urban microbiome

Urban microbiome research refers to the study of microorganisms (bacteria, viruses, fungi, and other microbes) in urban environments.

•  To understand the complex relationships between the urban environment, human health, and biodiversity and to catalogue microorganisms that exist in urban environments.

•  The largest urban microbiome research to date is the MetaSUB consortium

•  Sample collection

•  DNA and RNA extraction

•  Library preparation for sequencing (PCR)

•  Sequencing

•  Bioinformatics analysis

•  16S rRNA is the central structural component of the bacterial and archaeal'

DNA Isolation

•  Lyse the sample (w/detergents, mechanical forces, etc…)

•  Proteins are precipitated and PCR inhibitors are inactivated

•  DNA binds to the silica column

•  DNA is hydrophilic, and silica is also hydrophilic. In the presence of a chaotropic agent, water molecules are all occupied and DNA is firmly attached to silica.

•  DNA is not soluble in ethanol, at all! It does not like the presence of ethanol.

•  Washing with ethanol (no water) to get rid of other components accept DNA.

•  Ethanol dissolves away all the other biomolecules (proteins, lipids, carbohydrates) that may contaminate our DNA sample.

•  Dry the column completely, to avoid any negative effect for PCR reaction from ethanol residues

•  Eluting DNA with a water-based buffer.

PCR

•  Rapidly amplifying millions to billions of copies of a segment of DNA - exponentially

•  Very accurate

•  PCR can work even with a limited amount of DNA samples (one copy)

•  Susceptible to contamination

•  Needed prior knowledge of the sequence

•  Some inhibitors from the sample can affect the reaction

•  Errors in amplification may occur

DNA Sequencing

•  The process of determining the nucleic acid sequence – the order of nucleotides in DNA.

•  Similar to a PCR reaction

•  Chain terminating reaction

•  Need extra analysis