Separation science in my lab research

1. Name Mesele Tilahun Belete Course Instrumental analysis
Student ID 02013035 Campus/ Major KRIBB/ biotechnology
Topics: Separation science in my lab research
In my laboratory we have been performed separation of different biological molecules mainly
plant DNA and RNA viruses. Thus, early detection and identification of plant viral agents (virus
and viroid’s) remain important to the field of virology that aims to control and mitigate the
effect and spread of these pathogens. Typically, viral diagnostic techniques, such as enzyme-
linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR), are used to detect
plant viral pathogens. However, these methods are too specific and limited because they depend
on prior knowledge and reagents to detect potential viral pathogens, including antibodies and
sequencing. Hence they cannot identify previously unknown viruses or a combination of viral
agents. Mostly in my lab demonstrated a non-specific molecular diagnostic technique utilizing
next generation sequencing (NGS) to detect and identify viral populations including known
and unknown virus and viroid’s, in various plant in the ecosystem. To perform such activities,
we have followed the following procedural techniques.
1: Sample collection: Collect various plant that showed virus-like symptoms such leaf
deformation, mild mottling, severe stunting, leaf mosaic, and vein clearing in south Korea
various places and bring into the lab. Each individual samples crushed to a fine powder by
aliquot of the liquid-nitrogen-ground plant material in pestle and mortar.
Fig.1 Liquid N2-ground plant samples crushing methods

2. 2: Extraction of nucleic acids: The total RNA/ or the DNA was extracted from the sample
using commercially available extraction Kit or phenol-based extraction methods. In the
process centrifugation is an important procedural principle to separate biological molecules.
Centrifuge is a device that used to separate various components of
fluid. Achieved by spinning the fluid at high speed with in a
container, thereby separating fluids of different densities. In my
research laboratory the particles are usually cells, subcellular
organelles, or large molecules separated by centrifugation. A
laboratory centrifuge is a laboratory equipment driven by motor,
spine liquid at high speed separate substance of greater and lesser
density. Separating and purifying biological materials.
Fig.2 Centrifuge based separation
3: Amplification: Studying isolated pieces of DNA is nearly impossible. Large amounts of a sample of
DNA are necessary for molecular and genetic analyses. Sometimes called molecular photocopying,
conventional polymerase chain reaction (PCR) is a technique used to amplify (replicate) trace amounts of
DNA and RNA from a sample. A PCR thermal cycler is used to produce the large amounts required for
research. PCR is based on three simple steps required for any DNA synthesis reaction: (1) denaturation of the
template into single strands; (2) annealing of primers to each original strand for new strand synthesis; and (3)
extension of the new DNA strands from the primers.
Virus contain nucleic acid molecules (either DNA
or RNA). Infected individuals have been detected
these nucleic acid molecules in their bodies.
Polymerase chain reaction (PCR) is a robust
technique to selectively amplify a specific segment
of DNA in vitro. [1] PCR is performed on
thermocycler and it involves three main steps: (1)
denaturation of dsDNA template at 92–95°C, (2)
annealing of primers at 50–70°C, and (3) extension
of dsDNA molecules at approx
Fig.3 PCR based separation

3. 4: Gel Electrophoresis: Gel electrophoresis is a technique used to separate DNA fragments
according to their size. DNA samples are loaded into wells (indentations) at one end of a gel,
and an electric current is applied to pull them through the gel. DNA fragments are negatively
charged, so they move towards the positive electrode. An electric current is used to move the
molecules through a gel or other matrix. The gel acts as a sieve to retard the passage of
molecules according to their size and shape.
Fig. 4 Gel Electrophoresis based separation
 Negative charges (DNA) move toward the positive Electrode
 Small DNA  fast movement
 Large DNA  slow movement
5. Sequencing: DNA sequencing refers to the general laboratory technique for determining the
exact sequence of nucleotides, or bases, in a DNA molecule. The sequence of the bases (often
referred to by the first letters of their chemical names: A, T, C, and G) encodes the biological
information that cells use to develop and operate. In order to analyze the entire genome, focus
on regions of interest with whole-exome and targeted sequencing, or study DNA-protein
interactions, Currently, in my lab the second-generation NGS technologies are commonly used
approach because they remain the fastest and the cheapest form of gene sequencing.
Fig. 6 DNA Sequencing techniques

4. The General Work Flow for Studying Viral pathogens Separation in my lab (KRIBB).
Sample collection
and pooling
Total RNA
extraction
Generate cDNA
Library
illumine
NextSeq500
/HTS paired end
Materials
Depleted
rRNA
De novo
assembly to contig
Quality check
and filtering
NGS (RNA-seq)
raw reads
Align contig to
NCBI data base
Identify virus and virus
like seq fragments
(taxonomic classification
Data
analysis
-Remove adapter
seq & low quality
read
Sequencegenesis
assembled
software
Identified coting's were
blasted by hand again
(taxonomic classification)
Design primer
Confirm RT-PCR
RT-PCR products
were sequenced and
confirmed
Confirm Purpose
- Revealto viral
population in the sample
-Revealto unknown viral
in the ecosystem
- First and diversity
detected viral pathogen in
South Korea
- Identify to frequently
infected viral agent’s in
various crops.