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Metagenomic DNA Isolation

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Published in: Biology
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A metagenome consists of Genomic material of multiple organisms. Isolation and Amplification makes us understand the diversified microorganisms.

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  1. METAGENOMIC DNA ISOLATION MADE BY MUKUL DHINGRA 813/BT/12
  2. INTRODUCTION Metagenomics is the study of metagenomes, genetic material recovered directly from environmental samples. The term "metagenomics" was first used by Jo Handelsman, Jon Clardy, Robert M. Goodman, and first appeared in publication in 1998. This relatively new field of genetic research enables studies of organisms that are not easily cultured in a laboratory(99.8%) as well as studies of organisms in their natural environment.
  3. Metagenome The referenced the idea that a collection of genes sequenced from the environment could be analyzed in a way analogous to the study of a single genome. The sample may consist of different species like bacteria,archae,and fungi.
  4. METAGENOMIC DNA ISOLATION Sample collection Whole DNA extraction Whole DNA amplification Whole DNA sequencing Data analysis
  5. PCR AMPLIFICATION The bacterial 16S rRNA genes were amplified using universal bacterial primers(18-20 bp),27 F and 1392 R. The PCR consists of the following processes ' Denaturation(95 C,5 min) ' AnnealIing(MeIting Temperature) Extension(72 C) Maturation(72 C) The last three processes are repeated 29-30 times.
  6. QUANTIFICATION The Absorbance ratio of DNA is to Protein is given by A260/A280 > 107 ' But if our ratio comes out to be in between 1.8 to 1.9,then the results are taken to be satisfactory. The Absorbance ratio of DNA is to Humic substances is A260/A230 Humic Substances are polyphenolic compounds which might be present in the soil. ' Restriction Digestion is done with the help of enzymes(Rll) like TAQ DNA Polymerase,Mg++ ions are used to chelate the humus.
  7. BACTERIAL AMPLIFICATION (16S rDNA)
  8. FUNGAL AMPLIFICATION (INTER TRANSCRIBED REGION)
  9. RESTRICTION DIGESTION OF METAGENOMIC DNA ' Restriction enzyme used:- Sau3Al ' Recognition sequence:- 5'GATC 31CTAG Staggered cut (Sticky ends) :- 31---CTAG GATC---31 ---51
  10. Applications Growth Vaccine Protein Resistance Secondary Metabolite
  11. DISADVANTAGES Often fragmentary Often highly divergent Rarely any known activity No chromosomal placement No organism of origin Huge data 3

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