Question

1)Explain the principles of RAPD and rep PCR genotypic fingerprinting. 2)what are the different criteria used for naming bacterial species.

3)Illustrate how the ribosomal genes and ITS regions are present in the genome of bacteria and fungi 4).Explain how you would use sequences of genes or regions of the genome to identify microorganisms. 5)Why is 16 SrRNA gene an excellent molecular(chronometer).6)What is the minimum similarity level for a bacterial species and a bacterial genus based on 16S rRNA gene sequences.7)Why is 16S-23S ITS sequences more discriminatory at sub genus and sub species levels of bacterial taxa than 16S rRDNA sequencing.8)Can bacterial genera and species be named based on 16 S-23ITS gene sequences.9)What are the major database used for comparing 16S rRNA gene sequences.10)What are the problems with the present nomenclature of bacteria as it relates to identification of bacteria by 16 SrDNA gene sequences.11)How did 16 rRNA gene sequencing identification contributed to clinal microbiology.12)Explain how bacteriophage typing can be used to identify bacteria.13)What is FAME and how is it used to identify microorganisms.14)Explain how BIOLOG system is used to identify microorganisms. 15)What is IMViC.What is the typical IMVic pattern for E.coli.16) name the different types of vaccine.Explain how they are produced.17)explain the function of dendritic,macrophages,Th1 and Th2 lymphocytes, B lymphocytes.18)what is the role of acidity,complement system(enzymes)normal microbial flora in the innate defense system

Answer 1

1. Principle of RAPD-

(i) extraction of highly pure DNA, (ii) addition of single arbitrary primer, (iii) polymerase chain reaction (PCR), (iv) separation of fragments by gel electrophoresis, (v) visualization of RAPD-PCR fragments after ethidium bromide staining under UV light and (vi) determination of fragment size comparing with known molecular marker with the help of gel analysis software.

Principle of rep-PCR genomic fingerprinting. -

1. Conserved repetitive elements dispersed through most bacterial genomes.

2. Amplification of distinct genomic regions employing primers designed to anneal to naturally occurring repetitive BOX, ERIC and REP elements.

3. Electrophoresis

2. criteria for naming bacteria-

Bacteria are named so that investigators can define and discuss them without the necessity of listing their characteristics.To form new bacterial names and epithets, authors are advised as follows.

1. Avoid names or epithets that are very long or difficult to pronounce.
2. Make names or epithets that have an agreeable form that is easy to pronounce when latinized.
3. Avoid combining words from different languages, hybrid names (nomina hybrida).
4. Do not adopt unpublished names or epithets found in authors' notes, attributing them to the authors of such notes, unless these authors have approved publication.
5. Give the etymology of new generic names and of new epithets.
6. Determine that the name or epithet which they propose is in accordance with the Rules.

3. Ribosomal dna and ITR in bacterial and fungal genome-

Ideally, the barcode locus would be the same for all kingdoms. A region of the mitochondrial gene encoding the cytochrome c oxidase subunit 1 (CO1) is the barcode for animals and the default marker adopted by the Consortium for the Barcode of Life for all groups of organisms, including fungi. In Oomycota, part of the kingdom Stramenopila historically studied by mycologists, the de facto barcode internal transcribed spacer (ITS) region is suitable for identification, but the default CO1 marker is more reliable in a few clades of closely related species. In plants, CO1 has limited value for differentiating species, and a two-marker system of chloroplast genes was adopted based on portions of the ribulose 1-5-biphosphate carboxylase/oxygenase large subunit gene and a maturase-encoding gene from the intron of the trnK gene. This system sets a precedent for reconsidering CO1 as the default fungal barcode.

4. For bacteria, mycobacteria, and fungi, many gene targets have been recognized as useful tools for identification. Broad-range gene targets for viruses have not been developed, owing to their greater genetic diversity and our inability to find a common genetic link. The gene targets that are selected must be functionally constant, serving as molecular clocks of microbial evolutionary change (phylogeny). The gene should have a conserved segment that is common to all bacteria (or fungi) and that is flanked by variable or highly variable regions. Conserved regions are responsible for the “universality” of the gene target where PCR and DNA sequencing primers anneal. During cycle sequencing, variable or highly variable regions generate unique nucleotide base fragments or sequences that serve as “signatures” for different species. The sequence is then compared with reference sequences that are deposited in public or private sequence libraries to determine relatedness.