Advanced Environmental Biotechnology II

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Advanced Environmental Biotechnology II

• Week 10 Nucleic Acid Hybridization

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• 8Applications of nucleic acid hybridization in
  microbial ecology
• A.Mark Osborn, Vivien Prior and Konstantinos
  Damianakis

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8.1 Introduction

• Nucleic acid hybridization can be defined as the
  complementary base pairing between two nucleotide
  strands by hydrogen bond formation between
  individual nucleotides.

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• Nucleic acid hybridization is central to
  molecular biology, to detect specific DNA
  sequences (probes), and during the polymerase
  chain reaction (PCR) and DNA sequencing (using
  oligonucleotide primers).

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• In microbial ecology many nucleic acid
  hybridization methods have been developed. First
  was applying DNA probes to find particular genes
  in individual microorganisms and/or recombinant
  DNA constructs.

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• Then in detection of particular organisms or
  genes within environmental samples.
• Also to estimate the complexity in terms of
  species diversity of microbial communities.

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• One major application is fluorescent in situ
  hybridization (FISH) - detect and enumerate
  specific microbial taxa within environmental
  systems, using oligonucleotide probes. This
  important for investigating spatial distribution
  of microbial communities. We will look at this
  next week.

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• This week
• - in vitro applications of nucleic acid
  hybridization, using nucleic acids isolated
  either from individual microorganisms, or
  directly from environmental samples.
• - Key methods,
• - how to screen for the presence of genes in
  cultured bacteria, and subsequently in
  environmental samples, and
• - the application of microarrays to investigate
  gene distribution, diversity and expression both
  in cultured microorganisms, and in the
  environment.

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8.2 Fundamentals of DNA hybridization

• simplest level - generation of a single-stranded
  nucleic acid probe (most typically ssDNA) that is
  labeled (eg with a radioisotope) - subsequent
  detection when the labeled probe binds to a
  single-stranded nucleic acid molecule (the
  target) usually first been immobilized on a solid
  matrix, e.g. a nylon membrane.

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• If the probe and the target nucleic acid show
  complementarity, i.e. significant similarity at
  the nucleotide level that will allow a
  double-stranded hybrid molecule to be formed,
  then the probe will anneal to the target nucleic
  acid and can be detected (using autoradiography
  or other visualization approaches eg
  fluorescence).

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• If the probe sequence is not similar to the
  target NA then hybridization will not occur.
• - hybridization allows detection of a specific
  DNA sequence from a mixed nucleic acid sample
  (e.g. chromosomal DNA, or total environmental
  DNA).

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• Methods vary with respect to the type of nucleic
  acid being screened for, the sequence of
  interest, the extent to which a probe will
  provide unambiguous results, i.e. probe
  specificity, and the type of probe that will be
  utilized in the hybridization experiment.

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8.2.1 Probe design

• The choice and/or design of the probe is very
  important.
• In most applications, the probe will be a DNA
  molecule and these are of two main types
  fragment probes or oligonucleotide probes.

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• Fragment probes usually consist of a
  double-stranded nucleic acid molecule, for
  example a restriction fragment or a PCR
  amplification product.
• These probes are typically 200 bp in length, but
  may be several kilobases in size. Prior to
  hybridization, the double-stranded probe will be
  denatured eg. by boiling the probe for a few
  minutes.

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• Oligonucleotide probes typically consist of
  single-stranded DNA molecules of 20 nucleotides
  in length, with denaturation of the probe
  maintained by inclusion of formamide in the
  hybridization reaction.

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• A third, less commonly used type of probe, is the
  polynucleotide probe that consist of between 60
  and 350 nucleotides, either following chemical
  synthesis for shorter polynucleotides, or as RNA
  transcripts for larger probes.

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• Single mismatches between the polynucleotide
  probe and target sequence will not result in
  dissociation of the probe except under very high
  stringency conditions , and therefore such probes
  can be used for experiments where high but not
  complete specificity is required - ideal for use
  in group-specific detection of bacterial genera -
  use in microarray experiments by some commercial
  array suppliers.

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• The choice of whether to use fragment or
  oligonucleotide probes will be influenced in
  particular by whether the researcher is
  interested in detecting or identifying sequences
  that share similarity to the sequence of interest
  or near or absolute identity.

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• Similar but not necessarily identical sequences -
  larger fragment probes are used. - detection of
  sequences that are divergent (e.g. up to 70
  identity) - e.g. for detection of functional
  genes, for which there may be considerable
  divergence between different bacterial species

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• Very specific detection - oligonucleotide probes
  are used. - single base changes can result in
  release of the probe from the target - e.g. a
  particular 16S rRNA sequence representing a
  species

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• Decreasing similarities between the probe and
  target sequences will result in the hybrid
  molecule becoming unstable.

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• The choice of probe is absolutely critical to the
  design of hybridization experiments.
• - need to consider both probe specificity and the
  origin of the sequence being used as a probe in
  hybridization experiments.

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• It is relatively straightforward to determine the
  sequence of any particular probe. Having the DNA
  sequence enables an in silico hybridization
  experiment to be conducted by doing a FASTA (Fast
  All) or BLAST (Basic Local Alignment Search Tool)
  comparison with the GenBank databases. Sequences
  showing high identity to the probe sequence can
  be identified.
• Empirical testing via actual hybridization
  experiments using the probe with a number of
  sequences showing varying degrees of identity
  should be performed.

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• Another major consideration in the design of
  fragment probes is the length of the probe being
  used. For larger probes, defined here as those gt1
  kb in length, it is possible that a significant
  proportion of the probe will hybridize to a
  target sequence, but that in other regions of the
  probe no homology will be found to the target
  DNA.

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• Under stringent conditions, the probe may be
  removed during washing stages in the
  hybridization protocol that remove unbound or
  partially bound probes.
• To increase specificity in hybridization
  reactions, the use of shorter probes (lt1 kb) is
  recommended
• If the researcher is interested in detecting
  multiple genes a collection of probes can be
  used.

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• As a simple alternative to the use of restriction
  fragment-derived probes, the polymerase chain
  reaction can amplify the sequence to be used as a
  probe . This has the obvious advantage that the
  probe region is defined by the user and can be
  readily sequenced.

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• For oligonucleotide probes, sequence specificity
  is critical to the success of the hybridization
  experiments. As oligonucleotide probes are
  defined by the researcher, the specificity of
  such sequences can again be tested in in silico
  comparisons to the DNA sequence databases.

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• There is now a number of probe-analysis software
  packages, in particular for the design of
  ribosomal RNA-based probes, to identify
  particular phylogenetic lineages, e.g. Probe
  Match (http//rdp.cme.msu.edu/probematch/search.j
  sp), ProbeBase (http//www.microbial-ecology.de/pr
  obebase/index.html) and PRIMROSE .

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• Once the probe sequence, amplicon or DNA fragment
  has been chosen the researcher will need to
  decide upon the choice and placement of the label
  that will be used to enable detection of the
  probe following hybridization.

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• Digoxigenin (DIG) labeling coupled with
  fluorescence-based detection, chemiluminescent
  detection and colorimetric detection has been
  adopted for use in many hybridization assays
• (see http//www.roche-applied-science.com/fst/pro
  ducts.htm7/DIG).

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• Probe labeling needs choice of location to attach
  the label. For oligonucleotide or polynucleotide
  probes, end labeling is typically used with the
  label attached either to the 5' end of the
  oligonucleotide using T4 polynucleotide kinase
  or to the 3' end of the oligonucleotide using
  terminal deoxynucleotidyl transferase

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• Fragment probes can similarly be labeled either
  terminally, for example by end labeling , or more
  commonly by random priming where the detection
  label is incorporated along the entire length of
  a series of DNA probes generated using enzyme to
  produce a pool of labeled probe molecules that
  are all homologous to the original template DNA.

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• For PCR products the simplest method is to
  incorporate the label as a labeled nucleotide
  during the amplification reaction.

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• Before use in DNA hybridization experiments, any
  fragment or PCR-derived probes will require
  denaturation to generate single-stranded DNA
  probes, typically by heating the probe to 95C
  for a few minutes.

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8.2.2 Choice of nucleic acid template

• The design of successful hybridization
  experiments will also depend on the nucleic acid
  template to which the DNA probe will be
  hybridized. Simply, this may use genomic DNA
  extracted from individual bacterial isolates.

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• As an alternative to direct isolation of genomic
  DNA before hybridization analysis, a number of
  studies have used colony hybridization to screen
  collections of environmental isolates for
  functional genes and to identify common
  environmental species.

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• In colony hybridization, the hybridization
  membrane is laid over the bacterial colonies on a
  plate, the membrane is then taken off the plate
  and the colonies are lysed on the membrane. The
  resulting crude extract is then fixed to the
  membrane.

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• An adaptation of the colony hybridization method
  can be used to investigate the distribution of
  bacteria on soil surfaces. Rather than using
  spread plates made from diluents of suspended
  soil particles, researchers placed an agar plate
  onto soil surfaces and gently pressed down onto
  the agar plate so that the underlying soil would
  adhere to the agar surface.

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8.3 Hybridization applications in microbial
ecology

• Although DNA hybridization methodologies have
  been available for nearly 30 years, there are
  surprisingly few reviews discussing the
  application of in vitro DNA hybridization in
  microbial ecology. This is in sharp contrast to
  reviews describing fluorescent in situ
  hybridization (FISH)-based applications.

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8.3.1 Hybridization analysis of cultured bacteria

• Early applications of DNA hybridization to
  microbial ecology focused on screening
  collections of environmental bacterial isolates
  for particular functional genes of interest.

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• Heavy metal resistance genes and genes encoding
  biodegradation functions in bacteria from
  polluted habitats.
• Degradation of polyaromatic hydrocarbons (nah
  genes and ndoB) , 2,4-dichlorophenoxyacetic acid
  (-D) (tfdA) , dicyanide , and carbofuran (mcd
  gene) .
• Nitrogen cycling (denitrification) , and
  antibiotic resistance (aminoglycoside
  acetyltransferases)

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8.3.2 Hybridization analysis of total community
DNA

• Isolate DNA directly from environmental samples
  that would be representative of the total.
• Question is how many bacterial species are
  present within any given environment?

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• The principles of DNA hybridization were applied
  to provide estimates for bacterial species
  numbers in soils. Torsvik investigated
  heterogeneity between DNA molecules that were
  extracted from soil, subjected to thermal
  denaturation and then allowed to reassociate to
  form double-stranded hybrid molecules.

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• The degree of reassociation will depend upon the
  extent to which single-stranded molecules will
  anneal (hybridize) to their counterparts. In a
  very simple community, reassociation will be
  commonplace as single-stranded sequences
  hybridize to complementary sequences.

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• By contrast, in a complex community, the
  likelihood of sequences hybridizing to their
  complementary sequences will be reduced. Thus in
  such experiments by measuring the reassociation
  of DNA hybrids by spectrophotometric analysis to
  detect double-stranded molecules over time, the
  extent to which reassociation of the community
  DNA occurs can be determined, and used to provide
  estimates of overall species diversity.

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• Estimated that there were as many as 4000
  different genomes within 1 g of soil, and this
  estimate is now widely reported in research that
  describes the complexity of environmental
  bacterial communities.

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• Subsequently, variations on this approach have
  been developed to investigate similarities
  between bacterial communities from different
  environmental samples.

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• Community DNA is extracted from each sample and
  then denatured and cross-hybridized following
  randomly primed-based labeling of the
  restriction-digested community DNA . The extent
  to which the community DNAs cross-hybridize
  provides an estimate of the similarities of the
  communities. When combined with additional
  denaturation-reassociation analysis, this can
  generate rapid estimates of community complexity.

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• For example Griffiths et al. estimated that a
  series of four agricultural soils showed
  similarities ranging from 35 to 75.

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8.3.2.1 16S rRNA-based oligonucleotide probe
analysis of bacterial communities

• A number of studies have utilized the same series
  of probes as FISH to investigate relative
  abundances of the domains Bacteria, Archaea and
  Eukarya, or to detect and enumerate particular
  species of interest.

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8.3.2.2 Screening environmental community DNA
with functional gene probes

• The application of DNA probes to detect
  functional genes in community DNA isolated from
  environmental samples without prior seeding has
  focused on the detection and analysis of genes
  conferring metal resistance or biodegradation
  functions.

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8.3.3 Reverse sample genome probing (RSGP) and
microarray analysis of microbial communities.

• Conventionally, in most hybridization experiments
  the target DNA (i.e. the DNA sequence that is
  being screened for the presence of a particular
  gene of interest) is immobilized on a membrane,
  whilst the DNA probe is supplied in solution.

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• However, this limits the number of probes that
  can be screened in any single hybridization
  experiment and typically necessitates the
  requirement for stripping of the initial
  hybridized probe from the membrane with
  subsequent probing with a second probe.

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• In RSGP the opposite approach is taken, whereby
  the probe sequence is attached to the membrane
  and the community DNA being screened is labeled
  and supplied in solution.

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Microarray analysis

• Microarrays utilize essentially the same system
  as for RSGP, wherein it is the probe sequence
  that is immobilized on a solid support, either on
  a hybridization membrane or increasingly glass
  slides, as are routinely used in transcriptomic
  analysis of sequenced bacterial genomes.

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• There have been a number of recent reports
  describing array-based analysis of total
  community DNA. One of the earliest studies
  focused on the application of 16S rRNA
  oligonucleotide probes which was used to study
  nitrifying bacteria.

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The development of arrays to screen for
functional genes

• A simple macroarray was developed containing a
  series of nifH sequences to investigate
  abundances of particular components of
  diazotrophs in marine waters.
• (nifH is the marker gene which encodes
  nitrogenase reductase)

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• A biodegradation array consisting of gt1000
  oligonucleotide probes has been successfully
  tested with both bacterial cultures, microcosm
  enrichments and bulk community DNA.

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• This microarray has allowed detection of a wide
  range of biodegradation genes, and has
  demonstrated that specific detection of multiple
  targets from environmental community DNA is
  achievable.

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