Nucleic acid extraction method

To release the nucleic acid from the cell for use in subsequent procedures

Purpose of nucleic acid extraction

Protein

Carbohydrate

Lipids

Other nucleic acid

Target Nucleic acid that should be free in contamination

Lysis

It is must take place in condition it will not damage the nucleic acid

Miescher

Person who first isolated DNA from human cells in 1869 by precipitation, the early routine laboratory procedures for DNA isolation were developed from density gradient centrifugation strategies

Meselson and Stahl

Person who used a method in 1958 to demonstrate semiconservative replication of DNA

1. Breaking open the tissue and cells

2. Removing proteins, lipids, and other contaminants from the nucleic acids

3. transferring the nucleic acid to water or buffer solution that will preserve them without interfering with subsequent work

Steps for nucleic acid extraction

Bacteria and fungi

Viruses

Nucleated cells in suspension (blood and bone marrow aspirates)

Plasma

Tissue samples

types of Sample preparation

Enzymes

Glass beads

Detergent (1% sodium dodecyl sulfate)

Strong base (0.2M NaOH in the presence of tris EDTA)

Glucose

Some bacteria and fungi have tough cell walls that must be broken to allow the release of nucleic acid:

Boiling or alkaline (NaOH)

DNA extracted by

Viruses

Sample preparation that is held by viral dna

White blood cells

Nucleic acid in human blood or bone marrow comes mostly from

Serum

It is rich source of protein, lipids and other molecules but not nucleic acid

Plasma

Free wbcs carrying nucleic acids and cell free nucleic acids are available from

WBCs in blood or bone marrow

Specimen are purified of red blood cells and other blood components by either differential density-gradient centrifugation or differential lysis

Differential density-gradient centrifugation

for whole blood or bone marrow mixed with isotonic saline os overlaid with ficoll

Ficoll

Is highly branched sucrose polymer that does not penetrate biological membrane

Lysis of rbc before wbc

Incubation of whole blood or bone marrow in hypotonic buffer or water will result in the

Centrifugation

WBCs are palleted by

Plasma

Solid tumor and transplanted organ release cells, exosomes and nucleic acid in the bloodstream

Exosomes

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

Contain proteins, lipids, and nucleic acid

Liquid biopsy

These sources of circulating nucleic acid can be detected for purposes of diagnostic and prognostic analyses

May preclude surgical biopsies and allow serial biopsy testing

Tissue sample

Fresh or frozen tissue samples are dissociated before DNA isolation procedures can be started

Human

Fungal

Bacterial

Viral sources

Nucleic acid is routinely isolated from what in the clinical laboratory

Liquid Nitrogen

Grinding the frozen tissue in what to homogenizing the tissue or simply mincing the tissue using scalpel disrupts the whole tissue samples

Xylene

Fixed, embedded tissue may be deparaffinized by soaking in

The mixture if three isomers of dimethylbenzene

Decreasing concentration of ethanol

After xylene treatment, the tissue is rehydrated by soaking it in

Fixed tissue

Alternatively, it may be used directly without dewaxing

Organic isolation

Is accomplished using a combination of high salt, low pH, and an organic mixture of phenol and chloroform

Cetyltrimethylammonium bromide

Isolation of small amount of DNA from challenging samples such as fungi can be facilitated by pretreatment with —; a cationic detergent that efficiently separates DNA from polysaccharide contamination

RNase

An enzyme that degrades RNA can be added at this point, it may also added to the resuspended DNA at the end of the procedure

Biphasic emulsion

Forms when phenol and chloroform added to the hydrophilic suspension of lysed cells

Inorganic isolation

Organic DNA extraction is sometime called salting out, it makes se of low pH and high salt conditions to selectively precipitate proteins, leaving the DNA in solution

Solid phase isolation

More rapid and comparably effective DNA extraction can be performed using solid matrices to bind and hold the DNA for washing

Chelex

Is a cations chelating resin that can be used for simple extraction of DNA from minimal samples

This is most commonly used in forensic application

4-6 hours at 400 celsius to inactivate the RNases

Reusable glassware is seldom used for RNA work, it can be rended RNF, after cleaning glassware must be baked for

Diethyl pyrocarbonate (DEPC)

Can be added to water and buffers except for tris buffer to inactivate RNases permanently. It converts primary and secondary amines to carbamic acid esters

Ribonuclease inhibitor proteins

Can be added directly to RNA preparation, these proteins form a stable non covalent complex with RNases in solution

Ethidium bromide

Sybr green I

Fluorescent dyes bind specifically to DNA and are used to visualize the sample preparation

Ethidium bromide

Sybr green IIn

Dyes that used to detect RNA

Silver stain

Dyes that has been used to detect small amount of DNA by visual inspection

Fluorometry

Called fluorescent spectroscopy, that measures fluorescence related to DNA concentration in association with DNA specific fluorescent dyes

3,5 diaminobenzoic acid 2HCl (DABA)

This dye combines with alpha methylene aldehydes to yield a fluorescent product

Still used to detection of nuclei and as control for hybridization and spot integrity in microarray analyses

Microfluids

Lab on a chip technology has been appliedto nucleic acid quantification and analysis using this