Methods of Labelling Polynucleotides with Dibenzorhodamine Dyes

Abstract

Dibenzorhodamine compounds having the structure are disclosed, including nitrogen- and aryl-substituted forms thereof. In addition, two intermediates useful for synthesizing such compounds are disclosed, a first intermediate having the structure including nitrogen- and aryl-substituted forms thereof, and a second intermediate having the structure including nitrogen- and aryl-substituted forms thereof, wherein substituents at positions C14 to C18 taken separately are selected from the group consisting of hydrogen, chlorine, fluorine, lower alkyl, carboxylic acid, sulfonic acid, —CH 2 OH, alkoxy, phenoxy, linking group, and substituted forms thereof. The invention further includes energy transfer dyes comprising the dibenzorhodamine compounds, nucleosides labeled with the dibenzorhodamine compounds, and nucleic acid analysis methods employing the dibenzorhodamine compounds.

1 . A dibenzorhodamine compound having the structure  
     
       
         
         
             
             
         
       
     
     including nitrogen- and aryl-substituted forms thereof.  
   
   
       2 - 40 . (canceled)  
   
   
       41 . An intermediate useful for the synthesis of dibenzorhodamine compounds having the structure  
     
       
         
         
             
             
         
       
     
     including aryl- and nitrogen-substituted forms thereof.  
   
   
       42 - 69 . (canceled)  
   
   
       70 . An intermediate useful for the synthesis of dibenzorhodamine compounds having the structure  
     
       
         
         
             
             
         
       
     
     including nitrogen- and aryl-substituted forms thereof wherein: 
 R 1  taken together with the C-12-bonded nitrogen and the C-12 and C-13 carbons forms a first ring structure having from 4 to 7 members; and/or  
 R 1  taken together with the C-12-bonded nitrogen and the C-11 and C-12 carbons forms a second ring structure having from 5 to 7 members.  
 
   
   
       71 - 100 . (canceled)  
   
   
       101 . A labeled nucleoside/tide having the formula: 
       NUC-D wherein    NUC is a nucleoside/tide or nucleoside/tide analog;    D is a dibenzorhodamine dye compound of  claim 1 , NUC and D being connected by a linkage;    wherein if NUC comprises a purine base, the linkage is attached to the 8-position of the purine, if NUC comprises a 7-deazapurine base, the linkage is attached to the 7-position of the 7-deazapurine, and if NUC comprises a pyrimidine base, the linkage is attached to the 5-position of the pyrimidine.    
   
   
       102 - 103 . (canceled)  
   
   
       104 . A method of polynucleotide sequencing comprising the steps of: 
 forming a mixture of a first, a second, a third, and a forth class of polynucleotides such that:    each polynucleotide in the first class includes a 3′-terminal dideoxyadenosine and is labeled with a first dye;    each polynucleotide in the second class includes a 3′-terminal dideoxycytidine and is labeled with a second dye;    each polynucleotide in the third class includes a 3′-terminal dideoxyguanosine and is labeled with a third dye; and    each polynucleotide in the forth class includes a 3′-terminal dideoxythymidine and is labeled with a forth dye;    wherein at least one of the first, second, third, or forth dyes is a dibenzorhodamine compound of  claim 1;     the other of the dyes being spectrally resolvable from the dibenzorhodamine dye(s) and from each other;    electrophoretically separating the polynucleotides thereby forming bands of similarly sized polynucleotides;    illuminating the bands with an illumination beam capable of causing the dyes to fluoresce; and    identifying the classes of the polynucleotides in the bands by the fluorescence spectrum of the dyes.    
   
   
       105 . A method of fragment analysis comprising: 
 forming labeled polynucleotide fragments, the fragments being labeled with a dibenzorhodamine compound of  claim 1;     subjecting the labeled polynucleotide fragments to a size-dependent separation process; and    detecting the labeled polynucleotide fragments subsequent to the separation process.    
   
   
       106 . (canceled)