申请人:BBI BioSeq, Inc., Massachusetts corporation
公开号:US20010046676A1
公开(公告)日:2001-11-29
The invention is based on the discovery that the sequence of monomers in a polymeric biomolecule can be determined in a self-contained, high pressure reaction and detection apparatus, without the need for fluid flow into or out from the apparatus. The pressure is used to control the activity of enzymes that digest the polymeric biomolecule to yield the individual monomers in the sequence in which they existed in the polymer. High pressures modulate enzyme kinetics by reversibly inhibiting those enzymatic processes which result in a higher average activation volume, when compared to the ground state, and reversibly accelerating those processes which have lower activation volumes than the ground state. Modulating the pressure allows the experimenter to precisely control the activity of the enzyme. Conditions can be found, for example, where the enzyme removes only one monomer (e.g., a nucleotide or amino acid) from the biomolecule before the pressure is again raised to a prohibitive level. The identity of the single released nucleotide or amino acid can be determined using a detector that is in communication with a probe in the detection zone within the reaction vessel.
本发明基于以下发现:在一个独立的高压反应和检测装置中,可以确定聚合生物大分子中单体的序列,而不需要流体流入或流出该装置。压力用于控制酶的活性,这些酶可以消化聚合生物大分子,按照聚合物中单体存在的顺序产生单体。高压通过可逆地抑制那些导致平均活化体积高于基态的酶过程,以及可逆地加速那些活化体积低于基态的酶过程,从而调节酶的动力学。通过调节压力,实验人员可以精确控制酶的活性。例如,在压力再次升高到令人望而却步的水平之前,可以找到酶只从生物大分子中去除一个单体(如核苷酸或氨基酸)的条件。释放出的单个核苷酸或氨基酸的特性可通过检测器来确定,检测器与反应容器内检测区的探针相通。