Advanced Techniques in Biology & Medicine
Open Access

DNA Extraction by the Utilization of Phenol Chloroform

Sonal Singh^{* *}Correspondence: Sonal Singh, Department of Molecular Biology, University of Lucknow, Lucknow, India, Email:

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Introduction

Deoxyribonucleic acid is a molecule composed of two polynucleotide chains that coil around one another to make a helix carrying genetic instructions for the event, functioning, growth and reproduction of all known organisms and lots of viruses. DNA and RNA (RNA) are nucleic acids. Alongside proteins, lipids and sophisticated carbohydrates (polysaccharides), nucleic acids are one among the four major sorts of macromolecules that are essential for all known sorts of life. Eukaryotic organisms (animals, plants, fungi and protists) store most of their DNA inside the nucleus as nuclear DNA, and a few within the mitochondria as mitochondrial DNA or in chloroplasts as chloroplast DNA. In contrast, prokaryotes (bacteria and archaea) store their DNA only within the cytoplasm, in circular chromosomes. Within eukaryotic chromosomes, chromatin proteins, like histones, compact and organize DNA. the ability to separate DNA is of essential significance to concentrating on the hereditary reasons for sickness and for the occasion of diagnostics and meds . It’s also essential for completing forensic science, sequencing genomes, detecting bacteria and viruses within the environment and for determining paternity [1].

How to use Phenol/Chloroform for DNA purification

• Cells which are to be studied got to be collected.

• Breaking the cell membranes hospitable expose the DNA alongside the cytoplasm within (cell lysis). Lipids from the cell wall and therefore the nucleus are weakened with detergents and surfactants. Separating proteins by adding a protease (discretionary) & Separating RNA by adding an RNase (discretionary).

• The solution is treated with a concentrated salt solution (saline) to form debris like broken proteins, lipids and RNA clump together. • Centrifugation of the answer, which separates the clumped cellular debris from the DNA.

• DNA purging from cleansers, proteins, salts and reagents utilized during the phone lysis step.

The foremost commonly used procedures are: Ethanol precipitation usually by ice-cold ethanol or isopropanol. Since DNA is insoluble in these alcohols, it'll total together, giving a pellet upon centrifugation. Precipitation of DNA is improved by expanding of ionic strength, normally by adding sodium acetic acid derivation. Phenol–chloroform extraction during which phenol denatures proteins within the sample. After centrifugation of the sample, denatured proteins stay within the organic phase while the aqueous phase containing macromolecule is mixed with chloroform to get rid of phenol residues from the answer [2].

Minicolumn purification that relies on the very fact that the nucleic acids may bind (adsorption) to the solid phase (silica or other) counting on the pH and therefore the salt concentration of the buffer.

Materials required

• Glycogen (20 μg/μL)

• 7.5 M NH4OAc (ammonium acetate)

• Ice bucket • Phenol: Chloroform:isoamyl alcohol (25:24:1)

• 100% ethanol

• Dry ice or a –80°C freezer

• 70% ethanol Cellular and histone proteins sure to the DNA are often removed by having precipitated the proteins with extracted them with a use of phenol & chloroform mixture previous to the DNAprecipitation.

After detachment, the DNA is broken up during a marginally soluble cushion, ordinarily during a TE support, or in super unadulterated water [3].

References

1. Yoshikawa H, Dogruman-Ai F, Turk S, Kustimur S, Balaban N, Sultan N. Evaluation of DNA extraction kits for molecular diagnosis of human Blastocystis subtypes from fecal samples. Parasitol Res. 2011;109(4):1045-1050.
2. Thomas JO, Travers AA. HMG1 and 2, and related ‘architectural’DNAbinding proteins. Trends Biochem Sci. 2001;26(3):167-174.
3. Grosschedl R, Giese K, Pagel J. HMG domain proteins: architectural elements in the assembly of nucleoprotein structures. Trends Genet. 1994;10(3):94-100.