Method for measuring the concentration of polynucleotides

• US 6,043,034 A
• Filed: 10/21/1997
• Issued: 03/28/2000
• Est. Priority Date: 10/25/1996
• Status: Expired due to Fees

• Current Assignee: Kyoto Daiichi Kagaku Company Limited
• Original Assignee: Kyoto Daiichi Kagaku Company Limited
• Inventors: Dou, Xiaoming, Wang, Yung Xiang, Takama, Toshio, Kono, Masao
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US08/955,251
• Time in Patent Office: 889 Days
• Field of Search: 435/6, 536/23.1, 536/24.3, 935/76, 935/77, 935/78
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 2545/114 involving a quantitation step
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode
• CPC Class Codes: C12Q 2565/632 being a surface enhanced, e.g. resonance, Raman spectrometer

Abstract

A method for measuring the concentration of polynucleotide in a sample comprising the steps of: (1) preparing a sample, a Raman scattering active agent capable of bonding to a polynucleotide and a surface-enhanced Raman scattering (SERS) active substrate capable of capturing the agent; (2) mixing the sample, the agent and the substrate to capture the agent which has not been bonded to polynucleotide on the surface of the substrate; (3) irradiating the mixture; and (4) measuring the SERS radiation which is generated by the agent captured on the surface of the substrate.

Claims

1. A method for measuring the presence of polynucleotide in a sample, comprising the steps of:

preparing a sample, a Raman scattering active agent capable of bonding to a polynucleotide and a surface-enhanced Raman scattering (SERS) active substrate capable of capturing said agent;

mixing said sample, said agent and said substrate, whereby the surface of the substrate captures Raman scattering active agent that has not been bonded to polynucleotide in the sample;

irradiating said mixture; and

measuring SERS radiation which is generated by said agent captured on the surface of said substrate and determining whether polynucleotide is present in the sample from the result of the measurement of the SERS radiation.

2. A method as claimed in claim 1, wherein the concentration of said polynucleotide is measured.

3. A method as claimed in claim 1, wherein the polynucleotide is double stranded deoxyribonucleic acid (DNA).

4. A method as claimed in claim 3, wherein the sample is one or more of the samples selected from the group consisting of a sample treated by the polymerase chain reaction (PCR), a sample treated by the Strand displacement amplification (SDA) method and a sample treated by Ligase chain reaction (LCR) method.

5. A method as claimed in claim 1, wherein the polynucleotide is ribonucleic acid (RNA).

6. A method as claimed in claim 5, wherein the sample is treated by the Qβ

method with Qβ

-replicas.

7. A method as claimed in claim 1, wherein the polynucleotide is single stranded DNA.

8. A method as claimed in claim 1, wherein the polynucleotide is a complex of RNA and DNA.

9. A method as claimed in claim 1, wherein said agent is one or more of the agents selected from the group consisting of 4'"'"', 6-diamidino-2-phenylindole, ethidium bromide, bisbenzimide and acridine orange.

10. A method as claimed in claim 5, wherein said agent is one or more of the agents selected from the group consisting of ethidium bromide, thiazole orange, bisbenzimide and acnridine orange.

11. A method as claimed in claim 1, wherein said substrate is one or more of the substrates selected from the group consisting of a silver colloid, a gold colloid, a copper colloid, an electrode and a metal plate.