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Lecture 8

Lecture 8. Nucleic Acid-Based Measurements Text Chapter 13. Extract DNA from soil remove cells from soil separate cells from soil lyse cells separate DNA from cells purify DNA. Extract DNA from soil Extract DNA from cells in presence of soil Bead-beating

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Lecture 8

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  1. Lecture 8 Nucleic Acid-Based Measurements Text Chapter 13

  2. Extract DNA from soil remove cells from soil separate cells from soil lyse cells separate DNA from cells purify DNA Extract DNA from soil Extract DNA from cells in presence of soil Bead-beating chemical or enzymatic treatment Sodium dodecyl sulfate or lysozyme Total community DNA

  3. DNA purification • Cesium chloride gradient centrifugation • Kits Low density DNA High density

  4. Visualizing nucleic acids-Blotting • Southern blotting • DNA • Northern blotting • RNA

  5. Agarose gel electrophoresis - Stain gel with ethidium bromide +

  6. DNA purification Agarose gel verification

  7. Gene Probes • Phylogenetic probes • 16S rRNA • Functional gene probes • dsr (bisulfite reductase) sulfate reduction • nirS (nitrate reductase) nitrate reduction

  8. cDNA 16S rRNA gene probes Target region • Oligonucleotide primers for PCR • Oligonucleotide probes complementary to 16S rRNA molecule • no need for PCR because many copies in cells 16S rDNA clone library cDNA RNA ribosome

  9. Secondary Structure: 16S rRNA • Different locations • on the 16S rRNA • molecule offer • identity at different • phylogenetic levels • Domain EU338 • Phylum • Class • Family • Group CF319a • Genus • Species • subspecies

  10. Fluorescent in situ hybridization (FISH)

  11. Protein synthesis From: Curtis & Barnes, Biology, Pg 315.

  12. fixation/permeabilization hybridization washing Hybridization Assay fixative oligo probe substratum cells visualization mounting

  13. Assessment of abundance of different populations in a sample • Construct oligo probes for each population represented in clone library • Floc particle from • activated sludge process • wastewater treatment • Complex microbial community • What is the abundance of • each population?

  14. FISH detection and localization of flavobacteria-cytophaga populations in activated sludge flocs • CF319a oligonucleotide probe detects flavobacteria-cytophaga group in presence of other wastewater bacteria (Manz et al. 1996) • 92% of DAPI-stained cells probed positive with EU338 9.6 + 2.1% of DAPI-stained cells probed positive with CF319a

  15. Problems and Pitfalls of FISH • Autofluorescence • Microorganisms • Abiotic components associated with sample • Lack of probe specificity • Positive and negative controls • Closely-related microbes • Probes for negative strand • Dual probes for different target regions on 16S rRNA molecule

  16. Problems and Pitfalls of FISH • Lack of probe specificity (continued) • Inaccuracy of sequence information in databases • Control through stringency • Temperature • formamide concentration • False negatives • Insufficient probe penetration across cell envelope • Higher order structure of target or probe • Low rRNA content in cells (starved cells have fewer ribosomes) • photobleaching stopped

  17. FISH and Function • Combining FISH with assessment of cell function • Fluorogenic substrates for enzymes of interest • Floc particle from • activated sludge process • wastewater treatment • Complex microbial community • What populations are doing • what?

  18. N How is the enzyme activity distributed across populations in the microbial community? water soluble water insoluble light blue to yellow-green no fluorescence fluorescence Ex. 345 nm; Em. 530 nm O Cl Cl O N N Cl N Cl PO4 H O ELF-PO4 ELF

  19. Localization of enzyme activity in flocs • Phosphatase activity detected via yellow-green fluorescence of ELF • Activity is: • localized within floc matrix • not associated with protozoans 50 µm

  20. Combining ELF and CF319a probes to determine what portion of the PO4ase-active cells in floc fall within cytophaga-flavobacteria group ELFTM PO4ase FISH probe & activity PO4ase activity CF319a FISH probe 17% of total community PO4ase activity contributed by cytophaga

  21. Summary FISH provides information on • Presence of specific populations • Morphology of specific populations • Relative numerical contribution of specific populations to total community • Spatial relationships between populations • Functions associated with specific populations

  22. Probes range in size from 18-100bp ssDNA Digoxigenin (DIG) Denatured ssDNA from suspect bacterium Gene probe detection of a DNA sequencewithout separating out DNA from cell mass

  23. Alternative gene probe detection of a DNA sequence

  24. Exploring microbial activity through expression of mRNAReverse transcription PCR • Need to know sequence of gene being expressed • Alternatively, use random hexamer primers, then sequence • cDNA product to identify gene being expressed

  25. Reverse transcriptase PCR (RT-PCR) • Make single-strand cDNA from mRNA • downstream antisense primer or random hexamer and RT to make complete cDNA copy of RNA molecule • Use cDNA, DNA polymerase, and a downstream primer in conventional PCR • extension leads to double-stranded DNA • Regular PCR of dsDNA

  26. Applications of RT-PCR • Detection of RNA viruses in water and soils • avoids having to infect animal cell cultures which is time-consuming, labor intensive, and expensive. • Detection of genes suspected of being expressed in the environment • phenol-degrading genes (dmpN) • Amplification of ribosomal RNA and subsequent cloning and comparative sequence analysis for phylogenetic studies in soils, hot springs, and sediments.

  27. Microarrays to detect gene expression mRNA from cells RT-PCR with random hexamer primers and labelled nucleotides labeled cDNA hybridization probe (18mer of gene of interest) Microarrays allow evaluation of simultaneous expression of many genes in a population or community. no hybridization

  28. Semi-nested PCR • Increases sensitivity • Allows confirmation of original product

  29. Multiplex PCR • Uses several sets of primers in one reaction to generate several diagnostic bands • Allows detection of multiple areas of the genome or multiple organisms in a sample

  30. Multiplex PCR

  31. More sensitive and detects all organisms regardless of physiological state Fast and easy to perform Easy to pick up contaminants Cannot discriminate between viable and nonviable or infectious and noninfectious cells or viruses Subject to inhibition by chemicals in environmental samples humic substances metals Advantages & Disadvantages of PCR Approach Advantages Disadvantages

  32. Colored bars represent different sequences. Red spirals indicate fluorescent label. Colored circles, squares, and rectangles indicate different restriction enzyme sites and their location in each sequence. The above graphic shows the fragments in order on each sequence. PCR Fingerprinting Terminal Restriction Fragment Length Polymorphism (TRFLP) 16S rRNA gene

  33. Applications of DNA fingerprinting • Detection of different microbial populations present in a soil or hot spring community • PCR Ribotyping • use 16S rRNA gene • use universal primers for generating full-length 16S rDNA fragments from each population • one of the primers is labeled • use classic PCR to produce labeled dsDNA of 16S rRNA gene • subject mixture of labeled fragments to restriction enzymes • run restriction fragments out on a gel

  34. Full-length16S rDNA fragments TRFLP Restriction enzyme fragments • The numbers in graphic indicate the relative abundance of each sequence, and the fragments have been re-ordered according to size. The fragment analysis peaks would look something like the graph on the right.

  35. Banding patterns produced are unambiguous Choice of restriction enzymes is empirical if banding patterns are the same, it does not necessarily mean that populations are the same one enzyme may cut two preparations at the same location Advantages and Disadvantages of RFLP Analysis Advantages Disadvantages

  36. Denaturing Gradient Gel Electrophoresis (DGGE) GC clamp: high melt domain GCCGG 5’ Forward primer 5’ 3’ 3’ Reverse primer Prevents complete melting of pcr product

  37. Add different sample to each well Same population in different samples Increasing formamide & urea concentration Different band in a lane identifies a different population

  38. Once separated, specific fragments can be excised from gel and sequenced Band density offers insight to the dominant populations in the community sampled DNA extraction methods may be selective for some populations over others Single population may have multiple rRNA operons with different 16S rRNA gene sequences Does not offer information on activity of populations present Advantages & Disadvantages of DGGE Advantages Disadvantages

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