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Multiplex Amplification of Nucleic Acids April 8, 2010 BME 215 Applied Gene Technology
Multiplex Amplification of Nucleic Acids
April 8, 2010
BME 215Applied Gene Technology
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Number of targets (n)
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(2n^
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The major problem associated with multiplex PCR
n=1
A-AB-BA-B
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A-AB-BA-BC-CD-DC-DA-CA-DB-CB-D
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A-AB-BA-BC-CD-DC-DA-CA-DB-CB-DE-EF-FA-EA-FB-EB-FC-EC-FD-ED-FE-F
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Relative number of cross reactions
The relative number of cross reacted products in a conventional
50.000-plex PCR are
10.000.000.000
Molecular inversion probes Golden Gate
&
Other technologies
Multiplex Amplification of Nucleic acids
• Molecular Inversion Probes was one of the most successful technologies to simultaneously analyze over 20,000 SNPs in a multiplex fashion
• Human genome is over 3 billion base pairs with over 250,000 exons
• Genotyping of known polymorphisms is one way of reducing the complexity of the problem of understanding the variations between genomes – but resequencing would be the ideal method
• Currently there exist several resequencing technologies that require selective amplification of target DNA
• A logical extension of the MIP technology would be to use them for Multiplex PCR
Molecular inversion probes (MIP)
Molecular inversion probes (MIP)
Molecular inversion probes (MIP)
Molecular inversion probes (MIP)
Molecular inversion probes (MIP)
Molecular inversion probes (MIP)
Molecular inversion probes (MIP)
Molecular Inversion Probes
Very specific. High multiplexing capacity from unimolecular action and tag. High sensitivity due to amplification.
PathogenMiper
5’ 100 bp 3’P- H2 BARCODE U2 R U1 H1 -OH
5’- U1 H1 G H2 BARCODE U2 -3’
G
GP- -C
G
PADLOCK PROBESEnzymatic probe inversion• Mixture is heated to denaturate, and slowly cooled to let annealing take place• Free unlabeled dNTPs are added the gap is filled through polymerase activity• Ligase ligates the gap and a circular probe is formed.1) Free DNA is degraded by exonuclease activity2)To release probes, uracil-DNA-glycosylate is added.3) PCR amplification
PathogenMip Assay
Connector Inversion Probe (CIP)
H2 U1RU2 H2P5´ 3´
5´ U1 CLONE DNAH1 H2 3´U2 CLONE DNA H2
CLONE D N A
A C G T
CLONE D N A
MIPs for Larger Gapfills
3’ 5’5’
5’3’3’ 5’
3’
MIPs for Larger Gapfills
Designed test probes for amplifying targets from 160 to 400 bases
Will show data after the next slide
MIPs For Multiplex PCR
Spacer backbonetarget targetAP1 AP2
5’ 3’
5’ 3’
• Spacer Multiplex Amplification ReacTion
SMART Multiplex Amplification
A CB
250 bp
D FE
500 bp
A:150-190 bpB:190-220 bpC:220-240 bpD:240-280 bpE:280-300 bp, :400-460 bpF:300-390 bp
Genome-wide Analysis of Alternative Splicing using MIPs
Alternative Splicing• Alternative splicing is a major source of proteome
complexity
• As many as 74% of multi-exon human genes may have alternative splice forms
• Alternative splicing has been shown to affect numerous protein characteristics including binding properties, intracellular localization, enzymatic activity, stability, and post-translational modifications, and to create dominant negative isoforms
• Splice site selection is regulated during development and in a tissue-specific manner
• Errors in the regulation of splice site selection have been implicated in a wide set of human diseases, especially cancer
• Spliceosomal errors are a common characteristic of cancer cells, and splicing errors have been found in genes that affect both tumor progression and susceptibility
Splicing widespread. Shown to affect every protein characteristic you can think of. May be as important as gene expression in determining cell fate. 15% of disease-associated point mutations cause splicing defects.
Gene-CollectorAnother way to multiplex DNA amplification
Fredriksson et al NAR 2007
Analyzed on custom Affymetrix
resequencing array
all exons in 10 cancer genes
First pass 90% success
The Selector technology
Dahl, F., et al. (2005) Nucleic Acids Res 33, e71.
Illumina - Golden Gate Assay
Illumina - Golden Gate Assay
Illumina - Golden Gate Assay
Illumina - Golden Gate Assay
Illumina
A
G
T
TNT
A
G
C
TNT
A
T
C
TNT
G
T
C
TNT
A
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A
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A
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TNT
SNP Selection
SNPs
Duplicated
regions
Neighboring
SNPs
Low-complexity
regions
Low Allelic
Frequency
Filtering
TNT
Rank 1
for each SNP { }
2736 2396
2968 2086
Length
compensation
Primer
design
Ranking
64
8
2
16
Score
N
!!
!!
SoftwareTNT
Amplification
Cyclic Threading
gDNA
Purification
Excess
primer
In vitro TNT
Whole genome genotyping
Whole Genome AmplificationRandom priming
Adaptor ligation
Target capturing procedures
Gap-fill procedures
Target circularization procedures
The Selector technology
