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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 l.jpg

Lecture 8

Nucleic Acid-Based Measurements

Text Chapter 13


Total community dna l.jpg

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


Dna purification l.jpg
DNA purification

  • Cesium chloride gradient centrifugation

  • Kits

Low density

DNA

High density


Visualizing nucleic acids blotting l.jpg
Visualizing nucleic acids-Blotting

  • Southern blotting

    • DNA

  • Northern blotting

    • RNA


Agarose gel electrophoresis l.jpg
Agarose gel electrophoresis

-

Stain gel with

ethidium bromide

+


Dna purification6 l.jpg
DNA purification

Agarose gel verification


Gene probes l.jpg
Gene Probes

  • Phylogenetic probes

    • 16S rRNA

  • Functional gene probes

    • dsr (bisulfite reductase) sulfate reduction

    • nirS (nitrate reductase) nitrate reduction


16s rrna gene probes l.jpg

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


Secondary structure 16s rrna l.jpg
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


Slide10 l.jpg

Fluorescent

in

situ

hybridization (FISH)


Slide11 l.jpg

Protein synthesis

From:

Curtis &

Barnes,

Biology,

Pg 315.


Slide12 l.jpg

fixation/permeabilization

hybridization

washing

Hybridization Assay

fixative

oligo probe

substratum

cells

visualization

mounting


Assessment of abundance of different populations in a sample l.jpg
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?


Slide14 l.jpg

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


Problems and pitfalls of fish l.jpg
Problems and Pitfalls of FISH populations in activated sludge flocs

  • 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


Problems and pitfalls of fish16 l.jpg
Problems and Pitfalls of FISH populations in activated sludge flocs

  • 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


    Fish and function l.jpg
    FISH and Function populations in activated sludge flocs

    • 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?


    How is the enzyme activity distributed across populations in the microbial community l.jpg

    N populations in activated sludge flocs

    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


    Localization of enzyme activity in flocs l.jpg
    Localization of enzyme activity in flocs populations in activated sludge flocs

    • Phosphatase activity detected via yellow-green fluorescence of ELF

    • Activity is:

      • localized within floc matrix

      • not associated with protozoans

    50 µm


    Slide20 l.jpg

    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


    Summary l.jpg
    Summary the PO

    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


    Gene probe detection of a dna sequence without separating out dna from cell mass l.jpg

    Probes range in size the PO

    from 18-100bp ssDNA

    Digoxigenin

    (DIG)

    Denatured

    ssDNA

    from

    suspect

    bacterium

    Gene probe detection of a DNA sequencewithout separating out DNA from cell mass



    Exploring microbial activity through expression of mrna reverse transcription pcr l.jpg
    Exploring microbial activity through expression of mRNA the POReverse transcription PCR

    • Need to know sequence of gene being expressed

    • Alternatively, use random hexamer primers, then sequence

    • cDNA product to identify gene being expressed


    Reverse transcriptase pcr rt pcr l.jpg
    Reverse transcriptase the POPCR (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


    Applications of rt pcr l.jpg
    Applications of RT-PCR the PO

    • 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.


    Microarrays to detect gene expression l.jpg
    Microarrays to detect gene expression the PO

    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


    Semi nested pcr l.jpg
    Semi-nested PCR the PO

    • Increases sensitivity

    • Allows confirmation of original product


    Multiplex pcr l.jpg
    Multiplex PCR the PO

    • 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



    Advantages disadvantages of pcr approach l.jpg

    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


    Pcr fingerprinting l.jpg

    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


    Applications of dna fingerprinting l.jpg
    Applications of DNA fingerprinting 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.

    • 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


    Trflp l.jpg

    Full-length16S rDNA fragments 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.

    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.


    Advantages and disadvantages of rflp analysis l.jpg

    Banding patterns produced are unambiguous 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.

    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


    Denaturing gradient gel electrophoresis dgge l.jpg
    Denaturing Gradient Gel Electrophoresis (DGGE) 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.

    GC clamp: high melt domain

    GCCGG

    5’

    Forward primer

    5’

    3’

    3’

    Reverse primer

    Prevents complete melting of pcr product


    Slide38 l.jpg

    Add different sample to 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.

    each well

    Same population

    in different samples

    Increasing

    formamide

    & urea

    concentration

    Different band in a

    lane identifies a

    different population


    Advantages disadvantages of dgge l.jpg

    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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