System and method for determining changes in fluorescence of stained nucleic acid in electrophoretically separated bands

A system for determining the mass of fluorophore stained, electrophoretically separated nucleic acid fragments in at least one band in at least one gel sample, said system being useful to measure mass down to from about 1.0 to about 5.0 picograms of nucleic acid per band, said system comprising the following elements:

• (i) a visible light constant output laser;
• (ii) narrow band pass filter means for blocking incoherent light from the laser beam;
• (iii) a sample gel cassette, the gel in said cassette being less than 1 mm thickness, being positioned in contact with and between smooth parallel surfaces of the cassette and said gel being substantially free of particulates of size greater than about 0.20 microns,
• (iv) means for directing said laser beam at a preselected spot diameter and at an incidence of from about 0 to about 10 degrees on said gel in said cassette;
• (v) means for passing said gel sample in said cassette through said laser beam spot;
• (vi) lens means for collecting the fluorescent signal generated by the passage of said at least one nucleic acid fragment containing band in said gel through said laser beam spot, for passing said collected signal through filters for blocking light at the excitation wavelength and through an imaging lens onto a detector to measure the intensity of fluorescence from said at least one band, said lens means being shielded from ambient and stray light interference;
• and
• (viii) computer means for processing the fluorescence intensity data, said computer means truncating noise spikes at the base, when said noise spikes have a full width at half maximum in a plot of fluorescent intensity versus separation direction that is less than the width of a peak for a nucleic acid band on said same plot.

A method for determining the mass of fluorophore stained nucleic acid fragments in at least one band in at least one gel sample which comprises the following steps:

• (i) exposing said gel sample including said at least one nucleic acid fragment band to a focused, visible light, constant output laser beam
• said beam being substantially free of incoherent light and having a spot diameter that can be as small as about 0.005 mm and being directed on said band at an incidence of not more than about ten degrees;
• (ii) collecting the fluorescent signal generated by step (i);
• (iii) measuring the intensity of the fluorescence in the collected signal under conditions such that said signal is free of light at the excitation wavelength;
• (iv) processing the fluorescence intensity data from step (iii) to determining the mass of the nucleic acid fragments in said at least one band, said processing step including truncation at the base of noise spikes having a full width at half maximum in a plot of fluorescent intensity versus separation direction that is less than the width of the peak for a nucleic acid band of said same plot.

A method useful to determining the presence of from about 1.0 to about 5.0 picograms a nucleic acid in an electrophoretically separated band of fluorphore stained nucleic acid in a gel which comprises the steps of

• (i) providing a gel cassette including a slab gel less than 1.0 mm in thickness, said gel substantially free of particulates larger than about 0.20 microns and positioned in contact with and between the parallel surfaces of upper and lower plates
• said gel including at least one electrophoretically separated band containing fluorophore stained nucleic acid fragments;
• (ii) exposing said gel including said at least one band to a focused, visible light constant output laser beam
• said beam being substantially free of incoherent light and having a spot diameter that can be as small as about 0.005 mm and being directed on said gel at about the minimum incidence angle to suppress interference fringing;
• (iii) passing said at least one band through said laser beam;
• (iv) collecting the fluorescent signal generated in step (iii);
• (v) measuring the intensity of the fluorescence in the collected signal under conditions such that said signal is free of light at the excitation wavelength;
• (vi) processing the fluorescence intensity data from step (iv) to determine the mass of the nucleic acid fragments in said at least one band, said processing step including truncation at the base of noise spikes having a full width at half maximum in a plot of fluorescent intensity versus separation direction that is less than the width of the peak for a nucleic acid band on said same plot.