my research experience - isciiimy research experience margarita salas centro de biología molecular...
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![Page 1: My Research Experience - ISCIIIMy Research Experience Margarita Salas Centro de Biología Molecular “Severo Ochoa” CSIC–UA CNIC PhDay November 23, 2018 SEVERO OCHOA (1905-1993)](https://reader035.vdocuments.net/reader035/viewer/2022062919/5ee00099ad6a402d666b4462/html5/thumbnails/1.jpg)
My Research Experience
Margarita SalasCentro de Biología Molecular “Severo Ochoa”
CSIC–UA
CNIC PhDay
November 23, 2018
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SEVERO OCHOA
(1905-1993)
Premio Nobel de
Medicina 1959
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Alberto Sols1917-1989
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Eladio Viñuela
1937-1999
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Exonuclease
domain
TPR2
TPR1
Palm
Thumb
Fingers
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Exonuclease
domain
TPR2
TPR1
Palm
Thumb
Fingers
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Phage ø29 and Biotechnology
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1989. ø29 DNA polymerase patent(Inventors: Luis Blanco, Antonio Bernad, Jose M. Lázaro,
Margarita Salas)
Licensed to USB
2001. Comercialization of the ø29 DNA polymerase
Amersham Biosciences ––> GE Healthcare
Kit Templiphi: amplification of circular DNA
2003.Kit Genomiphi: amplification of linear genomic DNA
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A B
C
D
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Li, W. et al
Amersham Biosciences
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GenomiPhi
-Amplification of genomic DNA
- Genetic analysis
- Construction of libraries
-DNA archives
-Forensic medicine
-Archeological studies
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1989. Patent of ø29 DNA polymerase(Inventors: Luis Blanco, Antonio Bernad, Jose M. Lázaro,
Margarita Salas)
Licensed to USB
2001. Explotation:
Amersham Biosciences ––> GE Healthcare
Kit Templiphi: amplification of circular DNA
2003.Kit Genomiphi: amplification of linear genomic DNA
The patent has produced to the CSIC royalties of
6.624.118 euros
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Improvement of 29 DNA polymerase
amplification performance by fusion of
DNA binding motifs
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29 DNA polymerase (HhH)2
TPR2
downstream
template tunnel
product
double-stranded
tunnel
displaced
strand
template
strand
primer
strand
thumb
5´
5´
3´
3´
5´
N-terminus
C-terminus
linker
dsDNA
melting
Modelling of the chimerical DNA polymerase
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C
ssDNA M13
unit-length
60 30 15 7.5 60 30 15 7.5 60 30 15 7.5 [DNA pol], nM
29 DNAP 29-H 29-HI
ssDNA M13
unit-length
5 10 5 10 5 10 time, min
A 29 DNAP 29-H 29-HI B
29-H
29-HI
29DNAPSyn
thesiz
ed
DN
A (
ng
)
time (min)
Chimerical DNA polymerases show an enhanced Rolling Circle
Replication efficiency
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29 DNAP 29-H 29-HI
Reaction time, h. 1 2 4 6 1 2 4 6 1 2 4 6
4370-
2899-
1933-
1331-
759-
DNA polymeraseA
Reaction time, h. 1 2 3 4 1 2 3 4 1 2 3 4
4370-
2899-
1933-
1331-
759-
DNA polymeraseB
29 DNAP 29-H 29-HI
Multiply-primed Rolling Circle Amplification of plasmidic DNA by
ø29 DNA polymerase and chimerical DNA polymerases
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29 DNAP 29-H 29-HI
time, h. 2 4 8 16 2 4 8 16 2 4 8 16
4370-
2899-
1933-
1331-
759-
Multiply-primed Whole Genome Amplification of genomic DNA with
29 DNA polymerase and chimerical DNA polymerases
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Input DNA, fg 0 10 100 0 10 100
Present
conditions
New
conditions
4370-
2899-
1933-
1331-
759-
Effect of the new conditions on the 29 DNA polymerase
amplification capacity using plasmidic DNA
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Effect of the new conditions on the 29 DNA polymerase
amplification capacity
plasmidic DNA
genomic DNA
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CHIMERA OF Ø29 DNA POLYMERASE
Margarita Salas, Miguel de Vega, José
Mª Lázaro, Luis Blanco and Mario
Mencía
Patent N200930413
Licensed to Sygnis AG
Exploited by QIAGEN
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Grupo ø29, 2015-2016