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Multiplex PCR分享

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陳亮鈞
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8 views22 pages

Multiplex PCR分享

Uploaded by

陳亮鈞
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Case study

• To investigate feeding interactions in the


foreland of the glacier ‘Rotmoosferner’ (Tirol,
Austria) by multiplex PCR(MP) assay

Mitopus glacialis
Pardosa spp. Oreonebria castanea

Collembola

Nebria rufescens
Introduction
• Primers play a crucial role in MP.
• Primers’ performance is influenced by:
• Internal stability
• Build a balanced multiplex PCR system
Melting temperature
• Secondary structure
• Interference with each other
• The variations of amplification efficiencies can lead unbalanced
amplification
• Between different targets
• Between species
Materials and methods
Primer design
• Part of mitochondrial cytochrome c oxidase subunit one gene was
sequenced (710 bp) for all target species.
• Sequences were aligned using BioEdit, and specific primer pairs were
designed using Primer Premier 5.
• QIAxcel was used for separation and visualization of PCR products.
• Can separate products differ as little as 20 bp
• Give electropherograme in which the signal strength of segments are present
in RFU .
QIAxcel
High resolution capillary
electrophoresis system
• Can separate segments
differ as little as 1-20 bp

http://decodingdna.yolasite.com/capillary-
electrophoresis.php
Generating standardized DNA templates
• PCR products were cleaned with QIAquick PCR purification kit
• DNA quantity was measured using Quant-iT PicoGreen (q ng/uL)
• Can quantitate as little as 25 pg/mL of dsDNA
• Molecular weight of each dsDNA sequences were
calculated with following equation:

• The number of dsDNA segment for all targets was


calculated with:
(Copies/uL)
Adjusting multiplex system
• Standardized templates were used (single and mixture)
• Singleplex and multiplex test at the estimated optimal annealing
temperature
• 0.2 uM of primers
• Thermal cycle: 35 cycles
95℃ 94℃ 62.5℃ 72℃ 72℃
15 min 30 sec 90 sec 60 sec 10 min

• Checking there was no non-specific fragments produced


• Gradient multiplex PCR for single and mixture of templates
• Adjust concentration of primers respectively
• ± 0.1 uM
• The strength of signals for all targets were equal
Testing PCR sensitivity & specificity
• To estimate the sensitivity of primer pairs in multiplex system
• The minimum number of dsDNA template copies necessary to amplify
product that resulted in ≧0.1 RFU was identified for all targets
1. Single template
2. All targets mixture at equal concentrations
3. Single template with ~300 ng of non-target DNA
4. All targets mixture with ~300 ng of non-target DNA

• To test whether the primers cross-react with DNA from non-target


animal.
• Wide variety of ground-dwelling and arial arthropods live in that area
were collected for DNA extraction.
Results
Primer efficiency and multiplex PCR
• Primers’ concentration were adjusted respectively.
• Thermal cycle: 35 cycles
95℃ 94℃ 60℃ 72℃ 72℃
15 min 30 sec 3 min 60 sec 10 min
Primer efficiency and multiplex PCR
Single target Mix target
Sensitivity & Specificity
• Sensitivity: 20 – 30 copies of dsDNA templates were sufficient to
amplify detectable signal (≧0.1 RFU) for each target.
• This was independent of whether the target templates were single or mixed.
• Non-target DNA didn’t reduce assay sensitivity.
• Specificity: some non-target organism produced fragments at
expected length, and some individuals produced unspecific segments.
• It was caused by contamination happened in trap.
• Unspecific segments close to expected product can be tested by
corresponding primers.
Discussion
Discussion
Importance of standardization of DNA templates and sensitivity test
for primers
• Avoid biased results towards preferentially amplified targets.
• Allows comparison of results between primers and studies
Discussion
Amplicon size
• High resolution capillary electrophoresis can separate segments very
similar in size
• Agarose electrophoresis: >30 bp for fragments < 300 bp
• Capillary electrophoresis: 1-20 bp
• For fresh tissue samples, it’s not a problem to amplify longer
segments, but it becomes challenging for degraded samples.
• <300 bp for degraded samples
Discussion
Thermal cycle and assay sensitivity
• By doubling annealing time, an increased signal strength was
observed.

=
Signal strength
90 sec 3 min
1 unit template 10 unit template
• 7/1 Compare JEV, CD and other 4
sample amplification in single and mix
situation under multiplex PCR
• JEV and CD templates were amplified
separately, but showed no signal when
mixed with other templates
• Signal strength:
6/17 5/12

Ortho&Noro > Corona&Lyssa > Flavi&Para


End
Introduction
• Multiplex PCR(MP) is increasingly used in biological and medical
studies
• Amplify several DNA fragments within one reaction
• Save time/money
• Reduce possibility of contaminations

• Although this method is widely applied, few papers address


methodological issues and how to improve and standardize MP assay
Introduction
• Primers sensitivity needs to be balanced to avoid biasing detection
rates towards the most sensitive primer pairs.

• By using standardized DNA templates, it’s allowed to balance MP


system and furthermore to compare and standardize sensitivity
between assays.

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