# US 11,299,718 B2 · Methods for amplification and sequencing using thermostable TthPrimPol

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- Number: US 11,299,718 B2
- Document ID: US11299718B2
- Status: Granted
- Kind: B2
- Application number: 14/776,901
- Filed: 2014-03-14
- Published: 2016-02-11
- Granted: 2022-04-12
- Earliest claimed: 2013-03-15
- Assignees: 4Basebio Sl
- Applicants: 4basebio SL
- Inventors: Angel J. Picher; Luis Blanco
- Web page: https://patentgrep.com/patent/US11299718B2

## CPC

- C12N 9/1252: {DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase}
- C12N 15/102: {Mutagenizing nucleic acids}
- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- C12Q 1/686: Polymerase chain reaction [PCR]
- C12Y 207/07007: DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Y: ENZYMES

## Abstract

The present invention is directed to methods for replicating, amplifying, and sequencing of nucleic acids using the thermostable, bifunctional replicase “TthPrimPol” from Thermus thermophilus HB27. 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.

## Claims

### Claim 1

A method for replicating, amplifying or sequencing of DNA in the absence of a primer comprising the following steps
a) providing
i) a protein that has both polymerase and primase activity, and has a sequence that is at least 80% identical to SEQ ID NO: 1, or
ii) a conjugate comprising a protein moiety that has both polymerase and primase activity, wherein the protein moiety of the conjugate has a sequence that is at least 80% identical to SEQ ID NO: 1,
b) providing a template nucleic acid,
c) providing nucleotides and/or nucleotide derivatives for incorporation in a complementary strand of nucleic acids,
d) providing a suitable buffer,
e) providing a second polymerase, and
f) contacting the materials of steps a-e in the absence of a primer and without the presence of a protein having primase activity other than the protein or conjugate provided in step (a) for a suitable amount of time to replicate, amplify or sequence the template nucleic acid.

### Claim 2 (depends on 1)

The method of claim 1, wherein the second polymerase provided in step e) is selected from the group consisting of prokaryotic family A polymerases and prokaryotic family B polymerases.

### Claim 3 (depends on 2)

The method according to claim 2, wherein the second polymerase is a thermostable polymerase selected from the group consisting of Taq polymerase, Tth polymerase, Pfu polymerase, and Vent polymerase.

### Claim 4 (depends on 1)

The method of claim 1, wherein the second polymerase provided in step e) is a Phi29 type DNA polymerase or a conjugate thereof.

### Claim 5 (depends on 1)

The method according to claim 1, wherein the protein of a) i) has a sequence that is at least 95% identical to SEQ ID NO: 1, or the protein moiety of the conjugate of a) ii) has a sequence that is at least 95% identical to SEQ ID NO: 1.

### Claim 6 (depends on 1)

The method according to claim 1, wherein the protein of a) i) has a sequence that is at least 90% identical to SEQ ID NO: 1, or the protein moiety of the conjugate of a) ii) has a sequence that is at least 90% identical to SEQ ID NO: 1.

### Claim 7 (depends on 1)

The method according to claim 1, wherein the protein of a) i) has the sequence set forth as SEQ ID NO: 1, or the protein moiety of the conjugate of a) ii) has the sequence set forth as SEQ ID NO: 1.

### Claim 8 (depends on 1)

The method according to claim 1, wherein step (b) comprises providing a template nucleic acid which is damaged.

### Claim 9 (depends on 8)

The method according to claim 8, wherein the damaged template nucleic acid is characterized by:
a) the presence of at least one single strand or double strand breach, and/or
b) the presence of at least one chemically modified nucleotide, and/or
c) being derived from formaldehyde or para-formaldehyde embedded tissue.

### Claim 10 (depends on 1)

The method according to claim 1, wherein step c) comprises providing nucleotide derivatives for incorporation in a complementary strand of nucleic acids, and at least one species of the nucleotide derivatives is a chemically modified nucleotide.

### Claim 11 (depends on 1)

The method of claim 1, wherein step c) comprises providing nucleotide derivatives for incorporation in a complementary strand of nucleic acids, and at least one species of the nucleotide derivatives of step c) is selected from the group consisting of:
a) oxidized nucleotide derivative,
b) deaminated nucleotides, and
c) derivatives comprising at least one sterically demanding side group.

### Claim 12 (depends on 1)

The method according to claim 1, wherein the template nucleic acid of step b) derives from less than 100 cells, preferably from a single cell.

### Claim 13 (depends on 1)

The method according to claim 1, wherein the amplification of contaminant DNA is suppressed.

### Claim 14 (depends on 9)

The method according to claim 9, wherein a single strand breach is present in the damaged template nucleic acid.

### Claim 15 (depends on 9)

The method according to claim 9, wherein at least one chemically modified nucleotide is present in the damaged template nucleic acid, and the chemically modified nucleotide is selected from the group consisting of 7,8-dihydro-8-oxoadenine, 7,8-dihydro-8-oxoguanine, thymine glycol, 5-hydroxycytosine and 5-hydroxyuracil.

### Claim 16 (depends on 10)

The method according to claim 10, wherein the chemically modified nucleotide is selected from the group consisting of 7,8-dihydro-8-oxoadenine, 7,8-dihydro-8-oxoguanine, thymine glycol, 5-hydroxycytosine and 5-hydroxyuracil.

### Claim 17 (depends on 8)

The method according to claim 8, wherein step c) comprises providing nucleotide derivatives for incorporation in a complementary strand of nucleic acids, and at least one species of the nucleotide derivatives is a chemically modified nucleotide.

### Claim 18 (depends on 17)

The method according to claim 17, wherein the chemically modified nucleotide is selected from the group consisting of 7,8-dihydro-8-oxoadenine, 7,8-dihydro-8-oxoguanine, thymine glycol, 5-hydroxycytosine and 5-hydroxyuracil.

### Claim 19 (depends on 1)

The method according to claim 1, wherein the polymerase or the polymerase moiety of the polymerase conjugate is capable of replicating, amplifying or sequencing DNA when a mix of nucleotides and nucleotides derivatives is provided in step c).