Nucleic Acid Extraction and Quantification

Nucleic acid extraction

Process of isolating and purifying DNA or RNA from a sample

Nucleic acid extrcation

Typically involving cell lysis, protein removal, and purification of the nucleic acid

Nucleic acid extraction

Purpose: to release nucleic acid from the cell for use in other procedures

Nucleic acid extraction

Purpose: must be free from contamination with protein, carbohydrates, lipids, or other nucleic acids

Nucleic acid extraction

Use pure nucleic acids for testing

Molecular analysis

Diagnostic testing

Forensic science

Three purpose of nucleic acid extraction

Molecular analysis

Extracted nucleic acids are essential for various molecular biology techniques, including PCR, sequencing, and gene expression analysis

Diagnostic testing

Nucleic acid extraction is crucial for detecting pathogens and other genetic material in clinical samples

Forensic science

DNA extraction plays a vital role in forensic investigations for identifying individuals and linking them to crimes.

Isolation

Routinely isolated from human, fungal, bacterial, and viral sources

Isolation

Pre-treatment to make nucleated cells available

Isolation

Need sufficient sample for adequate yield

Organic isolation

Inorganic isolation

Methods of isolation

Organic isolation method

Purifying DNA by removing contaminants

Organic isolation method

Combination of high salt, low pH and organic mixture of phenol and chloroform

Phenol

Chloroform

Combination of high salt, low pH and an organic mixture of ____ and ______

Organic isolation method

To avoid RNA contamination, add RNAse enzyme that degrades RNA

RNAse enzyme

To avoid RNA contamination, add __________ that degrades RNA

Phenol/Chloroform

Biphasic emulsion forms

Phenol/Chloroform

Hydrophobic layer on bottom has cell debris

Phenol/Chloroform

Hydrophilic layer on top dissolves DNA

Cell debris

Hydrophobic layer on bottom has _______

DNA

Hydrophilic layer on top dissolves _____

Phenol/Chloroform

Remove top layer, add cold ethanol to extract the DNA

Cold ethanol

Remove top layer, add ________ to extract the DNA

Inorganic isolation method

Also called salting out

Inorganic isolation method

Uses low pH and high salt condition to selectively precipitate proteins

Low pH

High salt conditions

Inorganic isolation method uses _____ and _____ to selectively precipitate proteins

Whole blood

Tissue samples

Microorganisms

Sources

Lysis (cell disruption)

Purification (removing contaminants)

DNA recovery (elution)

Basic steps for DNA extraction

Lysis (cell disruption)

To break open cells and release the DNA, along with other cellular components

Mechanical disruption

Enzymatic digestion

Chemical lysis

3 methods of lysis

Mechanical disruption

Using methods like sonication or bead beating to physically break open cells

Enzymatic digestion

Using enzymes like Proteinase K to digest proteins and release the DNA

Chemical lysis

Using detergents or chaotropic agents to disrupt cell membrane

Purification (removing contaminants)

To remove cellular debris, proteins, and other unwanted materials from the DNA

Precipitation

Filtration/Centrifugation

Purification matrix binding

Magnetic bead capture

4 methods for Purification

Precipitation

Using alcohol (like ethanol) to cause the DNA to precipitate out of solution, while leaving other cellular components behind.

Filtration/Centrifugation

Separating the DNA from other cellular components through _____ or _______

Purification matrix binding

Using silica or other matrices to bind to the DNA, allowing for the removal of contaminants through washing and then eluting the DNA

Magnetic bead capture

Using magnetic beads coated with a binding matrix to capture DNA, allowing for the removal of contaminants through washing and then eluting the DNA

DNA recovery (elution)

To collect and store the purified DNA

Elution with buffer

Elution with water

2 methods of DNA recovery (elution)

Elution with buffer

Washing the purified DNA from the extraction matrix (e.g., silica beads) with a specific buffer solution

Elution with water

Washing the purified DNA from the extraction matrix with water

CTAB extraction

Chelex extraction

Proteinase K digestion

Phenol-Chloroform Extraction

Magnetic bead-based extraction

Bead beating

Most commonly used dna extraction procedures

CTAB extraction

Cetylteimethylammonium bromide (CTAB) is a cationic detergent used to extract DNA from plant tissues, effectively removing plant-based debris

Chelex extraction

Chelex resin is used for DNA extraction from various samples, including those with low cell numbers, and is known for its simplicity and efficiency

Proteinase K digestion

Proteinase K is a protease used to digest proteins, including those that may interfere with nucleic acid extraction or downstream analysis

Phenol-Chloroform extraction

Traditional method uses a mixture of ____ and _____ to denature proteins and separate them from nucleic acids

Magnetic bead based extraction

Utilizes _____ that bind to specific nucleic acid sequences, allowing for easy separation and purification using a magnet

Bead beating

Uses ____ to physically disrupt cells and release nucleic acids, particularly useful for samples that are difficult to lyse

Lysis buffers

These buffers disrupt cell membranes and nuclei, releasing DNA

Guanidine isothiocyanate (denaturant)

Sarkosyl (surfactant)

Mercaptoethanol (reducing agent)

Sodium citrate (pH 7.0)

EDTA (magnesium chelator)

Lysis buffer often contains:

Guanidine isothiocyanate (denaturant)

Helps to denature proteins and nucleic acids

Sarkosyl (surfactant)

Helps to solubilize proteins and lipids

Proteins and nucleic acid

Guanidine isothiocyanate helps denature ___ and ____

Proteins and lipids

Sarkosyl helps to solubilized ___ and ___

Mercaptoethanol (reducing agent)

Prevents oxidation of RNA and DNA

Sodium citrate (pH 7.0)

Helps to maintain the pH of solution

EDTA (magnesium chelator)

Prevents nucleases from degrading the DNA

Phenol and Chloroform

Solvents used to separate DNA from proteins and other cellular components

Ethanol or isopropanol

Alcohols used to precipitate (or make insoluble) the DNA, allowing it to be collected

Salt (NaCl)

Helps to stabilize the DNA and promote precipitation