These two images display relatively the same information within different formats; therefore viewers can obtain a better understanding of the Metagenomic Process.
Figure 1: The process of metagenomics listed in four steps:
1) isolation of DNA from environmental sample 2) manipulation of DNA then Ligation of fragments with vectors 3) Construct Library 4) Analysis
Figure 2: Flow diagram of the steps in metagenomic DNA library construction. DNA is isolated from the cells in the sample and then fragmented, inserted into vectors, cloned. The vectors are introduced into host cells. After the metagenomic libarary, the genomes undergo function of sequence analysis.
Step One: Genetic Material Extraction
first step of this procedure is to collect a sample from an environment
(such as soil, sand, or water). This sample contains numerous and
diverse microorganisms for analysis. The genetic material from all the
cells has to be extracted through physical or chemical methods (ex.
sonication or alkaline conditions). Once the cell is broken open and the
DNA is free; the genetic material from the sample is separated by
methods of DNA isolation including "density centrifugation, affinity
binding, and solubility or precipitation (Citation 9).
Step Two: Genetic Material Manipulation
genetic material from the microbes is essentially too large to work
with. Therefore, enzymes called "restriction endonucleases" cut the DNA
into particular sequences of base pairs. Sequentially, the DNA
fragments merge with vectors (Citation 9).
are small units of DNA that inserts itself into a cell and begin
replicating and producing the protein encoded in the DNA. The vector
also accommodates a "selectable marker" which provides a "growth
advantage" which the microorganism would not usually have, for instance
antibiotic resistance. In addition, selectable markers can recognize
which organisms contain vectors and which do not (Citation 9).
Step Three: Library Construction
vectors, fragments of extracted DNA, are introduced into the organism.
This allows the DNA that originated from organisms (that could not grow
under standard laboratory conditions) to be able to grow, studied, and
expressed. The DNA within the vector changes into the cells of the model
organism, most commonly Escherichia coli.
occurs when DNA is inserted into a cell. Then the DNA will produce stable
proteins. To determine which method of transformation to use (chemical,
electrical, or biological), analyze the type of sample under
investigation and determine "the required efficiency of the reaction" (Citation 9).
all the genetic material in the vectors is in the same sample. However,
vectors can only allow one type of DNA fragment from the sample to
thrive. Transformed cells grow on "selective media" meaning that
specific cells (ones caring vectors) will be the only ones to survive.
Therefore, the accumulation of growing cell is called a colony. Keep in
mind that the numerous cells within the colonies are cloned from one
single cell. These samples are called metagenomic libraries since they
consist of "metagenomic DNA samples on vectors" (Citation 9).
Step Four: DNA Analysis
creating the metagenomic libraries, the DNA from those libraries
must undergo analysis. DNA contains many genes; the expression or
in-expression of these genes contributes to the organisms properties.
genotype is an organism's complete genetic makeup; it contains the entire
cell's information. On the other hand, phenotypes are physical expression
of genes so they are observable traits. By using the organism phenotype,
metagenomic analysis searches for distinctions in the microorganism such
as shape or color.
most chemical properties of organisms cannot be seen by the naked eye,
metagenomic DNA must undergo a different method. Found in molecular
biology, an assay
is a procedure that analyzes the concentration of the organisms
produced within the organic sample. This method determines if the
microorganism had gain an "enzymatic function" that was not present
before "such as use of an usual nutrient source for growth under
conditions that limit normal nutrient availability" (Citation 9).
libraries contain genomes of the microorganism and acts as a database
to search for new different types of a gene. The metagenomic DNA is
first enters a microorganism that has a specific gene function
deficiency. By comparing the metagenomic DNA sequence to a database of
known DNA we receive information about the metagenomic DNA structure,
origin, organization and evolution. Comparisons of the base sequences
also can reveal how the gene protein functions. Frequently metagenomic
analysis involves a number of phenotypic and then genotypic analysis to
isolate specific genes from the environmental sample and to "effectively
characterize the information encoded by the DNA sequence" (Citation 9).