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