高等学校化学学报

高等学校化学学报 ›› 2000, Vol. 21 ›› Issue (S1): 346.

• Chemistry in Surface Science • 上一篇    下一篇

Simultaneous High Throughput Single Molecule Electrophoresis and Single Molecule Spectroscopy

MA Ym-Fa1, SHORTREED Michael R.2, YEUNG Edward S.2   

  1. 1. Department of Chemistry, Truman State University, Kirksville, MO 63501, USA;
    2 Department of Chemistry, Iowa State University, Ames, Iowa, 50011, USA
  • 出版日期:2000-12-31 发布日期:2000-12-31

Simultaneous High Throughput Single Molecule Electrophoresis and Single Molecule Spectroscopy

MA Ym-Fa1, SHORTREED Michael R.2, YEUNG Edward S.2   

  1. 1. Department of Chemistry, Truman State University, Kirksville, MO 63501, USA;
    2 Department of Chemistry, Iowa State University, Ames, Iowa, 50011, USA
  • Online:2000-12-31 Published:2000-12-31

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摘要：

Single molecule detection (SMD) has developed rapidly in recent years, especially high-throughput single molecule detection. Such research facilitated several fundamental studies at the single molecule level. In the fixture, SMD may be successfully applied to biological, clinical and medical research for DNA sequencing and single-molecule scans for disease detection. Presently, single-molecule identification of DNA and proteins is performed using fluorescence intensity, mobility or hybridization with a selective probe. In some cases, such methods are insufficient for confident single-molecule identification. Therefore, we invented a high-throughput combination single-molecule spectroscopy/imaging technique for monitoring the spectroscopic differences of several different individual molecules while they migrate in solution. The technique can offer three-dimensional data for each molecule:mobility, fluorescence intensity and spectroscopy information. Two sample systems were selected as test cases. In the first case, λ DNA is labeled with YOYO-Ⅰ,POPO-Ⅲ and a combination of the two dyes. Many individual λ DNA molecules are simultaneously imaged and identified by their spectroscopic differences. In the second case, a biotinylated 2.1 kb PCR product (also labeled with YOYO-Ⅰ) was reacted with avidin-conjugated R-phycoerythrin. The individual reactants and products are also simultaneously imaged and identified by their spectroscopic differences. This technique can be used for high-throughput DNA screening, molecular identification and monitoring intermolecular interactions with a speed of over 2,000,000 molecules per second. The existing method is the highest and most powerful single-molecule screening method available to date. Such technology is expected to have a great impact on single-molecule diagnosis and monitoring molecular interaction at the single molecule level and will be beneficial to early detection and diagnosis of disease (e.g. cancer, HIV). Furthermore, this technique allows one to directly observe and evaluate the data without any complicated calculations.

Abstract:

Single molecule detection (SMD) has developed rapidly in recent years, especially high-throughput single molecule detection. Such research facilitated several fundamental studies at the single molecule level. In the fixture, SMD may be successfully applied to biological, clinical and medical research for DNA sequencing and single-molecule scans for disease detection. Presently, single-molecule identification of DNA and proteins is performed using fluorescence intensity, mobility or hybridization with a selective probe. In some cases, such methods are insufficient for confident single-molecule identification. Therefore, we invented a high-throughput combination single-molecule spectroscopy/imaging technique for monitoring the spectroscopic differences of several different individual molecules while they migrate in solution. The technique can offer three-dimensional data for each molecule:mobility, fluorescence intensity and spectroscopy information. Two sample systems were selected as test cases. In the first case, λ DNA is labeled with YOYO-Ⅰ,POPO-Ⅲ and a combination of the two dyes. Many individual λ DNA molecules are simultaneously imaged and identified by their spectroscopic differences. In the second case, a biotinylated 2.1 kb PCR product (also labeled with YOYO-Ⅰ) was reacted with avidin-conjugated R-phycoerythrin. The individual reactants and products are also simultaneously imaged and identified by their spectroscopic differences. This technique can be used for high-throughput DNA screening, molecular identification and monitoring intermolecular interactions with a speed of over 2,000,000 molecules per second. The existing method is the highest and most powerful single-molecule screening method available to date. Such technology is expected to have a great impact on single-molecule diagnosis and monitoring molecular interaction at the single molecule level and will be beneficial to early detection and diagnosis of disease (e.g. cancer, HIV). Furthermore, this technique allows one to directly observe and evaluate the data without any complicated calculations.

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