BLAT: Molecular and Immunological Methods

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BLAT Molecular and Immunological Methods

• Lyle McMillen
• Contact lylemcm_at_bigpond.net.au

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Molecular and Immunological Methods

• 2 basic approaches will be covered, both based on
  specific interactions found in vivo
• Nucleic acid specificity (DNA and RNA binding)
• Antibody recognition and interactions

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Nucleic acid techniques

• These techniques all depend upon the specific
  nature of nucleic acid interactions.
• Namely, Adenosine forms 2 hydrogen bonds with
  Thymine (DNA) or Uracil (RNA). Guanine form 3
  hydrogen bonds with Cytosine.
• Purines hydrogen bond with pyrimidines.
• So, A is complemented with T/U, while G is
  complemented with C.
• Interactions outside of these specific pairings
  are not stable.
• Specific nature of these pairings allows one
  strand of DNA or RNA to specify the nucleic acid
  sequence of a complementary sequence.

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Base pairing
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PCR a quick review

• DNA is replicated and transcribed to RNA in vivo
  by DNA or RNA polymerases, which covalently bond
  single nucleotides (deoxynucleoside triphosphates
  dA, dC, dG, and dT or dU) into a complementary
  sequence to the single stranded DNA template.
• Each strand of DNA serves as a template for the
  synthesis of a second, complementary strand of
  DNA.

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PCR a quick review

• The use of DNA polymerases allowed duplication of
  DNA when used in conjunction with a pair of
  primers complementary to the ends of the target
  DNA sequence. Unfortunately the polymerase
  (isolated from E. coli) degraded rapidly at high
  temperatures, and high temperatures were needed
  to denature the double stranded DNA produced, and
  allow more DNA replication.
• The discovery of a thermostable DNA polymerase in
  Thermus aquaticus allowed its inclusion in a
  series of repeated thermal cycles, in which the
  DNA was denatured to single strands, the primers
  annealed, and the Taq polymerase allowed to
  synthesise new complementary DNA.
• Since the discovery of Taq DNA polymerase, a
  number of alternative thermostable DNA
  polymerases have been discovered or engineered to
  provide different characteristics and performance
  in PCR.

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Taq polymerase

• Taq polymerase activities
• Activity optimum at 75-80 ºC
• 5-3 DNA polymerase (100 bases/second)
• No 3-5 exonuclease activity (ie no
  proofreading, and an error rate of 1 in 9000
  bases)
• Low 5-3 exonuclease activity
• Polyadenylates at the 3 end, creating 3-dA
  overhang

Taq polymerase 94 kDa monomer
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PCR a quick review
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DNA sequencing a variant PCR

• As DNA polymerases synthesise a second strand of
  DNA complementary to the sequence of the template
  strand, dNTPs are covalently linked to the
  growing polymer in a specific order.
• A modified PCR reaction is used to determine the
  order in which these nucleotides are added to the
  DNA polymer DNA sequencing.
• Addition of dideoxynucleotides (ddNTPs, lacking
  the 3-OH required for formation of the
  phosphodiester bond between 2 nucleotides) in a
  low concentration to the mix terminates extension
  of the DNA polymer at random points. A series of
  fragments terminated at random points in the DNA
  sequence are generated.

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DNA sequencing a variant PCR
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Key concept Reporter molecules

• DNA and RNA are fairly hard to see in a research
  environment, particularly in low concentrations.
• A variety of reporter molecules, or labels, are
  used to make DNA/RNA easier to detect.
• Fall into 2 broad categories
• Molecules which bind to NAs and fluoresce (Dyes)
  used in agarose gels and some other
  applications. Examples Ethidium bromide, GelRed,
  SYBR green
• Modified nucleotides which have an integral
  label, which are incorporated into the DNA or RNA
  (labels). Examples Radioisotope (35S or 32P)
  labelled dNTPs, fluorescently tagged ddNTPs

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DNA sequencing a variant PCR

• Historically, radioactively labelled dATP was
  included in four separate sequencing mixes, along
  with one of four ddNTPs (which terminate
  extension when incorporated) in low
  concentrations. This was the Sanger, or dideoxy
  terminator, method, developed by Frederick Sanger
  and colleagues in the UK in 1975.
• Each mix generates a population of varying length
  DNAs, radioactively labelled, which start with
  the primer sequence.
• These mixed populations could be separated on the
  basis of size (and therefore number of bases) by
  gel electrophoresis on a denaturing
  polyacrylamide-urea gel, and the different sized
  fragments visualized on an autoradiograph.
• The terminal nucleotide for each fragment was
  determined by which ddNTP was incorporated into
  the reaction.

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DNA sequencing a variant PCR

• A number of limitations arise from this
  technique.
• 4 datasets per DNA fragment, which need to be
  intregrated.
• Data collected manually.
• Short lengths of sequence data - generally
  200-300 bases was as much as could be
  realistically achieved, although 500-800 bases
  were possible.
• Radioisotopes present a hazard to researchers and
  a problem for waste disposal.

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Reporter molecules Fluorophores, fluorescent
labels and dyes

• A fluorophore is a portion of a molecule which
  causes that molecule to be fluorescent. Its a
  functional group which absorbs a specific
  wavelength of light and re-emits the energy at a
  different, specific wavelength.
• The wavelength absorbed is the excitation
  frequency, while the wavelength emitted is the
  emission frequency.
• The wavelength shift is due to a loss in energy
  as heat, resulting in the emission of a longer
  wavelength photon. This is a Stokes shift.
• Fluorescent labels bind specifically to the
  target molecule, and include a fluorophore. They
  bind specifically to a target nucleic acid
  sequence.
• Fluorescent dyes bind to the target molecule type
  (eg. All DNA, or all double stranded DNA), but
  binding is not dependent on the target sequence.
  Dyes also include a fluorophore functional group.

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Reporter molecules Fluorophores, fluorescent
labels and dyes

• Examples include
• Fluorescein and the derivative Fluorescein
  isothiocyanate Excitation at 494 nm, emission at
  521 nm. Fluorescent dye or fluorophore used in
  immunohistochemistry and Fluorescent In-Situ
  Hybridisation (FISH)
• Ethidium Bromide (EtBr) A nucleic acid dye
  commonly used to stain DNA in electrophoresis.
• SYBR green A nucleic acid dye, that fluoresces
  when intercalated in double-stranded DNA.
  Typically excited at one of three wavelengths
  (290 nm, 380 nm, and 497 nm), and emits at 520
  nm.
• Dichlororhodamine A range of fluorophores with
  different emission spectra. Used to label dNTPs
• 6-carboxyfluorescein (6-FAM) Fluorophore used to
  label oligonucleotide in real time PCR.

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DNA sequencing current technologies
Dichlororhodamine dyes are used to label ddNTPs
in a dideoxy terminator reaction. Each ddNTP is
labelled with a particular variant dye, with
different emission wavelengths (i.e. Different
colour), resulting in a single reaction
generating random fragments, with each fragment
labelled with a dye that corresponds to the
terminal base.
Dichlororhodamine dye
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DNA sequencing current technologies

• These fragments can be separated on a gel or
  using capillary gel electrophoresis. Detection
  is via a laser filtered to the dye excitation
  wavelengths, with a corresponding emission
  wavelength filter to detect any fluorescence.
• Generates a chromatographic trace of the four
  emission wavelengths (corresponding to the four
  labelled ddNTPs).

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DNA sequencing current technologies
This trace is easily interpreted, with each peak
corresponding to the terminal base on the
labelled DNA fragment.
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DNA sequencing current technologies

• This technology presents a number of advantages
  compared to radioisotope labelling approaches
• Single tube reaction vs 4 reactions/sample.
• Automated data collection, into a single data set
  vs manual data collection, collating 4 data sets.
• Generally able to read 800-1200 bases/reaction vs
  200-300/reaction.
• No significant hazardous waste vs radioisotope
  waste.

A number of high throughput sequencing
technologies are being developed, with the goal
of sequencing millions of bases very rapidly.
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PCR end-point analysis

• Conventional PCR is typically analysed by
  electrophoresis and visualisation of the amplicon
  (PCR product) on an agarose gel. Visualisation
  is achieved through the use of a fluorescent dye
  such as ethidium bromide.
• This occurs at the end of the PCR reaction. This
  is an end-point analysis.

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PCR end-point analysis
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PCR kinetics

• Three distinct phases during a PCR reaction.
• Exponential phase exact doubling of product
  every cycle (assuming 100 efficiency). Very
  specific and precise.
• Linear phase highly variable, with reaction
  components starting to be consumed, products
  degrade, and the reaction is slowing. The extent
  of slowing will vary from replicate to replicate.
• Plateau/end-point the reaction has stopped, and
  no more products are being prepared. Product may
  begin to degrade. Final yield will vary
  significantly between replicates.

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PCR kinetics
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PCR kinetics

• So, conventional PCR (via end-point analysis) is
  not an accurate way to quantitate the PCR
  template. It is also limited in its ability to
  quantitate different yields of amplicon using
  staining.
• It would be preferable to measure the
  accumulation of amplicon during the exponential
  phase, when the rate limiting factors are the
  amount of template and efficiency of
  amplification.

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Real time PCR

• Also called quantitative or kinetic PCR (but not
  RT-PCR, which is Reverse Transcriptase PCR).
• Adds a reporter molecule to a PCR reaction,
  allowing detection of the amplicon through the
  course of the PCR. This is the most important
  difference to conventional PCR methodologies.
  These reporter molecules are attached to primers,
  oligonucleotide probes, or the amplicon,
  conferring fluorescent potential on these
  molecules.
• Reporter molecules are fluorescent molecules, and
  are detected using a fluorescent
  spectrophotometer in the real time PCR platform.
• Two broad categories of reporter molecule they
  interact either specifically (labels) or
  non-specifically (dyes) with the amplicons
  nucleotide sequence.
• Quantitative analysis is based on detection of
  the amplicon during the exponential phase of the
  PCR.
• Data is presented as the thermal cycle at which
  the level of fluorescence reaches an arbitrary
  threshold, set within the exponential phase of
  the PCR. This is referred to as the CT value.

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Real time PCR

• So, how does it work?
• Two commonly used approaches.
• Double stranded DNA detection
• This approach utilises a fluorescent dye which
  specifically binds to double stranded DNA
  (intercalating agent) SYBR green, and later
  derivatives such as SYBR greener, LC green 1,
  SYTO 9, EVA Green.
• The PCR proceeds as normal, and the dye
  intercalates into the double stranded amplicon.
• The more amplicon is produced, the more dye is
  intercalated.
• As these dyes intercalate, their emission
  intensity increases (over 100-fold for SYBR
  green), due to conformational changes on binding.
• It is worth noting that SYBR green is toxic to
  PCR, and is therefore used at extremely low
  concentrations. There are saturation dyes
  available that are not toxic, and can be used at
  higher concentrations giving stronger
  fluorescence.

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Real time PCR
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Real time PCR

• The second major approach utilises hydrolysis of
  a specific oligonucleotide containing a
  fluorescent label often called the TaqMan
  method, but also called 5 nuclease, Taq nuclease
  or dual-labelled probes.
• Taq polymerase has a 5-3 exonuclease activity.
  Hydrolyses DNA on the same strand as the newly
  synthesised DNA.
• The oligonucleotide probe contains 2 functional
  groups a 5 fluorophore, and a 3 fluorophore
  (e.g. TAMRA) or non-fluorescent quencher (NFQ).
  Energy generated by the excitation of the 5
  fluorophore is captured by the 3 quencher, and
  emitted as fluorescence or heat (NFQ). If a
  second fluorophore is the quencher, the emission
  wavelength is different to that of the 5
  fluorophore. This process is called Fluorescence
  Resonance Energy Transfer, or FRET.
• The probe anneals to the target region
  specifically. As the Taq polymerase synthesises
  DNA, it hydrolyses the probe. Cleavage of the 5
  fluorophore from the rest of the probe enables it
  to emit fluorescence, which can be detected.
• The level of fluorescence detected is
  proportional to amount of probe hydrolysis, and
  therefore the amount of amplicon synthesised.

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Real time PCR
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Real time PCR alternative probe strategies

• There are a number of other probe strategies
  available, many of which are patented.
• Hybridisation probing entail using two probes,
  each labelled with a different fluorophore
  (typically 6-FAM and a red fluorophore).
• These probes hybridise within 1-5 bases of each
  other on the amplicon.
• Excitation of the first fluorophore allows the
  excitation of the second fluorophore via FRET.
• Leads to fluorescence at the second fluorophores
  emission wavelength (610, 640, 670 or 705 nm,
  depending on fluorophore), while exciting using
  the wavelength of the first (470 nm).
• Detection occurs at the end of annealing step.
• Once detection is complete, an increase in
  temperature triggers DNA polymerase activity,
  displacing probe and amplifying the target
  region.
• Note that when not hybridised, the first
  fluorophore will emit fluorescence in its
  emission wavelength (530 nm for 6-FAM).

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Hybridisation probes
Annealing
Denaturation
Extension
Completion
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Real time PCR platforms

• The real time PCR platform consists of a few
  basic elements
• Thermal cycler basically a PCR machine, usually
  capable of rapid and precise variations in
  temperature (usually between 15 and 99 ºC).
• Excitation wavelength emitter, capable of
  transmitting the excitation wavelength of the
  fluorescent reporter to each sample.
• Emission detector, capable of precise
  quantitation of the amount of fluorescence being
  emitted by the sample at the fluorophores
  emission wavelength.
• Data recorder, recording the fluorescence from
  each sample at the end point of each thermal
  cycle (end of extension step).

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Real time PCR platforms

• There are a few major types of real time PCR
  platform from a range of suppliers, but they all
  perform the same function.
• Most are capable of managing multiple
  fluorophores simultaneously, allowing multiple
  amplicons to be probed in a multiplex assay
  (well discuss this in more detail later).
• All are also associated with sophisticated data
  management and analysis software, which makes
  data analysis easy, reliable and reproducible.
• Raw data integrity is always protected
  important for clinical and diagnostic
  applications.

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Data output
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Real time PCR applications

• The most obvious application of real time PCR is
  for detection and quantitation of a specific DNA
  sequence.
• May also be used for monitoring changes in gene
  expression, genotyping, or detection of genetic
  variations such as single nucleotide
  polymorphisms (SNPs).

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Quantitative real time PCR

• Real time PCR data is presented as CT (Cycle
  threshold) values, defined as the thermal cycle
  at which the fluorescence reaches an arbitrary
  threshold.
• If a series of samples with known concentrations
  of initial template DNA is included in the assay,
  a linear plot of CT vs log initial template may
  be generated.
• These standards can be a known number of cells, a
  defined number of copies of a plasmid, or any
  other defined, quantifiable and reproducible
  number of target templates.
• This plot permits linear regression analysis,
  allowing the calculation of the copy number of
  any unknown target relative to the standards.
• The plot also indicates amplification efficiency
  (slope) and some indication of sensitivity
  (y-intercept).

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Quantitative real time PCR
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Quantitative real time PCR

• There are 4 basic assumptions underlying
  quantitation by real time PCR
• The initial template is double stranded.
• When analysing RNA, reverse transcriptase produce
  single stranded cDNA, which is made double
  stranded in the first amplification cycle. True
  amplification begins in cycle 2.
• PCR efficiency is 100, and both strands of all
  templates are copied into full length copies each
  cycle.
• This never happens, due to inefficient primer
  hybridisation, template folding and probe and dye
  interference.
• PCR efficiency is constant throughout the
  amplification process.
• Secondary structures may inhibit amplification
  from long templates such as genomic DNA or from
  supercoiled plasmids, mitochondria and bacterial
  genomes.
• Compare with standards based on the same starting
  material.
• Fluorescence is proportional to the amount of
  template.
• This depends on the dye used, the sequence
  amplified, the length of the amplicon, the
  optical properties of the platform, data
  acquisition and instrument settings.

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Gene expression analysis

• One application of quantitative real time PCR is
  analysis of gene expression in different tissues
  or under different treatment regimes.
• mRNA expression from the gene of interest is
  quantitated from each sample using a real time
  RT-PCR.
• These are real time PCRs performed on cDNA,
  generated from RNA (extracted from the target
  tissue) by reverse transcriptase. The reverse
  transcriptase primer can be the same as one of
  the real time PCR primers, or be just outside the
  real time PCR amplicon. Only one primer is
  needed.
• Reverse transcriptase efficiency is a significant
  contributor to variability observed in real time
  RT-PCR, and needs to be taken into account when
  developing any real time RT-PCR method.
• Samples are normalised to the level of expression
  of a house-keeping gene. These are genes that
  are always expressed at constant levels in each
  cell, thought to be involved in routine cellular
  metabolism. e.g. glyceraldehyde-3-phosphate
  dehydrogenase (G2PDH or GAPDH), beta actin, some
  ribosomal proteins.
• This allows proportional comparison of target
  mRNA levels between samples.

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Genotyping

• A range of real time PCR methods can be used to
  determine genotype of a target amplicon.
• The simplest approach relies upon determining the
  melting temperature of the amplicon using a
  melting curve.
• The real time PCR is performed as normal,
  incorporating a non-hydrolysed probe or dye
  typically performed with SYBR Green or a
  saturation dye such as SYTO 9 or LC Green 1.
• Once the amplification program is complete (and
  quantitation data collected), the samples is
  heated through a gradient, with fluorescence data
  gathered at set temperature intervals (typically
  every 1 ºC, but can be as often as every 0.2 ºC
  in high resolution equipment). The gradient is
  typically from 50 ºC to 95 ºC, but can be refined
  to a narrower range.
• As the temperature increases, the amplicon will
  denature, unzipping from double stranded to
  single stranded. The fluorescence of the dyes
  will decrease as more of the amplicon denatures.
• The temperature at which this decrease in
  fluorescence is at its fastest is called the
  melting temperature (TM), and varies with the
  GC and sequence of the amplicon. Determined by
  plotting reduction in fluorescence against change
  in temperature (dF/dT).
• Different genotypes of the amplicon will have
  different TMs. TM analysis is also used to
  ensure that the desired target has been
  amplified.

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Genotyping
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SNP analysis a specific form of genotyping

• Single nucleotide polymorphisms (SNPs) are the
  most common form of genetic variation.
• SNPs are a single base variation at a specific
  locus within a gene, usually consisting of two
  alleles. The rare allele is generally present in
  1 of the population.
• The SNP may be in the coding sequence, non-coding
  region, or intergenic regions, and can have
  impacts on polypeptide sequence, gene splicing ,
  transcription factor binding or non-coding RNA
  sequence.
• They can be detected through melting curve
  analysis , with specific patterns being generated
  for each homozygote (both diploid alleles
  containing the wild type or mutant) and for a
  heterozygote (one wild type and one mutant
  allelle).
• Heterozygote amplifications include both alleles
  in the one reaction, and they will anneal, but do
  not match perfectly (a heteroduplex). As a
  consequence, the TM of the heteroduplex will be
  lower than that of the amplicons from the
  homozygotes.

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SNP analysis
NB This plot is normalised to the mutant allele
melting curve.
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SNP analysis

• A second option involves the inclusion of
  hydrolysis probes for each allele in the one
  reaction. Each hydrolysis probe contains a
  different fluorophore with distinct excitation
  and emission wavelengths. This is an example of
  a multiplex assay.
• If probe design is right and reaction conditions
  are stringent enough, a single base variation
  from the probe target will be sufficient to
  prevent annealing of the probe to the variant
  sequence.
• Amplification of each allele will be reported by
  the probe specific for that allele.
• Multiple SNPs can interrogated simultaneously,
  although this is limited by the number of
  distinct probes available, the optimisation of
  reaction conditions, and the capabilities of the
  real time PCR platform.

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Real time PCR design considerations

• In order to have the most efficient amplification
  and detection possible, a number of guidelines
  have been developed for optimum real time PCR
  assay design.
• The length and structure of the amplicon are
  fundamental to good real time PCR design. In
  general, real time PCR amplicons are very short
  compared to conventional PCR amplicons (70-300
  bp).
• GC of the amplicon should be between 30-80,
  and runs of identical nucleotides, particularly 4
  or more Gs, should be avoided.
• Primers should not be complementary to themselves
  or each other to avoid primer-dimer formation.
• Primer TMs should be within 2 ºC of each other
  (58-60 ºC is optimal), and primers should be
  18-22 nucleotides long.
• Number of Gs and Cs in the last 5 bases of the
  3 end of the primer should not exceed 2.
• Hydrolysis probes should have a TM 10 ºC higher
  than the primers (but not above 75 ºC), and
  should anneal close to the primer on the same
  strand.
• Hydrolysis probes should be 20-30 bases long,
  unless stabilised with a minor groove binder
  moiety (a tricyclic functional group that folds
  back into the minor groove of the probe-target
  duplex and stabilises the interaction). MGB
  probes may be only 13-18 bases long, and still
  achieve the desired TM.
• The probe should not have a G at the 5 end, to
  avoid unpaired Gs quenching fluorescence, and
  there should be more Cs than Gs (increases the
  change in fluorescence when the probe is
  hydrolysed).

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Real time PCR design considerations

• Fortunately, there are a number of software tools
  available (e.g. PrimerExpress, Oligo 6.0, Vector
  NTI) which can generate a number of design
  options from a DNA sequence. These possible
  designs will meet the basic rules for assay
  design, and will need to be checked manually by
  the researcher to ensure they are suitable for
  the specific application.

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Another use for fluorescent probes - microarrays

• A DNA microarray is a series of microscopic spots
  of specific oligonucleotides (typically stretches
  of a gene) covalently bound to a matrix (ie. A
  slide or chip).
• Under high stringency conditions, only a
  complementary sequence will bind to these probes.
• If the sample to be probed is fluorescently
  labelled, array sites containing probes that bind
  the sample will fluoresce.

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Another use for fluorescent probes - microarrays

• Typical uses include gene expression profiling,
  comparing genome content, and SNP detection.
• In gene expression profiling, mRNA is isolated
  from two samples, and cDNA is prepared by reverse
  transcriptase.
• During cDNA synthesis, fluorescently labelled
  nucleotides are incorporated different
  fluorophores are used in each sample. Cy3
  (emission at 570nm, or green) and Cy5 (emission
  at 670 nm, or red) are commonly used.
• The cDNA samples are then hybridised to the
  microarray, containing thousands of oligos
  specific to individual genes in known locations
  on the array.
• Fluorescence is measured at each location on the
  array, and variations in expressed genes are
  identified.
• Note that if both Cy3 and Cy5-labelled cDNA bind
  to an array location, the spot appears yellow.
  This gives four possible outcomes
• Black no expression of that gene in either
  sample.
• Red or green expression of that gene in only
  one of the samples.
• Yellow expression of the gene in both samples.
• Housekeeping genes are always included as a
  reference.

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Microarrays
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Microarrays
A 40000 spot two-colour oligo microarray.