False.

1. Most animal cells do not have a cell wall like microbial cells, and consequently, are easier to lyse and can be lysed using only detergents

2. Another major difference from prokaryotes lies largely on the absence of membrane-enclosed organelles in the cytoplasm

• Resists damage

• Neutralizes Nucleases

• Preserves integrity

• Accentuates lysis of reagents

Why is EDTA preferred over any other tubes in collecting blood samples?

False.

Oral swab typically yields 100 to 1500 ng of DNA per swab while tissue samples may yield 50 to 500 ng of DNA per gram of sample

Modified True or False.

Oral swab typically yields 1000 to 15000 ng of DNA per swab while tissue samples may yield 100 to 2500 ng of DNA per gram of sample

• 20000ng/mL - 40000ng/mL

• 250ng/cm2 - 500ng/cm2

• 150000ng/mL - 300000ng/mL

• 10ng/swab - 3000ng/swab

• 1ng/root - 750ng/root

• 1ng/root - 10ng/root

Typical DNA Amounts that may be extracted from Biological Materials: Amount needed?

• Liquid Blood

• Blood Stain

• Liquid Semen

• Post-coital Vaginal Swab

• Plucked Hair (w root)

• Shed Hair (w root)

• 1000ng/mL - 10000ng/mL

• 100ng/swab - 1500ng/swab

• 1ng/mL - 20ng/mL

• 3ng/mg - 10ng/mg

• 50ng/mg - 500ng/mg

Typical DNA Amounts that may be extracted from Biological Materials: Amount needed?

• Liquid Saliva

• Oral swab

• Urine

• Bone

• Tissue

Plasmid DNA

Used for a number of downstream procedures such as transfection, sequencing, screening, clones, restriction digestion, cloning, and PCR

• Transfection

• Sequencing

• Screening

• Clones

• Restriction digestion

• Cloning

• PCR

Plasmid DNA downstream procedures

Antibiotic Resistant Gene

Plasmids are typically designed to contain? allowing selection of bacteria containing the plasmids during growth of colonies or cultures

Extraction of Plasmids

Typically performed from bacterial liquid cultures and there are many methods available for plasmid DNA isolation that are capable of isolating large amounts of high-quality DNA

Cycle of Plasmid DNA

Alkaline Lysis Method

Where does the most common method for plasmids are based on?

To take advantage of the alkaline denaturation of plasmid and bacterial chromosomal DNA and the selective renaturation of plasmid DNA following neutralization of the solution

Principle of Alkaline Lysis Method?

The small-scale mini preparation

Plasmid of DNA that is commonly used to screen bacterial clones for the presence of recombinant DNA inserts?

• Ficoll-directed Density Gradient Through Centrifugation

• Quick Extraction Through Proteinase K and Phenol

DNA Extraction from Whole Blood

Ficoll-Directed Density Gradient Through Centrifugation

Fresh blood is collected in the presence of anticoagulants such as EDTA or citrate

Discarded

• Ficoll

• Erythrocytes/Granulocytes

Recovered for further analyses

• Plasma

• WBC

In Ficoll-Directed Density Gradient Through Centrifugation, What is usually discarded and recovered for further analyses after centrifugation of whole blood?

• Tris

• EDTA

• Sodium dodecyl sulfate (SDS)

• MgCl2

• Proteinase K

Quick Extraction Through Proteinase K and Phenol

For the proteinase K protocol, what are mixed with whole blood in the presence of high salt for overnight digestion at 37C

Proteinase K

This is a broad-spectrum serine protease.

Commonly used in molbio to digest protein and remove contamination from preparations of nucleic acid.

• Protease K

• Endopeptidase K

Other name of Proteinase K?

False.

The addition of proteinase K to nucleic acid preparations rapidly inactivates nucleases that might otherwise degrade the DNA or RNA during purification.

Modified True or False.

Addition of proteinase K to nucleic acid preparations rapidly activates nucleases that might otherwise degrade the DNA or RNA during purification.

SDS

What denaturant stimulates the activity of enzymes toward native proteins?

• SDS

• Urea

• Trypsin

• Chymotrypsin Inhibitors

What chemical/s denature proteins?

• Tris-HCl (pH 8)-saturated phenol and water

• 4hr at room temp

Quick Extraction Through Proteinase K and Phenol

For Phenol Method, Whole blood is mixed with? followed by shaking for how long and what temp?

Aqueous Phase

Quick Extraction Through Proteinase K and Phenol

After centrifugation, what is collected for further standard purification?

Phenol-chloroform extraction

Is a liquid-liquid extraction technique used in molecular biology for isolating DNA, RNA, and protein?

Equal volumes of a phenol:chloroform mixture and an aqueous sample are mixed, forming a biphasic mixture. This method may take longer than a column- based system such as• the silica-based purification, but has higher purity and the advantage of high recovery of RNA

Principle of Phenol-chloroform extraction

Phenol-chloroform extraction

This method relies on phase separation by centrifugation of a mix of the aqueous sample and a solution containing water- saturated phenol, chloroform and a chaotropic denaturing solution (guanidinium thiocyanate) resulting in an upper aqueous phase and a lower organic phase (mainly chloroform).

DNA Extraction from Dry Blood Spots:

Chelex-100 Soaking in Saponin Process?

DNA Extraction from Dry Blood Spots:

Chelex-100 Soaking in PBS Process?

DNA Extraction from Dry Blood Spots:

Chelex-100 No soaking Process?

DNA Extraction from Dry Blood Spots:

InstaGene Matrix Soaking in PBS process?