DNA isolation is a process that purifies DNA from a sample using physical and chemical methods. It generally aims to separate DNA present in the cell nucleus from other cellular components for purposes like genetic analysis. The main steps of DNA isolation are: 1) preparing a cell extract by lysing cells and removing debris, 2) purifying DNA from contaminants using techniques like ethanol precipitation or phenol-chloroform extraction, 3) concentrating the DNA samples, often via ethanol precipitation, and 4) measuring DNA purity and concentration using UV spectrometry.

1. PRINCIPLES OF DNA ISOLATION
MADE BY: AYESHA MUMTAZ
MSC MICROBIOLOGY

2. INTRODUCTION
 DNA isolation is a process of purification of DNA from sample using a
combination of physical and chemical methods.
 The first isolation of DNA was done in 1869 by Friedrich Miescher.
 Methods used to isolate DNA are dependent on the source, age, and size of
the sample.
 In general, they aim to separate DNA present in the nucleus of the cell from
other cellular components.
 Isolation of DNA is needed for genetic analysis, which is used for scientific,
medical, or forensic purposes.

3.  Scientists use DNA in a number of applications, such as introduction of DNA
into cells and animals or plants, or for diagnostic purposes, in medicine the
latter application is the most common.
 On the other hand, forensic science needs to recover DNA for identification of
individuals (for example rapists, petty thieves, accident, or war victims),
paternity determination, and plant or animal identification.
 Presence of proteins, lipids, polysaccharides and some other organic or
inorganic compounds in the DNA preparation can interfere with DNA analysis
methods.
 They can also reduce the quality of DNA leading to its shorter storage life

4. SOURCES
 Sources for DNA isolation are very diverse.
 Basically it can be isolated from any living or dead organism.
 Common sources for DNA isolation include whole blood , hair, sperm , bones,
nails, tissues, blood stains, saliva , buccal (cheek) swabs, epithelial cells, urine,
paper cards used for sample collection, bacteria, animal tissues, or plants.
 Stored samples can come from archived tissue samples, frozen blood or
tissue, exhumed bones or tissues, and ancient human, animal, or plant
samples.

6. PROCEDURE
 Isolation of DNA basically consists of four major steps.
i. Preparation of a cell extract.
ii. Purification of DNA from cell extract.
iii. Concentration of DNA samples.
iv. Measurement of purity of DNA concentration.

7. 1. PREPARATION OF A CELL EXTRACT:
 To extract DNA from a tissue/cells of interest, the cells have to be separated and the
cell membranes have to be disrupted.
 The "Extraction buffer" helps in carrying out these processes.
 Chemicals such as EDTA (Ethylene Diamine Tetra Acetate) which removes Mg2+ ions
that are essential for preserving the overall structure of the cell membrane, and SDS
(Sodium Dodecyl Sulfate) which aids in disrupting the cell membranes by removing
the lipids of the cell membranes are included in the extraction buffer.
 Having lysed the cells, the final step in the preparation of a cell extract is removal of
insoluble cell debris.
 Cell debris and partially digested organelles etc. can be pelleted by centrifugation
leaving the cell extract as a reasonably clear supernatant

8. 2. PURIFICATION OF DNA FROM CELL EXTRACT.
 In addition to DNA the cell extract will contain significant quantities of
detergents, proteins, salts and reagents used during cell lysis step and RNA.
 A variety of procedures can be used to remove these contaminants, leaving
the DNA in a pure form. • The most commonly used procedures are:
 i. Ethanol precipitation.
 ii. Phenol–chloroform extraction.
 iii. Minicolumn purification.

9. 1. Ethanol precipitation:
 Ethanol precipitation usually by ice-cold ethanol or isopropanol.
 Since DNA is insoluble in these alcohols, it will aggregate together, giving a
pellet upon centrifugation.
 Precipitation of DNA is improved by increasing of ionic strength, usually by
adding sodium acetate.

10. 2. Phenol–chloroform extraction:
 Phenol–chloroform extraction in which phenol denatures proteins in the
sample.
 After centrifugation of the sample, denaturated proteins stay in the organic
phase while aqueous phase containing nucleic acid is mixed with the
chloroform that removes phenol residues from solution
3. Minicolumn purification.
 Minicolumn purification that relies on the fact that the nucleic acids may
(adsorption) to the solid phase (silica or other) depending on the pH and the
salt concentration of the buffer.

11. 3. CONCENTRATION OF DNA SAMPLES
 The most frequently used method of concentration is ethanol precipitation.
 In the presence of salt and at a temperature of -20°C or less, absolute ethanol
will efficiently precipitate polymeric nucleic acids.
 With a concentrated solution of DNA one can use a glass rod to pull out the
adhering DNA strands while for dilute solutions precipitated DNA can be
collected by centrifugation and redissolving in an appropriate volume of
water.

13. 4. MEASUREMENT OF PURITY OF DNA CONCENTRATION.
 DNA concentrations can be accurately measured by UV absorbance
spectrometry.
 The amount of UV radiation absorbed by a solution of DNA is directly
proportional to the amount of DNA sample
 Usually absorbance is measured at 260 nm, at this wave length an absorbance
of 1.0 corresponds to 50 µg of double-stranded DNA per ml.

14.  With a pure sample of DNA the ratio of the absorbencies at 260 nm and 280
nm (A260/A280) is 1:8.
 Ratios of less than 1:8 indicate that the preparation is contaminated, either
with protein or with phenol.