Klausur Schwerpunkte

• ubiquitously distributed

• high copy number

• up t0 20.000 ribosomes per cell

• length 16S-RNA 1.500 bp

• eukaryotes (80S) & prokaryotes (70S)

• selection pressure independent from enviromental conditions (constant)

• secondary structure is highly conserved

• enables easy comparison of homologous sequence stretch

• rarely cases of horizontal gene transfer (HGT)

• changes in sequences develop with constant speed and slow enough to reflect the time of bacterial evolution

• in different regions of the 16S rRNA the “molecular clock” changes with different speed

• mutations in conserved parts are evolutionary older

• Chimera

  • composed of fragments of different 16S rRNA genes

  • sheared DNA increase in amount of chimera

  • 1-10% of 16S rDNA clones in enviromental libraries

• deletion mutants

• point mutants

• contamination

  
  

• diferences in lysis efficiency

• different GC-content of templates

• different binfing efficiency of degenerated primers

• secondary structure of templates

• cloning problems

• use of different DNA extraction techniques

• utilization of different “universal primers” (ideally non-degenerated)

• use of small number of PCR cycles, relativly high amounts of template DNA

• pooling of replicate PCRs

• mechanical micromanipulation

  • microcapillary glass of the micromanipulators is connected to syringe pump: controlled ejection

• microfluidics = sample fluids containing multiple cells is divided into single plugs that are stochastically encapsuled

• clonal cells can be further used for cultivation, cryopreservation, culture-independent analysis…

• enables single-cell genomics

• Phi29 DNA-Polymerase elongates DNA strands starting with random primers (Hexamers)

• if a double strand is reached it will be displaced and further amplified with random primers

• typically based on 16S-RNA (18S-RNA)

• good stability, large database, high copy number

Method

• fixation and permeabilization of samples

• hybridization

• washing steps to remove unbound probe

• detection via microscopy and flow cytometry

Applications

• in situ-detection of non-cultivable microorganisms, quantification in enviromental samples

• in situ-relation of metabolic processes with certain groups of microbes

• in situ-identification of symbiotic microbes in plants and animals

• is a technique used to amplify and simultaneously quantify a targeted DNA molecule

• this method allows for both detection and quantification of one or more specific sequences in a DNA sample

Method

• sample preparation: extract DNA/RNA, convert RNA to cDNA if needed

• reaction setup: mix DNA/cDNA template with primer, DNA polymerase, dNTPs, buffer, and fluorescent dye

• PCR ampification: denaturation (heat to seperate strands), annealing (cooling for primer binding), extension (synthesize new DNA strands and measure fluoresence)

• data analysis: determine cycle threshold values for quantification

Advantage

• high sensitivity

• quantification

• high specificity

• rapid results compared to traditional PCR methods

• dynamic range of quantification

Applications

• gene expression analysis: measure expression levels of specific genes

• pathogen detection: identify and quanttify pathogens in clinical, food and envirmental samples

• genetic variation analysis: detect single nucleotide polymorphism (SNPs) and mutations

• copy number variation (CNV) Analysis: quantify the number of copies of a particular gene in a sample

• DNA methylation analysis: assess methylation stats of specific DNA regions

• DNA Denaturation: heat DNA samples to seperate

• primer binding: add primer that bind to single-stranded template

• extension with DNA polymerase: DNA polymerase adds nucleotides to the growing DNA strand

• incorporation of ddNTPs: add mixture of normal dNTPs and fluorescently labled ddNTPs -> terminate extension

• DNA strands of various length are created

• fragment separation: ge electrophoresis to separate frgments by size

• detectio with laser

advantage/disadvantage

• + high accuracy

• - only one single DNA molecule

• - low thoughput

• expensive

• sample preparation (fragment DNA and add adapters)

• cluster generation (bind to flow cell, bridge amplification)

• sequencing by synthesis (add fluorescent lable reversible)

• detection (remove terminator group)

• data analysis (align signals)

Advantage/Disadvantage

• + high accuracy

• + high throughput

• low cost

• - short reads

• - expensive instrument

• sample preparation (extraction)

• library prep (add sequencing adapters)

• loading (introduce library into flow cell)

• sequencing ( apply electrical current across membrane, DNA pass through nanopore -> change in current)

• real time data (monitor current, use algorithm to translate signal)

Advantage/Disadvantage

• + no amplification or labeling

• + DNA, RDNA and protein sequencing

• + portable

• + real-time sequencing

• + unrestricted length

• - high error rate

WHY

• genome size large

• huge amount of data

• requires clever algorithm and find patterns

• store/search/compare

• visualize collection of data

overview of algorithm used for OUT analysis

• sequencing similarity-based clustering

• model based clustering based on statistical models

• density based clustering