Intermittent detection during analytical reactions

Abstract

Methods, devices, and systems for performing intermittent detection during analytical reactions are provided. Such methods facilitate collection of reaction data from disparate reaction times. Further, such methods are useful for reducing photo-induced damage of one or more reactants in an illuminated analytical reaction at a given reaction time. In preferred embodiments, the reaction mixture is subjected to at least one illuminated and non-illuminated period and allowed to proceed such that the time in which the reaction mixture is illuminated is less than a photo-induced damage threshold period.

1 .- 18 . (canceled) 
     
     
         19 . A method of controlling the progress of a polymerase on a template nucleic acid comprising:
 providing a template nucleic acid comprising a duplex fragment inserted between two hairpin linkers, wherein one or both of the hairpin linkers comprise a stop or pause point and wherein at least one of the hairpin linkers comprises a primer binding site;   providing nucleotides, a primer complementary to the at least one primer binding site, and a polymerase enzyme under conditions whereby nucleotides are incorporated by the polymerase enzyme in a template-directed primer extension reaction, wherein the primer extension reaction is stopped or paused when the polymerase enzyme encounters a stop or pause point.   
     
     
         20 . The method of  claim 19  wherein the one or both of the hairpin adaptors comprises a pause point, and the method further comprises reinitiating the extension reaction after the extension reaction is paused. 
     
     
         21 . The method of  claim 19  wherein the stop or pause point comprises a blocking moiety. 
     
     
         22 . The method of  claim 19  wherein the stop or pause point comprises a photolabile group. 
     
     
         23 . The method of  claim 19  wherein the stop or pause point comprises a strand binding moiety. 
     
     
         24 . The method of  claim 19  wherein the stop or pause point comprises a non-native nucleotide. 
     
     
         25 . The method of  claim 19  wherein the stop or pause point comprises an abasic site. 
     
     
         26 . The method of  claim 25 , wherein providing the template nucleic acid comprises treating the template nucleic acid with Uracil DNA glycosylase to introduce the abasic site at a site-specific deoxyuracil of one or both of the hairpin linkers. 
     
     
         27 . The method of  claim 25  wherein one set of conditions are provided whereby the primer extension reaction is stopped at the abasic site, and another set of conditions are provided whereby the extension reaction continues past the abasic site. 
     
     
         28 . The method of  claim 27  wherein the another set of conditions comprise the use of pyrene. 
     
     
         29 . The method of  claim 19 , wherein the stop or pause point is a site that causes a permanent cessation of nascent strand synthesis. 
     
     
         30 . The method of  claim 19  wherein the stop or pause point comprises a specific sequence to which a blocking group will bind. 
     
     
         31 . The method of  claim 30  wherein the blocking group is a reversibly bound blocking group 
     
     
         32 . The method of  claim 31  wherein the reversibly bound blocking group is removed and the primer extension reaction is reinitiated. 
     
     
         33 . The method of  claim 31  wherein the reversibly bound blocking group comprises a protein. 
     
     
         34 . The method of  claim 31  wherein the reversibly bound blocking group comprises a lac repressor protein and the specific sequence comprises a lac repressor recognition sequence. 
     
     
         35 . The method of  claim 19  wherein the stop or pause point comprises a pyrimidine dimer 
     
     
         36 . The method of  claim 24  wherein the non-native nucleotide is produced by a modification enzyme. 
     
     
         37 . The method of  claim 36  wherein the modification enzyme comprises a glycosylase, a methylase, or a nuclease. 
     
     
         38 . The method of  claim 19  wherein the duplex fragment comprises genomic DNA.