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DNA barcoding in Microorganisms. Alexandre Soares Rosado Institute of Microbiology UFRJ - Brazil. Bergey’s Manual = 4500 Species Some studies DNA reassociation= 10.000 genomes / g soil; 1% - 5% of microorganismos are culturable The majority = uncuturable .

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Dna barcoding in microorganisms
DNA barcoding in Microorganisms

Alexandre Soares Rosado

Institute of Microbiology

UFRJ - Brazil


Dna barcoding in microorganisms

Bergey’s Manual = 4500 Species

Some studies DNA reassociation= 10.000 genomes / g soil;

1% - 5% of microorganismos are culturable

The majority = uncuturable


Dna barcoding in microorganisms

BRAZILIAN BIODIVERSITYFonte: Lewinsohn & Prado, 2000


Dna barcoding
DNA barcoding

  • DNA barcoding is a methodology for identifying species using a short DNA sequence. It is purported to be a reliable, inexpensive and easily accessible tool for both taxonomic specialists and non-specialists (e.g., government officials, professionals in health and agriculture).

  • To date, rRNA genes are the most frequently used target for identifying microorganisms, because not only do they occur in all living organisms, but they typically are also present in several copies that are distributed over the genome.


Background information
Background information

  • Based on the information, many molecular tools (e.g. FISH, T-RFLP, PCR, RAPD, Sequencing etc.) have been developed to discriminate many microorganisms. Of the methods, DNA sequencing generally provides the most accurate means for identifying them.

  • For species identities, investigators generally compare their own DNA sequence to GenBank database by BLAST search. In each case, result quality depends mainly on the reference databank being used. However, each of these databases contains many errors, putting limits on their value for diagnosing differently originated organisms.

  • Further, in cases that DNA sequence matched is not found and hit score queried is low, it is difficult to identify them based on BLAST search.


Dna barcoding in microorganisms

Application of molecular fingerprinting techniques to study the

composition and dynamics of soil microbial communities

Advantages

Cultivation-independent analysis of large numbers

of samples

Top-to-bottom analysis:

Sequencing of differentiating bands

Taxon-specific primers

Use of probes to identify bacterial isolates

corresponding to differentiating bands



Dna barcoding in microorganisms

Ubiquitous distribution the

Functionally conserved in all forms of life

The rRNA-gene - an ideal molecular marker?

„The molecular clock“

Regions of different degrees of conservation

Disadvantages

Different number of ribosomal operons

Sequence heterogeneities

A given variable region allows a different resolution for

different taxa


Dna barcoding in microorganisms

Figure 1 the: gene rpoB


Dna barcoding in microorganisms

Figure 2: theDistance (p) among strains of Paenibacillus 16S and rpoB


Dna barcoding in microorganisms

„community“ DNA or RNA the

PCR amplification of 16S

or 18S rDNA fragments

Few prominent populations (low evenness): patterns with few bands

Molecular fingerprints of

microbial communities

Denaturing gradient gel electrophoresis

(DGGE, TGGE)

Many equally abundant populations

(high evenness):

patterns with many bands


Dna barcoding in microorganisms

Soil aggregation and bacterial community structure as theaffected by tillage and cover cropping in theBrazilian CerradosPeixoto et al., 2006

The Cerrados region in central Brazil occupies 22% of the country. It is characterized by high average temperature (22 - 270C), rainfall (800 - 1600 mm) and solar radiation (475- 500 Cal/cm2/day).



Dna barcoding in microorganisms

L TW1 TW2 T1 T2 NTW1 NTW2 NT1 NT2 F1 F2 L

L TW1 TW2 T1 T2 NTW1 F2 NT1 NT2 F1 NTW2 L

A

B

16S



Dna barcoding in microorganisms

Biodiversity F2 L

DGGE- Fingerprints 16S rDNA

Diversity of bacterial comunities

Identification/ 16S rDNA-Sequencing

1b Bacillus megaterium

2b Arthrobacter sp.

1s Sphinghomonas

2s Streptomyces galbus

3s Streptomyces sp.

4s Nocardia

5s Pseudomonas sp.

6s Pseudomonas

1p Bacillus megaterium

2p unknown Bacterium

3p unknown Bacterium

1r Pseudomonas sp.


Dna barcoding in microorganisms

IV F2 L

c

7

a

IV

6

IIIb

Cluster I

Cluster II

Cluster III

4

V

5

IIIb

IIIb

IV

Cluster

Cluster IV

Cluster V

IIIb

3

III

I

1

V

V

VI

Cluster

Cluster IV

V

Cluster

2

Comparison between Denaturing Gradient Gel Electrophoresis (DGGE) and Phylogenetic Analysis for characterization of A/H3N2 Influenza Samples detected during 1999-2004 epidemics in Brazil.

b


Dna barcoding in microorganisms

Mangroves F2 L

  • Our knowledge of mangrove associated bacteria has been limited by a pronounced sampling, culturing and experimental bias.

  • Prokaryotic organisms recognized so far are only few bacterial phyla

  • Further, most isolates/ or strains studied hitherto have yet been correctly identified to species-level.

  • Thus, the aim of this project is to determine accurate, precise 16S DNA sequences of more than 1,300 bp from sediments and rizosphere of mangrove plants tighter with traditional methods, and then to evaluate them to advance phylogentic relationships and DNA barcoding.


Dna barcoding in microorganisms

Important issues F2 L

  • Biodiversity and Systematics

  • Our knowledge of biodiversity needs to be greatly expanded by doubling the rate of taxonomic inventories and species discovery and description by 2015.

  • This will require a commensurate increase in taxonomic expertise and infrastructure.

  • The rapidly developing field of informatics and communications technology must be harnessed both to facilitate scientific work and to disseminate taxonomicproducts to all users, including the general public.

  • There are several issues and problems, however, that need to be addressed before barcoding can be instituted, especially for single-celled microorganisms . Linking barcodes to accurately identified species represents a large hurdle that must be overcome.


Dna barcoding in microorganisms

When the most efficient means of rapid barcode-based species identification is sought, a choice can be made either for one of these methodologies or for basic high-throughput sequencing, depending on the strategic outlook of the investigator and on current costs.

Arrays and functionally similar platforms may have a particular advantage when a biologically complex material such as soil or a human respiratory secretion sample is analysed to give a census of relevant species present.


Acknowledgments
Acknowledgments: identification is sought, a choice can be made either for one of these methodologies or for basic high-throughput sequencing, depending on the strategic outlook of the investigator and on current costs.

Laboratory of Molecular Microbial Ecology –LMME

Petrobras

CNPq