Detection and quantification of biomolecules using mass spectrometry

A technology of biomolecules and mass spectrometry, applied in biochemical equipment and methods, microbial measurement/testing, fermentation, etc., can solve time-consuming problems

Inactive Publication Date: 2009-12-16
SEQUENOM INC
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  • Application Information

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Problems solved by technology

Among various problems, fluorescent dyes offer only limited multiplexing options, and currently available methods that attempt to overcome these limitations (e.g. by using primer extension and ligation-based SNP analysis followed by universal PCR and hybridization to chip arrays) are often extremely limited. Time consuming (eg 1-2 days)
[0002] Due to the increased number of detection channels, the use of mass spectrometry may provide a solution to improve multiplexing, but the practical application of previously revealed mass spectrometry-based methods can still be further improved

Method used

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  • Detection and quantification of biomolecules using mass spectrometry
  • Detection and quantification of biomolecules using mass spectrometry
  • Detection and quantification of biomolecules using mass spectrometry

Examples

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example

[0144] The examples provided below illustrate but do not limit the invention.

example 1

[0145] Example 1: Detection of exon 10 of the RhD gene

[0146] Detection assays were performed to detect the exon 10 region of the rhesus monkey D gene. The design of PCR primers and detection oligonucleotides was carried out according to the specific instructions section. In a particular assay, the detection oligonucleotide carries a non-complementary 5' overhang consisting of 6 adenines. Due to the presence of the target sequence (exon 10 of RhD) in the sample, the PCR primers and detection oligonucleotides hybridize to the target. During amplification, the detection oligonucleotide is degraded by the 5' nuclease activity of the DNA polymerase extending upstream from the PCR primer. During degradation, mass-distinguishable products (MDPs) including the 5' polyA tag are released and unambiguously identified by mass spectrometry (see Figure 8 ). Detection of these mass signals confirms the presence of the target nucleic acid.

[0147] Primers and detection oligon...

example 2

[0164] Example 2: Detection of RhD gene exon 5

[0165] Detection assays were performed to detect the exon 5 region of the rhesus monkey D gene. The design of PCR primers and detection oligonucleotides was carried out according to the specific instructions section. In a particular assay, the detection oligonucleotide carries a non-complementary 5' overhang consisting of 8 adenines. Due to the presence of the target sequence (exon 5 of RhD) in the sample, the PCR primers and detection oligonucleotides hybridize to the target. During amplification, the detection oligonucleotide is degraded by the 5' nuclease activity of the DNA polymerase extending upstream from the PCR primer. During degradation, mass-distinguishable products (MDPs) including the 5' polyA tag are released and unambiguously identified by mass spectrometry (see Figure 9 ). Detection of these mass signals confirms the presence of the target nucleic acid.

[0166] Primers and detection oligonucleotide ...

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Abstract

The present invention is directed in part to a method for detecting a target nucleic acid using detector oligonucleotides detectable by mass spectrometry. This method uses the 5' to 3' nuclease activity of a nucleic acid polymerase to cleave annealed oligonucleotide probes from hybridized duplexes and release labels for detection by mass spectrometry. This process is easily incorporated into a PCR amplification assay. The method also includes embodiments directed to quantitative analysis of target nucleic acids.

Description

technical field Background technique [0001] Current methods for detecting and quantifying nucleic acids in multiplex assays, especially those employing fluorescent dyes for detection, have limitations. For example, White discusses the use of Problems of multiplex analysis of analytical methods (Trends in Biotechnology (1996) 14(12); 478-483). Among various problems, fluorescent dyes offer only limited multiplexing options, and currently available methods that attempt to overcome these limitations (e.g. by using primer extension and ligation-based SNP analysis followed by universal PCR and hybridization to chip arrays) are often extremely limited. Time consuming (eg 1-2 days). [0002] The use of mass spectrometry may provide a solution to improve multiplexing due to the increased number of detection channels, but the practical application of previously disclosed mass spectrometry-based methods can still be further improved. For example, Ross described the use of peptide ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12P19/34
CPCC12Q1/6823C12Q1/686C12Q1/6827
Inventor 迪尔克·约翰内斯·范登博姆
Owner SEQUENOM INC
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