Pulsed-Multiline Excitation for Color-Blind Fluorescence Detection

Abstract

The present invention provides a technology called Pulse-Multiline Excitation or PME. This technology provides a novel approach to fluorescence detection with application for high-throughput identification of informative SNPs, which could lead to more accurate diagnosis of inherited disease, better prognosis of risk susceptibilities, or identification of sporadic mutations. The PME technology has two main advantages that significantly increase fluorescence sensitivity: (1) optimal excitation of all fluorophores in the genomic assay and (2) “color-blind” detection, which collects considerably more light than standard wavelength resolved detection. Successful implementation of the PME technology will have broad application for routine usage in clinical diagnostics, forensics, and general sequencing methodologies and will have the capability, flexibility, and portability of targeted sequence variation assays for a large majority of the population.

1 . A device comprising:
 (a) one or more lasers having two or more excitation lines;   (b) one or more beam steering mirrors wherein said excitation lines each strike said mirrors;   (c) a first prism, wherein said two or more excitation lines strike one surface and exit from a second surface of said first prism; and   (d) a second prism at an angle relative to said first prism, wherein said two or more excitation lines strike one surface of said second prism after exiting said first prism and exit said second prism,   wherein said two or more excitation lines are substantially colinear or coaxial after exiting said second prism.   
     
     
         2 . The method of  claim 1 , wherein said two or more excitation lines are substantially coaxial after exiting said second prism. 
     
     
         3 . The method of  claim 1 , wherein said two or more excitation lines are substantially colinear after exiting said second prism. 
     
     
         4 . A method of illuminating a sample comprising:
 (a) steering two or more excitation lines onto a first surface of a first prism;   (b) steering two or more excitation lines from the second surface of said first prism to a first surface of a second prism; wherein said second prism is angled about 45° from said first prism;   (c) steering said two or more excitation lines onto a sample after exiting second surface of said second prism, wherein said two or more excitation lines are substantially colinear or coaxial after exiting said second prism.   
     
     
         5 . The method of  claim 4 , wherein said two or more excitation lines are substantially coaxial after exiting said second prism. 
     
     
         6 . The method of  claim 4 , wherein said two or more excitation lines are substantially colinear after exiting said second prism. 
     
     
         7 . A method of controlling a sequence of excitation lines comprising:
 obtaining a TTL circuit comprising an electronic stepper wherein said circuit is operationally connected to one or more lasers having two or more excitation lines; and   controlling the sequential firing of the one or more lasers having two or more excitation lines with a clock pulse from the circuit, wherein the frequency of firing one laser is equivalent to the frequency of firing a second laser, but phased shifted so that one or more lasers having two or more excitation lines can be sequentially pulsed.   
     
     
         8 . The method of  claim 7 , wherein the cycle time of one clock pulse is from 1 second to 5 seconds. 
     
     
         9 . The method of  claim 7 , wherein the length of time a first laser produces an excitation line is similar to the length of time a second laser produces an excitation line. 
     
     
         10 . The method of  claim 7 , wherein between 2-to-16 excitation lines are sequentially pulsed. 
     
     
         11 . The method of  claim 10 , wherein between 2-to-8 excitation lines are sequentially pulsed. 
     
     
         12 . A method of controlling a sequence of excitation lines comprising:
 obtaining a TTL circuit comprising an electronic stepper wherein said circuit is operationally connected to two or more lasers; and   controlling the sequential firing of the two or more lasers with a clock pulse from the circuit, wherein the frequency of firing a first laser is different from the frequency of firing a second laser.   
     
     
         13 . The method of  claim 12 , comprising between 2-to-16 lasers.