# US 8,465,925 B2 · Method for nucleic acid isolation and amplification

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- Number: US 8,465,925 B2
- Document ID: US8465925B2
- Status: Granted
- Kind: B2
- Application number: 12/098,197
- Filed: 2008-04-04
- Published: 2009-02-19
- Granted: 2013-06-18
- Earliest claimed: 2004-08-09
- Assignees: Generation Biotech, LLC; GenoVision Inc.; Qiagen GmbH
- Applicants: Johannes Dapprich; Nancy Murphy; Christian Korfhage
- Inventors: Johannes Dapprich; Nancy Murphy; Christian Korfhage
- Web page: https://patentgrep.com/patent/US8465925B2

## CPC

- C12Q 1/6846: {Common amplification features}
- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- 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
- 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)}

## Abstract

The present invention provides methods and compositions for sequence-specific isolation of polynucleotide molecules from nucleic acid populations and subsequent amplification of isolated polynucleotide molecules or fragments thereof.

## Claims

### Claim 1

A method for amplifying a polynucleotide molecule of interest or a fragment thereof, comprising:
(a) isolating a polynucleotide molecule from a nucleic acid population using an immobilizable separation group to provide an isolated polynucleotide molecule, and
(b) isothermally amplifying the isolated polynucleotide molecule or a fragment thereof, wherein
(i) the amplification is a sequence-biased amplification which is defined as a hybrid of generic amplification and sequence-specific amplification performed in the presence of both random primers and sequence-specific primers, or
(ii) the amplification is a sequence-specific amplification performed in the presence of center-specific primers and end-specific primers.

### Claim 2 (depends on 1)

The method of claim 1 wherein
(A) the nucleic acid population comprises the polynucleotide molecule of interest,
(B) one strand of the polynucleotide molecule comprises a target nucleic acid sequence and a distinguishing element,
(C) the target nucleic acid sequence is within 100 nucleotides of the distinguishing element in the one strand of the polynucleotide molecules, and
(D) step (a) comprises:
(i) contacting the nucleic acid population with a targeting element that binds specifically to the target nucleic acid sequence in the polynucleotide molecule,
(ii) selectively attaching the immobilizable separation group to the targeting element bound to the target nucleic acid sequence in the polynucleotide molecule to form a targeting element-separation group complex,
(iii) immobilizing to a substrate via the separation group the targeting element-separation group complex to which the target nucleic acid sequence in the polynucleotide molecule is bound, and
(iv) removing the immobilized targeting element-separation group complex to which the target nucleic acid sequence in the polynucleotide molecule is bound, thereby isolating the polynucleotide molecule from the nucleic acid population.

### Claim 3 (depends on 2)

The method of claim 2 wherein the polynucleotide molecule of interest is a genomic DNA molecule.

### Claim 4 (depends on 2)

The method of claim 2 wherein the target nucleic acid sequence is located immediately 3′ to the distinguishing element in the one strand of the polynucleotide molecule.

### Claim 5 (depends on 2)

The method of claim 2 wherein the targeting element comprises an oligonucleotide.

### Claim 6 (depends on 2)

The method of claim 2 wherein the separation group comprises an immobilizable nucleotide.

### Claim 7 (depends on 6)

The method of claim 6 wherein the immobilizable nucleotide is a terminating nucleotide.

### Claim 8 (depends on 6)

The method of claim 6 wherein the immobilizable nucleotide is a non-terminating nucleotide.

### Claim 9 (depends on 2)

The method of claim 2 wherein the distinguishing element is a polymorphic sequence.

### Claim 10 (depends on 2)

The method of claim 2 wherein the distinguishing element is a single nucleotide polymorphism.

### Claim 11 (depends on 2)

The method of claim 2 wherein
(1) the targeting element comprises an oligonucleotide,
(2) the separation group comprises an immobilizable nucleotide, and
(3) the separation group is attached to the targeting element by extending the oligonucleotide in the presence of the immobilizable nucleotide, thereby forming an extension product that comprises the immobilizable nucleotide.

### Claim 12 (depends on 11)

The method of claim 11 wherein
(4) the 3′ terminus of the oligonucleotide is complementary to the distinguishing element or a portion thereof in the polynucleotide molecule,
(5) the immobilizable nucleotide is non-terminating, and
(6) the extension product comprises multiple separation groups.

### Claim 13 (depends on 11)

The method of claim 11 wherein
(4) the target nucleic acid sequence is immediately 3′ to the distinguishing element, and
(5) the immobilizable nucleotide is terminating and complementary to the distinguishing element or a portion thereof.

### Claim 14 (depends on 2)

The method of claim 2 wherein the distinguishing element is haplotype-specific.

### Claim 15 (depends on 2)

The method of claim 2 wherein the distinguishing element is locus-specific.

### Claim 16 (depends on 2)

The method of claim 2 further comprising dissociating the isolated polynucleotide molecule from the substrate prior to step (b).

### Claim 17 (depends on 1)

The method of claim 1 wherein
(A) the nucleic acid population comprises the polynucleotide molecule of interest,
(B) one strand of the polynucleotide molecule comprises a target nucleic acid sequence and a distinguishing element,
(C) the target nucleic acid sequence is within 100 nucleotides of the distinguishing element in the one strand of the polynucleotide molecule, and
(D) step (a) comprises:
(i) contacting the nucleic acid population with a targeting element-separation group complex, wherein the targeting element-separation group complex binds specifically to the target nucleic acid sequence in the polynucleotide molecule,
(ii) selectively stabilizing the binding of the targeting element-separation group complex to the target nucleic acid sequence in the polynucleotide molecule,
(iii) immobilizing to a substrate via the separation group the stabilized targeting element-separation group complex to which the target nucleic acid sequence in the polynucleotide molecule is bound, and
(iv) removing the immobilized stabilized targeting element-separation group complex to which the target nucleic acid sequence in the polynucleotide molecule is bound, thereby isolating the polynucleotide molecule from the nucleic acid population.

### Claim 18 (depends on 17)

The method of claim 17 wherein
(1) the targeting element of the targeting element-separation group complex comprises an oligonucleotide, and
(2) the 3′ terminus of the oligonucleotide is complementary to the distinguishing element or a portion thereof in the polynucleotide.

### Claim 19 (depends on 17)

The method of claim 17 further comprising dissociating the isolated polynucleotide molecule from the substrate prior to step (b).

### Claim 20 (depends on 1)

The method of claim 1 wherein the polynucleotide molecule is at least about 10 kb in length.

### Claim 21 (depends on 1)

The method of claim 1 wherein the random primers are about 2 kb apart from their neighboring primers.

### Claim 22 (depends on 1)

The method of claim 1 further comprising:
(c) characterizing one or more polymorphic sites in the amplified polynucleotide molecule or fragment thereof, and optionally
(d) assembling information of the characterized sites.

### Claim 23 (depends on 1)

The method of claim 1 wherein the sequence-biased amplification is a locus-biased amplification which is defined as a hybrid of generic amplification and locus-specific amplification and is performed in the presence of both random primers and locus-specific primers.

### Claim 24 (depends on 23)

The method of claim 23 wherein the locus-specific primers bind to a common sequence within, or flanking, the locus shared by all or a majority of the alleles occupying the locus.

### Claim 25 (depends on 24)

The method of claim 24 wherein the sequence flanking the locus is located outside the locus but the distance between the sequence flanking the locus and the nearby terminus of the locus is within 500 nucleotides.

### Claim 26 (depends on 1)

The method of claim 1 wherein in the sequence-biased amplification the sequence-specific primers are end-specific primers.

### Claim 27 (depends on 26)

The method of claim 26 wherein the end-specific primers anneal to the isolated polynucleotide molecule or the fragment thereof within 500 nucleotides from an end of the isolated polynucleotide molecule or the fragment thereof.

### Claim 28 (depends on 1)

The method of claim 1 wherein in the sequence-specific amplification the center-specific primers are degenerate primers whose sequence has several possible bases at certain positions.

### Claim 29 (depends on 1)

The method of claim 1 wherein the end-specific primers anneal to the isolated polynucleotide molecule or the fragment thereof within 500 nucleotides from an end of the isolated polynucleotide molecule or the fragment thereof.

### Claim 30

A method for amplifying multiple polynucleotide molecules of interest from a population of nucleic acid molecules, comprising:
(a) isolating multiple polynucleotide molecules from a nucleic acid population using one or more immobilizable separation groups to provide isolated polynucleotide molecules of interest, and
(b) isothermally amplifying the isolated polynucleotide molecules or fragments thereof, wherein
(i) the amplification is a sequence-biased amplification which is defined as a hybrid of generic amplification and sequence-specific amplification performed in the presence of both random primers and sequence-specific primers, or
(ii) the amplification is a sequence-specific amplification performed in the presence of center-specific primers and end-specific primers.

### Claim 31 (depends on 30)

The method of claim 30 wherein step (a) comprises:
(A) contacting a nucleic acid population that comprises multiple polynucleotide molecules of interest with multiple targeting elements so that each targeting element binds specifically to a target nucleic acid sequence of its corresponding polynucleotide molecule, wherein
(i) the target nucleic acid sequence is located within 100 nucleotides of a distinguishing element in one strand of the polynucleotide molecule, and
(ii) the distinguishing element distinguishes the polynucleotide molecule from another nucleic acid molecule that is nearly identical to the polynucleotide molecule,
(B) selectively attaching separation groups to the multiple targeting elements bound to the target nucleic acid sequences of corresponding polynucleotide molecules to form targeting element-separation group complexes,
(C) immobilizing to substrate(s) via the separation groups the targeting element-separation group complexes to which the target nucleic acid sequences in the polynucleotide molecules are bound, and
(D) removing the immobilized targeting element-separation group molecules are bound to isolate the polynucleotide molecules from the population of nucleic acid molecules.

### Claim 32 (depends on 31)

The method of claim 31 wherein different separation groups are attached to different targeting elements.

### Claim 33 (depends on 31)

The method of claim 31 wherein identical separation groups are attached to different targeting elements.

### Claim 34 (depends on 31)

The method of claim 31 wherein at least three different polynucleotide molecules of interest are isolated.

### Claim 35 (depends on 30)

The method of claim 30 wherein step (a) comprises:
(A) contacting a nucleic acid population that comprises multiple polynucleotide molecules of interest with multiple targeting elements to which separation groups are attached so that each targeting element binds specifically to a target nucleic acid sequence of its corresponding polynucleotide molecule, wherein
(i) the target nucleic acid sequence is located within 100 nucleotides of a distinguishing element in one strand of the polynucleotide molecule, and
(ii) the distinguishing element distinguishes the polynucleotide molecule from another nucleic acid molecule that is nearly identical to the polynucleotide molecule,
(B) selectively stabilizing the binding of the targeting elements to the target nucleic acid sequences of their corresponding polynucleotide molecules to form stabilized targeting element-separation group complexes to which the target nucleic acid sequences in the polynucleotide molecules are bound,
(C) immobilizing to substrate(s) via the separation groups the targeting element-separation group complexes to which the target nucleic acid sequences in the polynucleotide molecules are bound, and
(D) removing the immobilized targeting element-separation group complexes to which the target nucleic acid sequences in the polynucleotide molecules are bound to isolate the polynucleotide molecules from the population of nucleic acid molecules.

### Claim 36

A method for amplifying a genomic DNA molecule of interest or a fragment thereof, wherein the genomic DNA molecule of interest comprises a polymorphic sequence, comprising:
(a) contacting a genomic DNA population that comprises the genomic DNA molecule of interest with an oligonucleotide, wherein
(i) the oligonucleotide comprises a sequence at least substantially complementary to a target nucleic acid sequence in one strand of the genomic DNA molecule of interest,
(ii) the target nucleic acid sequence is located immediately 3′ to the polymorphic sequence in the one strand of the genomic DNA molecule of interest, and
(iii) the 3′ portion of the oligonucleotide is complementary to the polymorphic sequence or a portion thereof when annealing to the one strand of the genomic DNA molecule of interest,
(b) extending the oligonucleotide in the presence of an immobilizable nucleotide using the one strand of the genomic DNA molecule of interest to which the oligonucleotide anneals as a template to provide an extension product,
(c) immobilizing to a substrate via the immobilizable nucleotide the extension product to which the genomic DNA molecule of interest is bound,
(d) removing the immobilized extension product to which the genomic DNA molecule of interest is bound to thereby isolate the genomic DNA molecule of interest from the genomic DNA population,
(e) optionally elute the genomic DNA molecule of interest from the substrate, and
(f) isothermally amplifying the isolated or eluted genomic DNA molecule of interest or a fragment thereof, wherein
(i) the amplification is a sequence-biased amplification which is defined as a hybrid of generic amplification and sequence-specific amplification performed in the presence of both random primers and sequence-specific primers, or
(ii) the amplification is a sequence-specific amplification performed in the presence of center-specific primers and end-specific primers.