The Process of Metagenomics

 

The metagenomic field encompasses researching, classifying, and manipulating  genetic material derived from environmental samples. In result, the Metagenomic Process can be explained within 4 steps.

 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.

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Step One: Genetic Material Extraction

   The 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

   The 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).

   Vectors 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

   The 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.

   Transformation 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).

   Initially 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

   After 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.

   A 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.

   Since 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).

   Metagenomic 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).

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How to study the Metagenomic Libraries' DNA

After construction of the metagenomic library, the genetic material of the content must undergo analysis.  Two approaches to study these library have developed: Sequence-based analysis and function-based analysis (Citation 1).

Figure 3: An diagram showing the module that has undergone sequence and structural based analysis.

Sequence-based analysis:

   Researchers analyze the DNA and identify the genes and "metabolic pathways"  by comparing the DNA with genes found in other  samples (Citation 2).

   Since one of the challenges of metagenomics is to conclude where the DNA fragment has originated from, sequence-based analysis has developed a technique to solve this issue. This technique finds "phylogenetic anchors," which matches the microorganism to the DNA. (Citation 1).

Function-based analysis:

   Researchers screen the DNA library for a certain function, such as antibiotic resistance or vitamin production. When a function is found, the DNA coding for that function is sequenced then compared with DNA other organisms or communities (Citation 2).

   Function-based analysis has the ability to discover new classes of genes and proteins since it detect genes that could not be detected on the DNA sequence; it has  led to the discovery of "new antibiotic, antibiotic resistance genes, metabolic enzymes, and signal molecules" (Citation 1).

   The challenges of this approach includes the "genes of interest must be expressed in the bacterial host cell used to maintain the metagenomic library" and the assay (procedure analyzes the concentration of the organisms produced  within the organic sample) used to find the active clones must be sensitive enough to detect the clones (Citation 1).

 

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