Other PCR Applications

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Other PCR Applications

• cDNA / Reverse Transcription PCR
• Simple
• mRNA / poly-A viral RNA
• PCR-generated RFLPs
• Ancient DNA PCR
• Real-Time PCR
• Random Amplification of Polymorphic DNA
• mRNA Representational Display

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cDNA / Reverse Transcription PCR

• Amplification of DNA from an RNA template via
  cDNA made using reverse transcriptase
• Simple any RNA - use specific primer to make
  cDNA amplify using two specific primers

5-SPECIFIC
PCR amplify DNA
3-SPECIFIC
Denature, anneal 2ndspecific primer to cDNA
Anneal primerto RNA
Elongate withRT

• Eukaryotic mRNA / poly-A virus RNA specific use
  non-specific (anchored) primer to make cDNA
  amplify using one specific primer

Amplify cDNAby PCR
5-SPECIFIC
NTTTTTTTT-5
Anneal primerto RNA
Elongate withRT
Denature, anneal 2ndspecific primer
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• Examples of PCR-RFLP for Nematode Diagnostics
• Nematode ITS1 Size Variation
• ITS1 Size Polymorphism 11 Tylenchid Genera
• Nematode rDNA ITS1 Size Variation
• PCR-RFLP Variation Among Several Cyst Nematode
  Species
• PCR-RFLP Variation Among Several Cyst Nematode
  Species
• PP Presentation
• PCR-RFLP Variation Between Potato Cyst and
  Tobacco Cyst Nematodes
• PCR-RFLP Variation Between Soybean Cyst Nematode
  (SCN) and Sugar Beet Cyst Nematode (SBCN)
• PCR-RFLP Variation Among Individual Sugar Beet
  Cyst Nematodes (SBCN)
• PCR-RFLP Variation of Corn Cyst Nematode,
  Heterodera zeae

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• PCR-RFLP
• Generate PCR products of defined size from
  related DNA / RNA sequences
• Cut with a panel of restriction endonucleases
• Run on gels, stain
• Recognise distinct restriction patterns
• Group samples accordingly

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scientists are starting to ask new questions of
ancient DNA (aDNA) that are revealing how the
genetic make-up of prehistoric populations
changed through time. These findings look set to
trounce assumptions about how evolution really
unfolded.
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Introducing the
Next-Generation Performance and More Capabilities
www.corbettlifescience.com
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SYBR Green I

• Generic dsDNA intercalation dye
• Inexpensive simple
• Used for real-time PCR product detection
• Used for DNA dissociation (melt) analysis
• Widely used

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Cross-section of rotary optics
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Rotary optics 3D animation
rotor spins tubes at 400 rpm
filter set (rotates for each channel)
sensitive PMT (photomultiplier) detector
lens
LED light source (rotates for each channel)
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Heating mechanism
Heater elements switch on
Centrifugal fan drives air around chamber
Chamber vent seals to contain air
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Cooling mechanism
Heater elements switch off
Centrifugal fan drives air around chamber
Chamber vent opens expelling hot air
Centrifugal fan Drives air into chamber
Cool air in
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Real-Time PCR
Fluorescence of 10 samplessimultaneously
Cycle number
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DNA Concentration Measurement

• The Rotor-Gene is fully equipped to do DNA
  concentration measurement using fluorescent dyes
• Using a standard run protocol and integrated
  analysis software, the concentration of unknown
  samples is determined from a standard curve.

Fluorescence
Concentration pg/µL
A DNA standard curve with replicates is shown.
Curve interpolated using a spline curve fit
(Rotor-Gene analysis software). Data was obtained
using reagents in the Quant-iT PicoGreen dsDNA
Kit (Invitrogen Corp., Carlsbad CA). Standard
Rotor-Gene concentration analysis run protocol
was used. 10 µL PicoGreen (diluted1/200 in 1? TE
buffer) was combined with 10 µL of each standard
(diluted in 1? TE buffer). Final volume 20 µL.
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SYBR melt analysis of a DNA fragment
Raw data plot Fluorescence drops as DNA melts and
SYBR is released
Derivative data plot This rate curve peaks at
maximum dissociation rate which is indicative of
the Tm (temperature of melting)
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SYBR melts can reflect product size
500 bp fragment
500 bp fragment
250 bp fragment
250 bp fragment
Raw data plotfluorescence vs. temp.
Derivative data plotdF/dT vs. temp
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SYBR Green I melts can reflect
sequencedetection of alleles
Allele A
Allele A
Allele B
Allele B
Primer Dimer
High primer conc (900 nM) Primer-dimer appears
as a third species
Low primer conc (50 nM) Single band contains two
species (alleles)
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Random Amplification of Polymorphic DNA

• RAPD (Random Amplified Polymorphic DNA) is a
  polymorphism assay based on the amplification of
  random DNA segments using sets of short primers
  of arbitrary nucleotide sequence.
• "poly" "many"
• "morphic" "shapes"
• The polymorphisms are detected as DNA segments
  which amplify from one parent but not from the
  other
• They can most usefully be used to fingerprint
  individuals, groups of individuals, whole species
  or genera.
• They can be used to construct genetic maps in a
  variety of species.
• They can also be used for differentiation of
  prokaryotic species.

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• So how does it WORK??
• Random decamers (10-mer oligos) are used
  SINGLY
• Complexity 410 1 048 576. If primers anneal
  perfectly
• 4 probable sites in E coli genome
• 4000 probable sites in Zea mays genome

2. Anneal primer, PCR
3. Run gel, analyse
1. Extract genomic DNA
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4. Repeat for new species DNA
2. Anneal primer, PCR
3. Run gel, analyse
1. Extract genomic DNA
5. Polymorphisms detected
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mRNA Representational Display

• Also known as Differential Display a powerful
  means of analysing differences in gene expression
  in terms of mRNA between
• The same cells / tissues under different stimuli
• Different cells / tissues under the same stimuli
• And for isolating the polymorphic cDNA afterwards
  for further analysis
• Identification of and whole-gene isolation
• Microarray screening / Protein expression / etc
• Arthur Pardee and Peng Liang, 1992. Science
  257967 U.S. Patent 5,262,311).

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How does it work

• Uses the same principle as both poly-A
  RNA-specific cDNA PCR and RAPDs
• Non-sequence-specific degenerate anchored
  oligo-dT-dependent cDNA synthesis
• PCR using oligo-dT and short random second-strand
  primers
• Degenerate anchored oligo-dT primers
• 3OH-NNTTTTTTTTTTTTTTTTTTTT-5 (18-20 Ts) or

AATTTTTT. GATTTTTT. CATTTTTT. TATTTTTT.
AGTTTTTT. GGTTTTTT. CGTTTTTT. TGTTTTTT.
ACTTTTTT. GCTTTTTT. CCTTTTTT. TCTTTTTT.
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Amplify cDNAby PCR
5-RANDOM
NNTTTTTTT-5
Run on acrylamide gelwith labelled
NN-oligo-dTor Southern blot
Anneal primerto RNA
Elongate withRT
Denature, anneal 2ndNON-specific primer
Liang et al. 2007
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Proc Natl Acad Sci U S A. 2001 February 27
98(5) 26462651. High-sensitivity array
analysis of gene expression for the early
detection of disseminated breast tumor cells in
peripheral bloodKatherine J. Martin, Edgard
Graner, Yi Li, Laura M. Price, Brian M.
Kritzman, Marcia V. Fournier, Esther Rhei, and
Arthur B. Pardee
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