2. Microbial Census - Tagged.pdf

Microbial Census DR KAYE Friend or Foe?  The flora of the human body is collectively termed microbiota. The majority is resident, and rarely pathogenic. Because of the effect of age, diet, hormones, contact, there is a great variation. The issue concerns identification of friend...

Microbial Census DR KAYE Friend or Foe?  The flora of the human body is collectively termed microbiota. The majority is resident, and rarely pathogenic. Because of the effect of age, diet, hormones, contact, there is a great variation. The issue concerns identification of friend or foe methods. Bye culture, hello Nucleic acid- based methods  Today, culture-independent, nucleic acid-based methods for characterizing bacterial populations is leading the way into broad and rapid surveys of microbiota.  Culture (few grow) and staining (kills organism)  Susceptibility tests are often not reproducible in vivo  Immune tests are often delayed  Chemical: Proteins ,lipid and CHO presence  Genetic – NA tests. PCR required to generate enough copies  Qualitative tests: Specific presence of one organism  Quantitative tests: Volume of organismic NA present Nucleic Acid Based Approaches  Nucleic acid–based methods detect organism-specific DNA or RNA sequences extracted from the microorganism. Sequences may or may not be amplified in vitro. Tests are qualitative>quantitative  Rapid  Specific  Sensitive  Differentiates between organisms (multiple assay) ex. law enforcement  Unamplified Testing: organism is cultured or is present in high concentration  Amplified Testing: Tiny volumes required. Good for organisms that don’t culture well Amplified tests  Target amplification  Polymerase chain reaction [PCR], reverse transcriptase–PCR [RT-PCR  Signal amplification  Branched DNA assays, hybrid capture  Probe amplification  ligase chain reaction  Postamplification analysis  Sequencing of the amplified product, microarray analysis The 16s Ribosomal RNA region  A 16S rRNA gene is used as the standard for classification and identification of microbes  16S rRNA gene sequences for most bacteria and archaea are available on public databases, such as NCBI  The gene is highly conserved since evolution - a "molecular fossil".  The 16s gene has two different domains, a larger one conserved domain, and a hypervariable region.  hypervariable region provides species-specific signature sequences useful for identification of bacteria  V1-V9 regions (V1-V8 are disease specific  The study of this region is called Phylogenetics  Pyrosequencing, Sanger sequencing of the 1500 bases  Illumina is faster and cheaper by 50x  Bonds the 50S and 30S rRNA units The 16s rRNA Nucleic Acid Based Approaches Use: Disease tracing and the effect on metabolism  An early study that used 16S rRNA gene profiling found that the microbiota of obese mice and humans differed from that of lean mice and humans. Moreover, when germfree mice (mice lacking any intestinal bacteria) were colonized with an “obese microbiota,” those mice gained more fat than germfree mice colonized with a “lean microbiota.”127 16s rRNA Sequencing Microbial rRNA 16s Analysis: 1. Basic Sanger: Uses ddNTPs* Maxam-Gilbert chemical terminators 2. Advanced (RNA-seq technology) Shotgun RNA overlapping and electrophores is from short to long sequences with photo color ID 3. Next Generation SoLiD (accurate bead method nucleotide +adapter colonies), Illumina(adapter+oligo+unknown BP sequence) into bridges Pyro sequencing (flurescent dye+nucleotide) *Dideoxynucleotides are chain-elongating inhibitors of DNA polymerase, used in the Sanger method for DNA sequencing. They are also known as 2',3' because both the 2' and 3' positions on the ribose lack hydroxyl groups, and are abbreviated as ddNTPs. 1.Basic: Sanger 2.Advanced: Shotgun 3.Next Generation: Illumina 3. Illumina explained  Illumina – Pyrosequencing is a method of DNA sequencing based on the "sequencing by synthesis" principle, in which the sequencing is performed by detecting the nucleotide incorporated by a DNA polymerase. Pyrosequencing relies on light detection based on a chain reaction when pyrophosphate is released Illumina and Libraries  To complete a census of the species, a sequence analysis of all the 16S rRNA genes present must be performed. The establishes library of 16S rRNA gene clones are then transformed into Escherichia coli so that comparing each of the sequences obtained to the database sequences can identify the nearest relatives and provide an immediate identification of the organism from which the sequence originated.104  The r16s System – what's next? Disadvantages of the 16sRNA system is that it does not provide information about which genes are being expressed. The Future: Gene expression can be measured using techniques that detect and quantitate  Assignment Q2.1 Infants in the first years of life are often more susceptible to certain bacterial infections than older children. Q2.2 How can you explain this? What function of the microbiota does this illustrate? Q2.3 Remember to merge or collate each homework question into an assignment folder in PDF format