Basic On Molecular Genetic- Isolation and Purification Of DNA; Part 4 (Plant Cells).

DNA Purification In Plants.

By Sekou SesayPublished 5 months ago • 4 min read

Basic On Molecular Genetic- Isolation and Purification Of DNA; Part 4 (Plant Cells).

INTRODUCTION

Similar to animal cells, plant cells are also living things with a nucleus containing genetic material; some main differences that distinguish a plant cell from an animal cell are: plant cell has a cellulose cell wall, large vacuoles, and chloroplasts, to mention but a few, but animal cells do not have these. Due to the presence of a cell wall in plant cells, different enzyme is used to disrupt its cell wall when isolating and purifying DNA, and instead of the chemical method that is used during DNA extraction and purification in animal cell, a mechanical method is used for plant cells.

1. Enzymatic Method

In this method, cellulose-pectinases are used to facilitate the disruption of the cellulose cell wall of plant cells and to some extent the protoplasm. Cetyltrimethylammonium bromide (CTAB), beta-mercaptoethanol, phenol, and NaCl can also be used in the process; listed below are the various steps involved in DNA isolation and purification from plant cells.

* Simple DNA Extraction ProcessThe prepared plant cell extract is placed in a test tube containing the necessary tool, a tris buffer, the enzyme cellulose-pectinases, the salt NaCl, and the agent cetyltrimethylammonium bromide (CTAB).

* The cellulose-pectinases disrupt the cell wall and the proteoplast membrane surrounding the plant cell's nucleic content, releasing the nucleic acid into the tris buffer.

* Because DNA is pH sensitive, a tris buffer is added to maintain the solution's pH preventing DNA's denaturation; cetyltrimethylammonium bromide (CTAB) is added.

* The main purpose of CTAB is to form a CTAB+ Nucleic acid complex; the complex precipitates and forms a coagulant at the bottom of the test tube while carbohydrate and protein form a complex at the top.

* Phenol is added to the mixture, it precipitates the carbohydrate and protein components at the top of the test tube which can then easily be collected and discarded since we are interested in the DNA.

* With the CTAB + Nucleic acid complex at our disposal, NaCl is added to dissolve the complex.

* Ribonuclease treatment is performed to get rid of the RNA molecules and ethanol is added to precipitate and collect the DNA molecules; because DNA-ethanol concentration is high, DNA is watched in an aqueous solution to dilute it leaving pure DNA for collection.

2. Mechanical Method

This is one other method used by laboratories to extract and purify DNA molecules from plants. A mortar and pestle, test tubes, tris buffer, liquid nitrogen, and lysis agents are used during this process. Below are the steps involved when extracting and purifying DNA from plant cells.

* In this process, let us consider the leaflets of a plant; fresh leaflets are collected from a plant of interest and washed under running water.

* The washed leaflets are stored in liquid nitrogen for some time after which they are grinded in a mortar using a pestle; the main purpose for storing leaflets in liquid Nitrogen is to soften them for easy grinding.

* During the process of grinding, liquid nitrogen is also added in intervals; this helps to facilitate the grinding process of the leaflets into a powdery form and also to deactivate DNAase.

* When leaflets are properly grinded, a lysis agent is added to the leaflets while still being grind; lysis agent such as carbohydratenases disrupts and breaks the cell walls of the leaflets thereby releasing their genetic materials.

* The plant cell extract is then placed in a test tube containing a tris buffer; tris buffer is to maintain the pH of the solution to prevent DNA from denaturing since DNA is pH sensitive.

* The test tube containing the grinded plant cell extract is centrifuged; cell debris settles at the bottom of the tube while nucleic acid components and proteins settle at the top of the separated mixture.

* Nucleic acids together with proteins are collected in a separate test tube and cell debris is discarded.

* Phenol is added to the collected mixture thereby coagulating proteins at the bottom of the tube.

* DNA and RNA are collected and transferred into a separate test tube; ribonucleases are added to separate and remove RNA and ethanol is added to precipitate the DNA components that remain. Concentrated DNAs are diluted several times in an aqueous solution and pure DNAs are obtained.