# US 8,465,950 B2 · Global amplification using a randomly primed composite primer

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- Number: US 8,465,950 B2
- Document ID: US8465950B2
- Status: Granted
- Kind: B2
- Application number: 13/349,927
- Filed: 2012-01-13
- Published: 2012-07-26
- Granted: 2013-06-18
- Earliest claimed: 2003-04-14
- Assignees: NuGEN Technologies, Inc.
- Applicants: Nurith Kurn; Shenglong Wang
- Inventors: Nurith Kurn; Shenglong Wang
- Web page: https://patentgrep.com/patent/US8465950B2

## CPC

- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- C12Q 1/6844: Nucleic acid amplification reactions
- C12Q 1/6853: using modified primers or templates
- 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

## Abstract

The invention relates to the field of polynucleotide amplification. More particularly, the invention provides methods, compositions and kits for amplification of (i.e., making multiple copies of) a multiplicity of different polynucleotide template sequences using a randomly primed RNA/DNA composite primer.

## Claims

### Claim 1

A method for amplification of a template DNA, comprising:
(a) incubating the template DNA with
(i) a pool of first composite primers, each first composite primer comprising
(1) a 5′ RNA portion that is not hybridizable to the template DNA and
(2) a 3′ DNA portion, wherein the 3′ DNA portion of each of the first composite primers comprises a random sequence that is different from the 3′ DNA portion of the other first composite primers in the pool, and
(ii) a DNA polymerase having strand displacement activity,
whereby a complex comprising an RNA/DNA heteroduplex is generated;
(b) incubating the complex with an amplification primer, wherein the amplification primer is a composite primer comprising a 5′ RNA portion and a 3′ DNA portion, whereby multiple copies of an amplification product are generated, wherein the incubation of the complex with the amplification primer is under conditions that permit RNA cleavage, primer hybridization, primer extension, and displacement of the primer extension product when the RNA of the amplification primer is cleaved and another amplification primer binds to the complex and is extended by strand displacement, whereby multiple copies of a polynucleotide amplification product are generated.

### Claim 2 (depends on 1)

The method of claim 1, wherein the amplification primer comprises some of the sequence of the first composite primers in the pool.

### Claim 3 (depends on 1)

The method of claim 1, wherein each of the first composite primers in the pool and the amplification primer are different primers.

### Claim 4 (depends on 1)

The method of claim 1, step (a), wherein the template DNA is further incubated with an auxiliary primer comprising DNA, wherein the auxiliary primer is designed to randomly hybridize to a multiplicity of sites on the template DNA.

### Claim 5 (depends on 1)

The method of claim 1, wherein the DNA polymerase comprises a DNA-dependent DNA polymerase.

### Claim 6 (depends on 1)

The method of claim 1, wherein the DNA polymerase comprises an RNA-dependent DNA polymerase.

### Claim 7 (depends on 1)

The method of claim 1, wherein the incubation in step (a) is under conditions that permit hybridization of the pool of first composite primers to the template DNA and first primer extension, whereby the complex comprising an RNA/DNA heteroduplex is generated.

### Claim 8 (depends on 1)

The method of claim 1, wherein the amplification products are generated using a DNA-dependent DNA polymerase and an agent that cleaves RNA from an RNA/DNA heteroduplex.

### Claim 9 (depends on 6)

The method of claim 6, wherein the DNA polymerase comprises a DNA-dependent DNA polymerase and the RNA-dependent DNA polymerase in the same enzyme.

### Claim 10 (depends on 8)

The method of claim 8, wherein the agent is an enzyme.

### Claim 11 (depends on 10)

The method of claim 10, wherein the enzyme is RNase H.

### Claim 12 (depends on 1)

The method of claim 1, wherein the 5′ RNA portion of the amplification primer is adjacent to the 3′ DNA portion.

### Claim 13 (depends on 1)

The method of claim 1, wherein the 5′ RNA portion of each of the first composite primers consists of about 7 to about 50 nucleotides.

### Claim 14 (depends on 13)

The method of claim 13, wherein the 3′ DNA portion of each of the first composite primers consists of about 5 to about 20 nucleotides.

### Claim 15 (depends on 1)

The method of claim 1, wherein the 5′ RNA portion of the amplification primer consists of about 7 to about 50 nucleotides.

### Claim 16 (depends on 15)

The method of claim 15, wherein the 3′ DNA portion of the amplification primer consists of about 5 to about 20 nucleotides.

### Claim 17 (depends on 1)

The method of claim 1, wherein the incubation of step (b) further comprises a non-canonical nucleotide.

### Claim 18 (depends on 17)

The method of claim 17, wherein the non-canonical nucleotide is dUTP.

### Claim 19 (depends on 1)

The method of claim 1, wherein the incubation of step (b) further comprises a labeled nucleotide.

### Claim 20 (depends on 1)

The method of claim 1, wherein the amplification comprises global amplification.

### Claim 21 (depends on 20)

The method of claim 20, wherein the global amplification comprises amplification of genomic DNA or cDNA.

### Claim 22 (depends on 20)

The method of claim 20, wherein the global amplification comprises amplification of mitochondrial DNA, microbial DNA, viral DNA, eukaryotic DNA or mixtures thereof.

### Claim 23 (depends on 1)

The method of claim 1, wherein the pool of first composite primers comprises at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, or at least 100 different sequences.

### Claim 24 (depends on 1)

The method of claim 1, wherein each of the first composite primers is hybridizable to a multiplicity of template polynucleotide sites.

### Claim 25 (depends on 1)

The method of claim 1, wherein at least 50, at least 100, at least 200, at least 300, or more distinct sequences are amplified.

### Claim 26 (depends on 1)

The method of claim 1, wherein the template DNA comprises target polynucleotides, wherein the target polynucleotide sequences are unknown.

### Claim 27 (depends on 1)

The method of claim 1, wherein the template sequence is unknown.