NUCLEIC ACID ISOLATION

DNA

present on the nucleus

RNA

mostly present on the cytoplasm

NUCLEIC ACID EXTRACTION

Main goal: purification of the cell

NUCLEIC ACID EXTRACTION

The initial release of the cellular material is achieved by breaking the cell wall (if present) and cell and nuclear membranes (cell lysis)

NUCLEIC ACID EXTRACTION

Target material is purified

NUCLEIC ACID EXTRACTION

Concentration and purity determination

Miescher (1869)

first isolation DNA from human cells

Meselson and Stahl (1958)

Demonstrate semi conservative replication of DNA

ALKALINE LYSIS

Procedures were used extensively for extraction of 1- to 50-kb plasmid DNA from bacteria during the early days of recombinant DNA technology

ALKALINE LYSIS

can only extract large DNAs

• Viruses

• Bacteria

• Nucleated Cells

Sample types and nucleic acid sources:

Bacteria and Fungi

have tough cell walls that must be broken to allow the release of nucleic acid

Proteinase K

• disintegrated protein; proteins are considered as major contaminant in DNA extraction and isolation

• with the addition of alkaline extraction

Lysozyme

Lyse cell wall; used for oxidative bursting of cells

Lysozyme

After using this, boiling extraction is to be done [chemical treatment]

1% Sodium Dodecyl Sulfate

• Acts as a detergent that reduces the surface tension of the cell; if the surface tension is reduced, the cell can easily be lysed

MECHANICAL DISRUPTION

• Bones or teeth are being physically destroyed

MECHANICAL DISRUPTION

If muscle tissue is used as the sample, you have to do ???

Grinding using mortar and pestle

Examples of Mechanical Disruption

BOILING EXTRACTION

• Diluted Sucrose

• Triton X-100 detergent

• Tris Buffer

• EDTA

Exosomes

Small vesicles form by invagination and budding from the inside of cellular endosome vesicles and are secreted by living cells

Exosomes

Used for the detection of mRNA

mRNA

found on exosomes

Liquid Biopsy

Sources of circulating nucleic acids

Liquid Biopsy

Isolation of cell-free nucleic acid requires procedures to concentrate the target nucleic acid before isolation [ex: SARS-CoV 2; nasopharyngeal specimens]

Saliva

can also be used if there’s high viral load

Differential density-gradient centrifugation

whole blood or bone marrow mixed with isotonic saline is overlaid with Ficoll

Ficoll

highly branched sucrose polymer

Ficoll

between the red cell and buffy coat; creating 4 layers; for easy isolation of WBCs; base on density-gradient

Differential osmotic fragility of RBCs and WBCs

Incubation in hypotonic buffer or water will result in the lysis of the RBCs

Fresh or Frozen Tissue

these samples are dissociated before DNA isolation

Frozen Tissue

• Grinding

• Mincing

Fixation

is the preservation of tissue – clearing, dehydration, paraffinization

Rehydration

using Ethanol [in decreasing concentration or amounts until water is back]

1. Organic Isolation Methods

2. Inorganic Isolation Methods

3. Solid-Phase Isolation

DNA Isolation Chemistries

Proteolytic Lysis of Fixed Material

additional methods; done on fixed tissues; after doing this on those kind of samples, pick isolation methods listed.

Rapid Extraction Methods

used for forensic applications [ex. Blood spots]

ORGANIC ISOLATION METHODS

Combination of high salt, low pH, and an organic mixture of phenol and chloroform [equal amounts of phenol and chloroform

ORGANIC ISOLATION METHODS

Isolation of small amounts of DNA from challenging samples such as fungi can be facilitated by pre treatment with Cetyltrimethylammonium bromide (CTAB)

Cetyltrimethylammonium Bromide

Isolation of small amounts of DNA from challenging samples such as fungi can be facilitated by pre treatment with ??

Cetyltrimethylammonium Bromide

Used to degrade the cell wall, particularly the polysaccharide in the cell wall [fungal or bacterial sample]

RNAse

an enzyme that degrades RNA

RNAse

is a common enzyme used in the lab; considered as the common contaminant in the lab

Guanidinium Thiocyanate

lysis buffer contains this to inhibit RNAse

Tris EDTA Buffer

used for storage of DNA for long term storing

deionized water or PCR-grade water

storage solutions used for short-term (24 hrs)

Phenol

extract hydrophilic components; top layer is the aqueous layer

Chloroform

organic layer; extract hydrophobic

DNA

it is hydrophilic because of the sugar phosphate backbone

Also called as “SALTING OUT”

Using high salt solutions instead of phenol and chloroform

Uses low-pH and high salt conditions to selectively precipitate proteins to isolate DNA

INORGANIC ISOLATION METHOD

• DNA can then be precipitated using isopropanol

• Resuspended in TE buffer or water

SOLID-PHASE ISOLATION METHOD

Better to use; extract good quality DNA and safe

SOLID-PHASE ISOLATION METHOD

More rapid and comparably effective DNA extraction

SOLID-PHASE ISOLATION METHOD

Uses Silica-based products - effectively bind DNA in high-salt conditions.

Diatomaceous Earth

source of silica particle

Columns, Beads

They are also used routinely for isolation of cellular DNA in genetics and oncology

Columns, Beads

commonly used to isolate viral and bacterial DNA from serum, plasma, or cerebrospinal fluid.

Elution Buffer

also acts as storage solution

PROTEOLYTIC LYSIS OF FIXED MATERIAL

Isolation of DNA from limited amounts of starting material

PROTEOLYTIC LYSIS OF FIXED MATERIAL

uses fixed tissues and Paraffin-embedded tissues

Chelex

Cation-chelating resin

Rapid Extraction Method

Used for simple extraction of DNA from minimal samples

suspension of 10% chelex resin beads

Acidify the proteins to make the proteins positively charged

Rapid Extraction Method

This method is most commonly used in forensic application [if there’s small amount of sample]

Detergent

It is used in the Lysis in Isolation of Mitochondrial DNA

Proteinase

Treatment used in Isolation of Mitochondrial DNA

Cold Ethanol

Precipitation used in Isolation of Mitochondrial DNA

Water/Buffer

Resuspension used in Isolation of Mitochondrial DNA

Isolation of Total DNA

Chemical extraction or Proteolytic Lysis