Overview

• Post By : Sonali Pandey


• Source: Microbioz India


• Date: 02 Sep,2021

A biological sample obtained in the form of a blood or semen or tissue sample from a known individual contains a number of substances beside DNA. DNA molecules must be separated from its other cellular components. Cellular proteins that package and protect DNA in the environment of the cell can inhibit the ability to analyse the DNA. Therefore, DNA extraction methods have been developed to separate proteins and other cellular materials from the DNA molecules.

Choosing the appropriate protocol

The ideal extraction method should fit the following criteria-

1. It should be- sensitive, consistent, quick and easy to use.
2. It should also possess- minimum risk to users, avoid possible cross contamination of samples and
3. Most importantly, The DNA extraction technique chosen should be able to deliver pure DNA samples ready to be used in downstream molecular applications.
4. The quality and quantity of genomic DNA extracted from blood samples is a key feature most facilities consider when choosing a protocol.

Some primary techniques for DNA Extraction used in DNA laboratories are-

1. Organic extraction (Chloroform-Phenol DNA extraction method).
2. Chelex extraction.
3. FTA Paper extraction protocol.
4. Solid phase extraction etc.

Experimental work – Where many things can go wrong

Some common mistakes that we do during DNA extraction are-

General laboratory safety measures, settings and ergonomics

Regardless of the biosafety level of the laboratories it is important for a person to always ensure that safety comes first. This includes wearing protective gloves, disposing off contaminated gloves after use in accordance with applicable laws and good practices, use of appropriate personal protective equipment (PPE) as a bare minimum such as lab coats.

Lessen the opportunity for cross contamination in extraction procedure-

All samples must be carefully handled regardless of the DNA extraction method to avoid sample to sample contamination or introduction of extraneous DNA.

Equipments and errors that could be possible during DNA extraction-

All the equipment should be calibrated as per the Laboratory guidelines. Pipettes are the key instrument that is used in DNA Extraction and it can cause error while Pipetting the samples as it can cause sample to sample contamination if not properly handled even if we cannot concentrate towards pipetting it can cause errors like skipped or double loading of chemicals to the sample.

To have appropriate knowledge about chemical’s property that can cause harms to oneself like-

1. Phenol- Corrosive, irritant and combustible.
2. Chloroform- Irritant, reproductive toxin and Carcinogen.
3. Isoamyl alcohol- Irritant and combustible.
4. Isopropanol- Irritant and flammable.

Knowledge of cautious handling of chemicals-

1. During work there is something that you should be cautious of are
2. Avoid inhalation of chemicals and use face masks as Chloroform is toxic if inhaled, high levels of Chloroform can cause damage to CNS, Liver and kidneys.
3. Avoid contact with the chemicals, double gloves are highly recommended and skin exposure to large amounts of chloroform can cause sores. Phenol is readily absorbed through the skin and causes severe burns to the eyes and skin.
4. If a pregnant woman is exposed to Chloroform, she may be at higher risk of miscarriage or birth of a child with birth defects.

Factors that can affect the DNA yield-

1. There are some factors which on being avoided by Laboratory personnel can be the reason for lower yield of DNA quantity like:
2. Incomplete lysis of the sample,
3. Loss of DNA during phase separation if you are using phenol chloroform extraction procedure
4. And if you are using a spin column kit, proteinase-k digestion and incubation temperature/duration is critical.

Proper storage of extracted DNA-

Extracted DNA is typically stored at -20°C or even at -80°C for long term storage to prevent nuclear activity. Nucleases are the protein enzymes found in cells that degrade DNA to allow for recycling of the nucleotide components.

References:

1. http://www.ehs.harvard.edu
2. http://ehs.berkeley.edu
3. https://www.google.com/url?sa=t&source=web&rct=j&url=https://www.uthsc.edu/research/safety/phenol-chloroform-trlzol-waste-disposal.
4. https://www.thermofisher.com › How to Use Phenol / Chloroform for DNA Purification | Thermo Fisher Scientific – IN