# US 7,611,871 B2 · Method for the specific determination of DNA sequences by means of parallel amplification

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- Number: US 7,611,871 B2
- Document ID: US7611871B2
- Status: Granted
- Kind: B2
- Application number: 10/363,161
- Filed: 2001-09-04
- Published: 2004-03-11
- Granted: 2009-11-03
- Earliest claimed: 2000-09-05
- Assignees: BioChip Technologies GmbH
- Inventors: Patric Zeltz; Stephan Schneider
- Web page: https://patentgrep.com/patent/US7611871B2

## CPC

- C12Q 1/6837: using probe arrays or probe chips (C12Q1/6874 takes precedence)
- 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

## Abstract

The invention concerns a general method for the specific determination of DNA sequences by means of parallel amplification through nested polymerase chain reaction in a combined liquid-phase/solid-phase-DNA-microarray system, together with modifications derived therefrom to determine, for example, point mutations and to sequence DNA sub-regions, which according to a particular embodiment can also be unknown.

## Claims

### Claim 1

Method for the specific determination of DNA sequences by means of parallel amplification in a combined liquid-phase/solid-phase-DNA-microarray system by performing a nested polymerase chain reaction using x PCR primer sets each of at least 3 PCR primers P11, P21, P31, P12, P22, P32, P13, P23, P33, . . . P1x, P2x, P3x, whereby x signifies a positive integer and corresponds to the number of DNA sequences to be determined, and whereby for each of the x PCR primer sets
(a) two outer PCR primers P1, P2 are chosen in such a way that they hybridise onto DNA sub-sequences lying upstream and downstream of the target DNA sequence that is to be amplified,
(b) one inner PCR primer P3 is chosen in such a way that the one inner PCR primer P3 hybridises onto a DNA sub-sequence lying within the target DNA sequence that is to be determined, and is able to form a P3 extension product, and
(c) the x outer PCR primers P11, P21, P12, P22, P13, P23, . . . P1x, P2x are present in liquid phase in excess relative to the x inner PCR primers P31, P32, P33, . . . P3x, and
(d) the x inner PCR primers P31, P32, P33, . . . P3x are present irreversibly bonded to a solid phase at x spatially separated defined positions, forming a DNA microarray; the method comprising the steps of:
(1) providing the x PCR primer sets;
(2) performing simultaneous amplification, involving a nested polymerase chain reaction, using said primers in both the liquid and solid phase in order to produce a P3 extension product irreversibly bonded to the solid phase, wherein the x outer PCR primers P11, P21, P12, P22, P13, P23, . . . P1x, P2x in the liquid phase are present in a 102 to 1012 -fold excess relative to the x inner PCR primers P31, P32, P33, . . . P3x in the solid phase; and
(3) ascertaining the presence of the P3 extension product at one or more defined positions of the DNA microarray.

### Claim 2 (depends on 1)

A method according to claim 1 for determining point mutations by means of parallel amplification, in which
(a) a polymerase chain reaction, through which all of the x variants to be determined are are amplified, is carried out, whereby
(b) at least P3x primer families are used, whereby each individual member P3x-N of the primer family differs from each other member with regard to the nucleotide at its 3′ terminus, whereby N describes the different nucleotides A, C, G and T, and the annealing temperature of the amplification reaction is chosen in such a way that the desired extension products are formed at only those defined positions of the DNA microarray that are characterized by the presence of the primer that is essentially exactly complementary to the point mutation to be determined,
(c) a DNA polymerase without any 3′→5′ exonuclease activity is used for amplification, and
(d) the determination of the point mutation takes place according to Step (e) of claim 1.

### Claim 3 (depends on 1)

A method according to claim 1 for the determination of the sequence of DNA sub-regions by means of parallel amplification in which
(a) a polymerase chain reaction, through which all of the x sequences to be determined are amplified, is carried out, whereby
(b) at least P3x primer families are used, whereby each individual member P3x-N, . . . P3x-Nn of the primer family differs from each other member with regard to its 1 to n nucleotides at the 3′ terminus, whereby N describes the different nucleotides A, C, G and T, and n indicates the length of the sequence(s) to be determined, and the annealing temperature of the amplification reaction is chosen in such a way that the desired extension products are formed at only those defined positions of the DNA microarray that are characterized by the presence of the primer that is essentially exactly complementary to the sequence that is to be determined,
(c) a DNA polymerase without any 3′→5′ exonuclease activity is used for amplification, and
(d) the detection of the sequence(s) to be determined takes place according to Step (e) of claim 1.

### Claim 4 (depends on 1)

A method to according to claim 1 to determine the sequence of unknown DNA sub-regions by means of parallel amplification in which
(a) a polymerase chain reaction is carried out through which the sequence of all of the n nucleotides of the sequences to be determined is ascertained, whereby
(b) a set of inner primers P3 have a defined length n and encompassing all of the permutations, whereby several primers can hybridise exactly with and can form extension products of a defined length with in each case a different region of the sequence to be determined, and each individual one of these primers is situated at a different defined position of the DNA microarray,
(c) a DNA polymerase without any 3′→5′ exonuclease activity is used for amplification, and
(d) the determination of the sequence takes place by ascertaining the extension products formed in Step (b), assigning these determined values to the defined positions of the DNA microarray, and compilation of the sequence to be determined in the context of a combinatorial analysis, computer-assisted if necessary, whereby the actual alignment of the individual data to the overall sequence is performed using the respective overlapping sequence regions.

### Claim 5 (depends on 1)

Method according to claim 1, whereby the melting temperatures of the two outer PCR primers P1 and P2 are different from the melting temperature of the inner primer P3.

### Claim 6

Method according to one of the foregoing claims, whereby the solid phase is chosen from the group consisting of metal surfaces, metal surfaces vapour-deposited with SiO2, metal/semimental oxide surfaces, glass surfaces, polymer surfaces, nylon surfaces and nitrocellulose membranes.

### Claim 7 (depends on 6)

Method according to claim 6, whereby the metal surface is made of aluminium or gold, the metal/semimetal oxide surface is made of Al2O3 or SiO2, the glass surface is made of quartz glass and the polymer surface is made of polypropylene, polymethylmethacrylate or cycloolefine copolymers.

### Claim 8 (depends on 7)

Method according to claim 7, whereby the x inner PCR primers P31, P32, P33, . . . P3x are bonded to a metal/semimetal oxide surface or glass surface via a bifunctional silane that has one, two or three hydrolysable atom(s) or group(s) on the silicon atom.

### Claim 9 (depends on 8)

Method according to claim 8, whereby the hydrolysable atoms or groups comprise halogen atoms, C1-C4-alkoxy-, C1-C4-acyloxy- or amino groups.

### Claim 10 (depends on 8)

Method according to claim 8, whereby the second functional group of the bifunctional silane can take part in nucleophilic substitution reactions, nucleophilic addition reactions, Diels-Alder reactions or radical substitution reactions.

### Claim 11 (depends on 10)

Method according to claim 10, whereby the second functional group of the bifunctional silane involves a reactive double bond, diene group, dienophile group, epoxy, aldehyde, hydroxy, carboxylic acid, active ester, amino, disulphide, thiol, aziridine, azlactone, isocyanate, isothiocyanate or azide group or reactive leaving group.

### Claim 12 (depends on 1)

Method according to claim 1, whereby the x outer PCR primers P11, P21, P12, P22, P13, P23, . . . P1x, P2x are present in a 104 -fold excess relative to the x inner PCR primers P31, P32, P33, . . . P3x.

### Claim 13 (depends on 9)

Method according to claim 9, whereby the second functional group of the bifunctional silane reacts to take part in nucleophilic substitution reactions, nucleophilic addition reactions, Diels-Alder reactions or radical substitution reactions.

### Claim 14

Method for the specific determination of DNA sequences by means of parallel amplification in a combined liquid-phase/solid-phase-DNA-microarray system by performing a nested polymerase chain reaction using x PCR primer sets each of at least 3 PCR primers P11, P21, P31, P12, P22, P32, P13, P23, P33, . . . P1x, P2x, P3x, whereby x signifies a positive integer and corresponds to the number of DNA sequences to be determined, and whereby for each of the x PCR primer sets
(a) two outer PCR primers P1, P2 are chosen in such a way that they hybridise onto DNA sub-sequences lying upstream and downstream of the target DNA sequence that is to be amplified,
(b) one inner PCR primer P3 is chosen in such a way that the one inner PCR primer P3 hybridises onto a DNA sub-sequence lying within the target DNA sequence that is to be determined, and is able to form a P3 extension product, and
(c) the x outer PCR primers P11, P21, P12, P22, P13, P23, . . . P1x, P2x are present in liquid phase in excess relative to the x inner PCR primers P31, P32, P33, . . . P3x, and
(d) the x inner PCR primers P31, P32, P33, . . . P3x are present irreversibly bonded to a solid phase at x spatially separated defined positions, forming a DNA microarray; the method comprising the steps of
(1) providing the x PCR primer sets;
(2) performing simultaneous amplification, involving a nested polymerase chain reaction, using said primers in both the liquid and solid phase in order to produce a P3 extension product irreversibly bonded to the solid phase, wherein the x outer PCR primers P11, P21, P12, P22, P13, P23, . . . P1x, P2x in the liquid phase are present in a 102 to 1012 -fold excess relative to the x inner PCR primers P31, P32, P33, . . . P3x in the solid phase; and
(3) ascertaining the presence of the P3 extension product at one or more defined positions of the DNA microarray,
wherein the solid phase, to which the x inner PCR primers P31, P32, P33, . . . P3x are irreversibly bonded, is a surface.

### Claim 15 (depends on 1)

Method according to claim 1, wherein nucleotides used to produce the P3 extension product are labeled radioactively or by fluorescence so that ascertaining the presence of the P3 extension product involves detecting radioactivity or fluorescence of the P3 extension product.

### Claim 16 (depends on 15)

Method according to claim 15, wherein confocal laser scanning microscopy is used to detect the P3 extension product.

### Claim 17 (depends on 14)

Method according to claim 14, wherein nucleotides used to produce the P3 extension product are labeled radioactively or by fluorescence so that ascertaining the presence of the P3 extension product involves detecting radioactivity or fluorescence of the P3 extension product.

### Claim 18 (depends on 17)

Method according to claim 17, wherein confocal laser scanning microscopy is used to detect the P3 extension product.

### Claim 19

Method for the specific determination of DNA sequences by means of parallel amplification in a combined liquid-phase/solid-phase-DNA-micro array system by performing a nested polymerase chain reaction using x PCR primer sets each of at least 3 PCR primers P11, P21, P31, P12, P22, P32, P13, P23, P33, . . . P1x, P2x, P3x, whereby x signifies a positive integer and corresponds to the number of DNA sequences to be determined, and whereby for each of the x PCR primer sets
(a) two outer PCR primers P1, P2 are chosen in such a way that they hybridise onto DNA sub-sequences lying upstream and downstream of the target DNA sequence that is to be amplified,
(b) one inner PCR primer P3 is chosen in such a way that the one inner PCR primer P3 hybridises onto a DNA sub-sequence lying within the target DNA sequence that is to be determined, and is able to form a P3 extension product, and
(c) the x outer PCR primers P11, P21, P12, P22, P13, P23, . . . P1x, P2x are present in solution in excess relative to the x inner PCR primers P31, P32, P33, . . . P3x, and
(d) the x inner PCR primers P31, P32, P33, P3x are present irreversibly bonded to a solid phase at x spatially separated defined positions, forming a DNA microarray; the method comprising the steps of
(1) providing the x PCR primer sets;
(2) performing simultaneous amplification, involving a nested polymerase chain reaction, using said primers in both the solution and the solid phase in order to produce a P3 extension product irreversibly bonded to the solid phase, wherein the x outer PCR primers P11, P21, P12, P22, P13, P23,. . . P1x, P2x in the solution are present in a 102 to 102-fold excess relative to the x inner PCR primers P31, P32, P33, . . . P3x in the solid phase; and
(3) ascertaining the presence of the P3 extension product at one or more defined positions of the DNA microarray,
wherein the solid phase, to which the x inner PCR primers P31, P32, P33, . . . P3x are irreversibly bonded, is a non-porous surface.

### Claim 20

Method for the specific determination of DNA sequences by means of parallel amplification in a combined liquid-phase/solid-phase-DNA-microarray system by performing a nested polymerase chain reaction using x PCR primer sets each of at least 3 PCR primers P11, P21, P31, P12, P22, P32, P13, P23, P33, . . . P1x, P2x, P3x, whereby x signifies a positive integer and corresponds to the number of DNA sequences to be determined, and whereby for each of the x PCR primer sets
(a) two outer PCR primers P1, P2 are chosen in such a way that they hybridise onto DNA sub-sequences lying upstream and downstream of the target DNA sequence that is to be amplified,
(b) one inner PCR primer P3 is chosen in such a way that the one inner PCR primer P3 hybridises onto a DNA sub-sequence lying within the target DNA sequence that is to be determined, and is able to form a P3 extension product, and
(c) the x outer PCR primers P11, P21, P12, P22, P13, P23, P1x, P2x are present in solution in excess relative to the x inner PCR primers P31, P32, P33, P3x, and
(d) the x inner PCR primers P31, P32, P33, . . . P3x are present irreversibly bonded to a solid phase at x spatially separated defined positions, forming a DNA microarray; the method comprising the steps of
(1) providing the x PCR primer sets;
(2) performing simultaneous amplification, involving a nested polymerase chain reaction, using said primers in both the solution and the solid phase in order to produce a P3 extension product irreversibly bonded to the solid phase, wherein the x outer PCR primers P11, P21, P12, P22, P13, P23, . . . P1x, P2x in the solution are present in a 102 to 1012-fold excess relative to the x inner PCR primers P31, P32, P33, . . . P3x in the solid phase; and
(3) ascertaining the presence of the P3 extension product at one or more defined positions of the DNA microarray,
wherein the solid phase, to which the x inner PCR primers P31, P32, P33, . . . P3x are irreversibly bonded, is a non-porous surface, wherein the non-porous surface is a glass surface and the x inner PCR primers P31, P32, P33, . . . P3x are irreversibly bonded to the glass surface via a bifunctional silane that has one, two or three hydrolysable atom(s) or group(s) on the silicon atom.