Describe the Methods Available for Determining Copy Number Changes of Genes or Fragments of Genes. Briefly Describe the Relative Advantages and Disadvantages of these Methods.

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Describe the Methods Available for Determining
Copy Number Changes of Genes or Fragments of
Genes. Briefly Describe the Relative Advantages
and Disadvantages of these Methods.

• 20/12/2007

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Methods include

• MAPH (multiplex amplifiable probe hybridisation)
• MLPA (multiplex ligation-dependant probe
  amplification)
• Microarrays
• QPCR (quantitative PCR)
• Realtime PCR
• Long-range PCR
• Southern blotting
• PFGE (Pulsed field gel electrophoresis)
• Linked markers (minisatellites/SNPs)

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MAPH

• Genomic DNA from the patient is hybridised to a
  (small!) membrane eg filter paper and excess
  probe washed off
• Probes are generated which correspond to the
  sequences being interrogated (eg exon sequences).
  They are constructed by cloning or PCR and have
  common flanking sequences at each end (universal
  primer tags) they are also of different lengths
• Probes are hybridised to the target DNA overnight
• Membrane is washed to release the unbound probes.
  (The remaining bound probes are present in the
  same copy number as the target sequence of the
  patient DNA)
• First round multiplex PCR carried out with
  membrane bound DNA as the template. Product from
  the first round PCR then used to seed a multiplex
  PCR reaction in solution, using universal primers
  (PCR products labelled eg with fluorescent
  primers)
• PCR products are then separated by
  electrophoresis. A relative comparison is made
  between the peak heights/intensities/areas of PCR
  products compared to internal copy number
  controls. A reduction of peak height/area/intensit
  y indicates a deletion, an increase of peak
  height/area/intensity indicates a duplication

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MAPH, advantages/disadvantages

• Advantages
• Can analyse upto 40 probes in one reaction
• Does not require specialised equipment
• Cheap
• Disadvantages
• Method is crude
• (MAPH was the forerunner of MLPA)
• Will only detect rearrangements involving copy
  number changes of probe binding sites

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MLPA

• A set of probes is generated complementary to the
  sequences being interrogated. Each probe is
  flanked by universal primer sequences and
  consists of 2 halves which will eventually become
  ligated. Each probe pair is a different length
• The probes are hybridised to genomic (patient)
  DNA in solution for several hours (upto 16)
• A ligase enzyme is added to ligate each probe
  pair, whilst still hybridised to the target DNA
• The genomic DNA and probes are denatured to
  produce single stranded DNA
• Multiplex PCR carried out with universal primers
  which amplify only from ligated probes (no
  product produced from unbound or unligated
  probes). The amount of PCR product from each
  probe will be proportional to the copy number of
  the target sequence originally present
• Universal primers are labelled with a fluorescent
  dye. PCR products are separated by capillary or
  gel electrophoresis. The results are analysed by
  comparing peak heights/areas to internal controls
  to detect copy number changes. Several templates
  available for analysis including those designed
  by Andrew Wallace (MNGRL), coffalyser etc

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MLPA
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• Insert MLPA results

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MLPA

• Advantages
• Many kits commercially available (quality
  control)
• Does not require any specialised equipment
• Can analyse upto 40 target sites simultaneously
• Relatively quick and simple
• Can be adapted to be methylation-sensitive eg
  PWS/AS
• Disadvantages
• Ligation-dependant (unlike MAPH)
• Will only detect copy number changes involving
  probe binding sites

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Microarrays

• Probes complementary to sequences being
  interrogated are attached to a glass slide,
  (produced by PCR or cloning)
• Patient genomic DNA and control DNA are sheared
  into smaller fragments and labelled with
  different coloured fluorophores, eg patient
  green, control red
• Patient DNA and control DNA are mixed with COT-1
  to block all repetitive sequences
• Patient and control DNA are co-hybridised to the
  array, and excess DNA washed off
• Array is scanned and ratio of redgreen
  fluorescence for each target is measured.
  Deletions show up as a ratio of 21 redgreen and
  duplications show up as a ratio of eg. 23
  redgreen. 11 ratio (normal shows up as orange)

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Microarrays

• Scan in array picture page 332 armour paper

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Microarrays

• Advantages
• Can analyse thousands of loci in one reaction,
  arrays typically have 10, 000-15,000 targets per
  slide
• Results can be gained in duplicate in one
  reaction (gt1 spot for each target sequence)
• Relatively quick way of screening several genes
  for a diagnosis for eg dev. delay
• Disadvantages
• Copy number changes of one have only recently
  been achieved
• Requires expensive specialised equipment to
  produce arrays, scan etc. Commercial arrays also
  expensive to buy in
• Not an efficient method for single gene screens

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Quantitative PCR (QPCR)

• PCR theoretically amplifies DNA exponentially,
  doubling the number of molecules present with
  each amplification cycle (log phase). After
  approximately 20 cycles (varies according to
  reagent concentration) the DNA amplification
  plateaus (lag phase). When the PCR is in the log
  phase the amount of product present should be
  proportional to the amount of template. The
  amount of PCR product can be measured in several
  ways
• Labelling PCR primers with fluorophores (QFPCR)
  and separating the products by electrophoresis
  (capillary or gel) and measuring the relative
  peak height or area produced (most common method)
• Labelling Primers or PCR product with radioactive
  isotopes and performing densitometry on the bands
  on an autorad (older method, cruder and less
  favoured nowadays!)

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QPCR

• PCR plot

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QPCR

• Advantages
• Cheap, quick and easy
• Reliable
• Disadvantages
• will only detect rearrangments involving copy
  number changes involving primer binding sites

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Real Time PCR

• PCR is carried out in real time allowing the
  increase of DNA to be visualised as the reaction
  takes place
• Several types of real time technology are now
  availaible
• 1. TaqMan
• A probe is constructed which is labelled with two
  fluorophores, (fluorescent parts of reporter
  proteins eg green fluorescent protein GFP). One
  fluorophore is a reporter dye (usually long wave
  length dye eg red) and the other is a quencher
  (short wave colour dye eg green)
• While both dyes are attached to the probe in
  close proximity, the quencher (Q) reduces the
  fluorescence of the reporter (R) by fluorescence
  (Forster) Resonance Energy Transfer (FRET). The
  reporter is normally at the 5 end of the probe
  and the quencher at the 3 end.
• Target DNA is denatured and the TaqMan probe
  binds to its complimentary sequence. PCR primersa
  are then annealled to the target DNA. PCR is
  undertaken. As the reaction continues, the taq
  polymerase displaces the TaqMan probe from the
  template. This separates the quencher from the
  reporter allowing it to fluoresce. The
  fluorescence is measured and is quantified as the
  reaction continues.The fluorescence is shown on a
  graph display (PCR cycle X axis, Log of
  intensity Y axis)

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Real time PCR

• Figure 1. The Taqman probe. The red circle
  represents the quenching dye that disrupts the
  observable signal from the reporter dye (green
  circle) when it is within a short distance.

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Real time PCR

• Figure 2. The TaqMan probe binds to the target
  DNA, and the primer binds as well. Because the
  primer is bound, Taq polymerase can now create a
  complementary strand.  

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Real time PCR

• Figure 3. The reporter dye is released from the
  extending double-stranded DNA created by the Taq
  polymerase. Away from the quenching dye, the
  light emitted from the reporter dye in an excited
  state can now be observed.

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Real time PCR

• Figure 4. A graph printout of actual data found
  using the TaqMan probe. Courtesy
  www.biotech.uiuc.edu.

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Real time PCR
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Real Time PCR

• 2. Molecular Beacons
• Uses a reporter probe that forms a hairpin
  structure. As in TaqMan there is a reporter and
  quencher fluorophore on either end which are
  brought into close proximity by the hairpin
  formation.
• The probe hairpin is complementary to the target
  sequence
• PCR is carried out and the newly synthesised
  strands and the hairpin probe are denatured and
  the molecular beacon binds to the PCR product.
  This physically separates the quencher from the
  reporter, allowing it to fluoresce. Unlike
  TaqMan, molecular beacons remain intact. The
  emission is measured as for TaqMan, as the no.
  PCR cylcles increases, the amount of fluorescence
  increases.

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Molecular Beacon

• Figure 1. Diagram of molecular beacon. This
  beacon is 33 nucleotides long with a reporter dye
  attached to the 5' end and a quencher attached to
  the 3' end. The nine 5' bases are able to form
  base pairs with the nine 3' bases which brings
  the reporter and quencher in very close
  proximity. Therefore, when the reporter is
  excited by the appropriate light, its emission is
  absorbed by the quencher and no fluorescence is
  detected. The pink lines represent nucleotides
  that can form base pairs with the PCR product
  under investigation.

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Molecular Beacon

• Figure 2. PCR product of interest. The two
  primers are show as purple arrows and the base
  pairing between the two strands are shown in
  pink.

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Molecular Beacon

• Figure 3. Detection of PCR product by molecular
  beacon. When the beacon binds to the PCR product,
  it is able to fluoresce when excited by the
  appropriate wavelength of light. The amount of
  fluorescence is directly proportional to the
  amount of PCR product amplified.

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Real Time PCR

• 3. SYBER green Probes
• SYBER green binds to double stranded DNA and
  emits DNA when excited. However, SYBER green can
  also bind to template (genomic) DNA, which can
  result in inaccurate data, therefore it has
  limited use in diagnostics

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Real time PCR

• Advantages
• Quick and relatively cheap
• Several reactions can be multiplexed by using
  different fluorecsnt dyes
• Disadvantages
• Requires specialist Real time PCR equipment

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Long Range PCR

• PCR is carried out across a large portion of DNA,
  (upto 27kb can be amplified, but 10-20kb is
  routine) and the products run out on an agarose
  gel. Any deletions or duplications show up as a
  change in expected product size. The reaction
  usually uses a combination of taq polymerase and
  a DNA polymerase with 3-5 proofreading ability
  eg Pwo. This allows longer extension than is
  achievable with taq on its own. Used in the past
  for eg DMD and Mitochondrial analysis

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Long Range PCR

• Advantages
• Quick, cheap,
• Requires no specialised equipment
• Best suited for detecting common large
  deletions/duplications, eg mito deletions, BRCA1
  exon 13 duplication
• Disadvantages
• Some re-arrangments may be missed due to none
  resolution of bands on agarose gel
• Will only detect deletions/duplications resulting
  in gain or loss of DNA (within limits of PCR
  raection) ie not inversions etc

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Southern Blotting

• Genomic DNA is digested with one or more
  restriction enzymes and the resulting fragment
  separated by gel electrophoresis. The DNA is then
  denatured in situ and transferred from the gel to
  a nitrocellulose filter or nylon membrane by
  capillary action. The relative position of the
  DNA bands remains the same during transfer. The
  DNA attached to the filter is hybridised to
  radiolabelled DNA or RNA probes and
  autoradiography is used to locate the band
  positions. Deletions or rearrangemnts are
  detectable by a change in expected band size.

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Southern assay
Methylated smear (full mutation)
M1 F1 M2 F2 F3 M3
Unmethylated X (active) 2.8kb
M1 F1 M2 F2 F3 M3
Hind III/ Eag I
Bgl II
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Southern Blotting

• Advantages
• Can detect rearrangements not involving copy
  number changes
• Disadvantages
• Requires use of radioactivity
• Takes a long time (3-4 weeks)

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PFGE

• Pulsed Field Gel Electrophoresis (PFGE) is a
  technique used to separate especially long
  strands of DNA by length to eg detect
  deletions. In general small fragments can wind
  their way through the gel matrix more easily than
  large DNA fragments, a threshold length exists
  where all large fragments will run at the same
  rate. But with a continuous changing of
  directions every few seconds or fraction of a
  second, the various lengths of DNA react to the
  change at differing rates. That is, larger pieces
  of DNA will be slower to begin moving in the
  opposite direction while smaller pieces will be
  quicker to change direction. Over the course of
  time with the consistent changing of directions,
  each band will begin to separate more and more
  even at very large lengths. Thus separation of
  very large DNA pieces using PFGE is possible.
• DNA is visualised by Southern blotting of the
  agarose gel

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PFGE

• Advantages
• Can detect rearrangements which do not result in
  copy number changes (if the rearrangemnt results
  in a change in digest pattern)
• Disadvantages
• Specialized equipment is required, consisting of
  a gel rig with clamped electrodes in a hexagonal
  design, a chiller and pump, and programmable
  power supply
• Requires high molecular weight DNA which needs a
  gentle extraction method (DNA usually stored in a
  PFGE block)
• Time consuming
• Requires radioactivity

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Linked markers

• Standard linked markers or SNPs can be used to
  detect deletions or copy number changes, by
  comparing the markers/SNPs present in a test DNA
  sample to a control DNA eg LOH studies of
  tumours, where the haplotype of tumour DNA is
  compared to somatic DNA, or SNP analysis used for
  the detection of trisomies in foetuses

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Other methods

• FISH (cytogenetic methods)
• Quantitative dHPLC
• Competitive PCR
• etc, etc, etc!