# US 2016/0068903 A1 · Selective Amplification of Nucleic Acid Sequences

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- Number: US 2016/0068903 A1
- Document ID: US20160068903A1
- Status: Published application
- Kind: A1
- Application number: 14/554,966
- Filed: 2014-11-26
- Published: 2016-03-10
- Earliest claimed: 2013-11-26
- Assignees: LC Sciences LC
- Inventors: Xiaochuan Zhou; Qi Zhu; Nijing Sheng
- Web page: https://patentgrep.com/patent/US20160068903A1

## CPC

- C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q 1/6848: characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
- C12Q 1/6853: using modified primers or templates
- C12Q 1/686: Polymerase chain reaction [PCR]
- C12Q 2525/155: incorporating/generating a new priming site
- C12Q 2525/161: incorporating target specific and non-target specific sites
- C12Q 2527/101: Temperature
- C12Q 2527/113: Time
- C12Q 2527/143: Concentration of primer or probe
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- 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
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The present invention relates to the field of nucleic acid sequence replication including PCR. Specifically, the present invention relates to methods and compositions for amplifying one or more target sequences from one or more template sequences. In particular, the present invention provides novel primer designs to enhance specificity of PCR reactions. The present invention also provides methods and compositions to perform the selection of specific sequence sections using specific primers and the amplification of all selected sequence sections using a pair of common primers in a single reaction tube.

## Claims

### Claim 1

An oligonucleotide primer comprising a 3p arm having a 3′ end and a 5′ end, a loop section and a 5p arm having a 3′ end and a 5′ end, wherein the 5p arm hybridizes to a DNA template and wherein the 3p arm hybridizes to the DNA template and provides sequence specificity for polymerase extension and wherein the loop section is located between the 5p arm and the 3p arm and does not bind the DNA template.

### Claim 2 (depends on 1)

The primer of claim 1 wherein the DNA template is substantially complementary to the 5p arm and the 3p arm.

### Claim 3 (depends on 1)

The primer of claim 1 wherein the 5p arm is from 10 to 100 nucleotides in length.

### Claim 4 (depends on 1)

The primer of claim 1 wherein the 3p arm is from 6 to 60 nucleotides in length.

### Claim 5 (depends on 1)

The primer of claim 1 wherein the loop section is from 12 to 50 nucleotides in length.

### Claim 6 (depends on 1)

The primer of claim 1 wherein the 5p is from 25 to 60 nucleotides in length and wherein the 3p arm is from 10 to 20 nucleotides in length and wherein the loop section is from 15 to 40 nucleotides in length.

### Claim 7 (depends on 1)

The primer of claim 1 wherein the 5′ end of the 3p arm and the 3′ end of the 5p arm are adjacent each other when bound to the DNA template.

### Claim 8 (depends on 1)

The primer of claim 1 wherein the 5p arm has higher binding energy than the 3p arm when hybridized to the DNA template.

### Claim 9 (depends on 1)

The primer of claim 1 wherein the 5p arm has a binding energy higher than twice that of the 3p arm.

### Claim 10 (depends on 1)

The primer of claim 1 wherein the 5p arm has a binding energy higher than three times that of the 3p arm.

### Claim 11 (depends on 1)

The primer of claim 1 wherein the loop section is a bulge loop.

### Claim 12 (depends on 1)

The primer of claim 1 wherein the loop section is a hairpin loop.

### Claim 13 (depends on 1)

The primer of claim 1 wherein the loop section is an internal loop.

### Claim 14

A hybridization structure for assay use comprising a probe and a target wherein the hybridization structure has one or more single stranded loops and two or more duplex segments wherein each loop is located between the duplex segments.

### Claim 15 (depends on 14)

The hybridization structure of claim 14 wherein the single stranded loop is in the probe and comprises one or more non-nucleotide moieties.

### Claim 16 (depends on 14)

The hybridization structure of claim 14 wherein the probe comprises a spacer.

### Claim 17 (depends on 14)

The hybridization structure of claim 14 wherein the hybridization structure is used for polymerase extension.

### Claim 18 (depends on 14)

The hybridization structure of claim 14 wherein the hybridization structure is used for hybridization detection.

### Claim 19 (depends on 18)

The hybridization structure of claim 18 wherein the loop contains 12 to 50 nucleotides.

### Claim at position 20

20-60. (canceled)