IDENTIFICATION OF BACTERIAL SPECIES BY COMBINED BIOINFORMATIC AND POLYMERASE CHAIN REACTION

 

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About Authors:
Emanual Michael Patelia, Bushra Ahmed, Muhammad Javaid, Jayesh Patel, Rakesh Thakur, Mishra Trinath
Department of Pharmacology, University of Bedfordshire, England,
United Kingdom, LU1 3JU
ricky.emanual@gmail.com

Abstract
The identification will be carried out by comparing a potential PCR product obtained from an unknown species with other PCR products specific to 5 known species, using the same five set of pairs of primers specific of these five species respectively. This implies that each PCR product obtained for each species has to be specific to each species and can be considered as a marker in this exercise. This specificity will be based on the uniqueness of the chosen template that is to be used for each PCR. In addition, since the PCR products will not be sequenced, they will be differentiated by their size, which will be identified by agarose gel electrophoresis. The PCR product can only be used as a marker that defines a species if the amplified sequence is unique to this species. Therefore, the first step in this exercise will be to identify a suitable sequence to amplify for each species. Following the identification of a suitable template, the size of the PCR will have to be defined; since the comparative analysis will be based not only on the presence of a product but also its size, all PCR products should have different sizes identifiable on agarose gel. Since the size of a PCR product is defined by the location of primers along the sequence, the second step in this exercise will be to design suitable primers. Finally, having defined specific template and primers for each species, PCR will be carried out using the DNA of unknown species as a template with all five sets of primers so that a successful PCR product and its size would identify the unknown species as one of the five species.

REFERENCE ID: PHARMATUTOR-ART-2147

PharmaTutor (ISSN: 2347 - 7881)

Volume 2, Issue 4

Received On: 04/03/2014; Accepted On: 15/03/2014; Published On: 01/04/2014

How to cite this article: EM Patelia, B Ahmed, M Javaid, J Patel, Identification of Bacterial Species by Combined Bioinformatic and Polymerase Chain Reaction, PharmaTutor, 2014, 2(4), 8-30

Introduction (Dale J W et al., 2012):
To design suitable primers for identification, the following procedure will be followed:
1. Definition of the five marker organisms
2. Identification of the availability of their genome
3. Definition and design of primers

1) Definition of the five marker organisms
The five organisms to which the unknown organism will be compared are:
Pseudomonas fluorescens (1), E. Coli (2), Micrococcusluteus (3), Bacillus subtilis (4) and Bacillus cereus (5)

2) Identification of the availability of their genome
Since in this instance the PCR product has to be specific to the species, the gene sequence chosen as a template has to be unique to this species. Although the gene of a protein that would be unique to each species would be ideal, in practice such a gene is not always easy to identify; genes of proteins which are conserved and found in several species might be suitable as long as the degree of identity is low enough so that the PCR products can be specific of the species. In practice this means that the primers should be chosen within sequences, or part of sequences, unique to the species.

In order to check for the specificity of a sequence, it is essential that the whole genome of the species be known. To find out, go to NCBI website and check within the list of known genome that the genome of each species is available
1) Enter the following address: ncbi.nlm.nih.gov/
2) In the menu on the left, choose: Genomes & Maps
3) Go down on this new page and choose the section entitled “genome
4) At the top of this new page, there is a search window, into which you should type the name of one of your 5 micro-organism, for example “Pseudomonas Fluorescens”. If the genome of this species is available, a new page will open that will give a summary of the genome.

Repeat the same procedure for each of the five species to check that their genome is available. You should keep a copy of each page to be included in the appendices of your report.

3)    Definition and design of primers
The sequence of the primers will define the uniqueness of the product. It is not necessary for the whole sequence of the product to be unique, as long as the sequence of the five set of primers are different.

To design such primers, different methods can be used. The most laborious methods would be to choose a gene for each organism and to compare its sequence to homologous gene in the other genomes; the suitable gene or sequence would have to include part of the sequences unique to this single organism.Although this empirical method is still in practice and acceptable, some tools have been designed that do this work for you.

Such primers can be designed using asoftware available on the same NCBI website. As an example, the procedure for the 1st microorganism, Pseudomonas fluorescens is described below.

From the Genome summary page you will have obtained in the previous section, scroll down the page to the last figure, and click on “GENBANK”; this will lead you to the page that describes the details of the genomic sequence.

On this new page that describes the genomic sequence, choose in the menu on the right, “Pick primers

A new page entitled “Primers BLAST” will open. It is in this page that all instructions that will define the primers and therefore the PCR product should be entered.

To keep all the results consistent and comparable, the size of the PCR products will be different for each species but with a given size for each species so that different groups will be able to compare their results.

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