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My Research Experience
Margarita SalasCentro de Biología Molecular “Severo Ochoa”
CSIC–UA
CNIC PhDay
November 23, 2018
SEVERO OCHOA
(1905-1993)
Premio Nobel de
Medicina 1959
Alberto Sols1917-1989
Eladio Viñuela
1937-1999
Exonuclease
domain
TPR2
TPR1
Palm
Thumb
Fingers
Exonuclease
domain
TPR2
TPR1
Palm
Thumb
Fingers
Phage ø29 and Biotechnology
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
A B
C
D
Li, W. et al
Amersham Biosciences
GenomiPhi
-Amplification of genomic DNA
- Genetic analysis
- Construction of libraries
-DNA archives
-Forensic medicine
-Archeological studies
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
Improvement of 29 DNA polymerase
amplification performance by fusion of
DNA binding motifs
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
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
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
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
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
Effect of the new conditions on the 29 DNA polymerase
amplification capacity
plasmidic DNA
genomic DNA
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
Grupo ø29, 2015-2016
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