Digital amplification

Abstract

The identification of pre-defined mutations expected to be present in a minor fraction of a cell population is important for a variety of basic research and clinical applications. The exponential, analog nature of the polymerase chain reaction is transformed into a linear, digital signal suitable for this purpose. Single molecules can be isolated by dilution and individually amplified; each product is then separately analyzed for the presence of pre-defined mutations. The process provides a reliable and quantitative measure of the proportion of variant sequences within a DNA sample.

1 - 103 . (canceled)  
     
     
         104 . A molecular beacon probe comprising: 
 an oligonucleotide comprising a stem and a loop structure and having a photoluminescent dye at one of the 5′ or 3′ ends and a quenching agent at the opposite 5′ or 3′ end, wherein the loop consists of 14-26 bases and has a Tm of 50-51° C., and wherein the stem consists of 4 base pairs having a sequence 5′-CACG-3′.    
     
     
         105 . The molecular beacon probe of  claim 104 , wherein the probe detects a wild-type nucleic acid better than a mutant nucleic acid.  
     
     
         106 . The molecular beacon probe of  claim 104 , wherein the probe detects a mutant nucleic acid better than a wild-type nucleic acid.  
     
     
         107 . A molecular beacon probe comprising: 
 an oligonucleotide comprising a stem and a loop structure and having a photoluminescent dye at one of the 5′ or 3′ ends and a quenching agent at the opposite 5′ or 3′ end, wherein the loop consists of 14-26 bases and has a Tm of 54-56° C., and wherein the stem consists of 4-6 base pairs comprising a sequence 5′-CACG-3′.    
     
     
         108 . A pair of molecular beacon probes comprising: 
 a first oligonucleotide comprising a first stem and a first loop structure and having a photoluminescent dye at one of the 5′ or 3′ ends and a quenching agent at the opposite 5′ or 3′ end, wherein the first loop consists of 14-26 bases and has a Tm of 50-51° C., and wherein the first stem consists of 4 base pairs having a sequence 5′-CACG-3′; and