Consejo Superior De Investigaciones Cientificas

4Basebio Sl

Centre Tecnologic De Telecomunicacions De Catalunya (Cttc)

Expedeon, S.l.

Luis Blanco

Antonio Bernad

Margarita Salas

Angel J Picher

Farhad Rezazadeh

Josep Mangues

Sergio Barrachina

Engin Zeydan

Luca Vettori

The present invention is directed to methods for replicating, amplifying, and sequencing of nucleic acids using the thermostable, bifunctional replicase 'TthPrimPol' fromHB27. The TthPrimPol enzyme is extremely tolerant to alterations of the nucleotides of the template nucleic acid. Therefore, in one aspect the invention discloses methods for replicating, amplifying and sequencing of damaged polynucleotide templates.

Methods for amplification and sequencing using thermostable TthPrimPol

An improved method for determining the nucleotide base sequence of a DNA molecule employs a .phi.-29 type DNA polymerase modified to have reduced or no exonuclease activity. The method includes annealing the DNA molecule with a primer molecule able to hybridize to the DNA molecule; incubating the annealed mixture in a vessel containing four different deoxynucleoside triphosphates, a DNA polymerase, and one or more DNA synthesis terminating agents which terminate DNA synthesis at a specific nucleotide base, wherein each the agent terminates DNA synthesis at a different nucleotide base; and separating the DNA products of the incubating reaction according to size, whereby at least a part of the nucleotide base sequence of the DNA can be determined. The improvement is provision of a DNA polymerase which is a .phi.29-type DNA polymerase.

.phi.29 DNA polymerase

An improved method for determining the nucleotide base sequence of a DNA molecule. The method includes annealing the DNA molecule with a primer molecule able to hybridize to the DNA molecule; incubating the annealed mixture in a vessel containing four different deoxynucleoside triphosphates, a DNA polymerase, and one or more DNA synthesis terminating agents which terminate DNA synthesis at a specific nucleotide base, wherein each the agent terminates DNA synthesis at a different nucleotide base; and separating the DNA products of the incubating reaction according to size, whereby at least a part of the nucleotide base sequence of the DNA can be determined. The improvement is provision of a DNA polymerase which is a .phi.29-type DNA polymerase.

PHI29 DNA polymerase

An improved method for determining the nucleotide base sequence of a DNA molecule. The method includes annealing the DNA molecule with a primer molecule able to hybridize to the DNA molecule; incubating the annealed mixture in a vessel containing four different deoxynucleoside triphosphates, a DNA polymerase, and one or more DNA synthesis terminating agents which terminate DNA synthesis at a specific nucleotide base, wherein each the agent terminates DNA synthesis at a different nucleotide base; and separating the DNA products of the incubating reaction according to size, whereby at least a part of the nucleotide base sequence of the DNA can be determined. The improvement is provision of a DNA-polymerase which is a .phi.29-type DNA polymerase.

PH.phi.29 DNA polymerase

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Madrid, Spain

Luis Blanco