Methods and devices for hemogeneous nucleic acid amplification and detector
AssigneeRoche Molecular Systems, Inc.
InventorRussell G. Higuchi
Life of this patent
Estimating the term…
Abstract
This invention relates to improved methods for nucleic acid detection using methods such as the polymerase chain reaction (PCR). More specifically, the invention provides methods for simultaneous amplification and detection to enhance the speed and accuracy of prior methods. The methods involve the introduction of detectable DNA binding agents into the amplification reaction, which agents produce a detectable signal that is enhanced upon binding double-stranded DNA. In a preferred embodiment, the binding agent is a fluorescent dye. The methods also provide means for monitoring the increase in product DNA during an amplification reaction.
Claims22
A method for detecting the presence of a target nucleic acid in a sample by amplification, said method comprising:
- (a) providing a nucleic acid amplification reaction mixture that comprises said sample and a fluorescent dye, whose fluorescence intensity is increased upon intercalation into a double-stranded nucleic acid, wherein said reaction mixture is contained within a container which container is operably linked to a device suitable for inputting excitation light into said reaction mixture and conveying fluorescent light emitted by said reaction mixture to a detector;
- (b) measuring said emitted light produced by the mixture of step (a);
- (c) treating said mixture under conditions for amplifying said target nucleic acid to produce amplified double-stranded nucleic acid;
- (d) measuring said emitted light produced by said mixture of step (c);
- and
- (e) determining if amplification has occurred.
A method for monitoring the increase in the amount of double-stranded DNA during amplification of a target nucleic acid in a sample, wherein said method comprises the steps of:
- (a) amplifying a region of said target nucleic acid by polymerase chain reaction method in the presence of a fluorescent pigment whose fluorescence intensity is increased upon intercalation into a double-stranded nucleic acid;
- which method comprises locating said sample in a container, which container is suitably located for inputting excitation light into said sample and conveying emission light to a detector for measuring said emissions, and wherein said method comprises the steps of:
- (b) monitoring the fluorescence intensity of said fluorescent pigment during said polymerase chain reaction,
- (c) determining the amount of fluorescence emission that is emitted by said sample.
A method for detecting a target nucleic acid in a sample, comprising the steps of:
- (a) amplifying a region of said target nucleic acid by a polymerase chain reaction method in the presence of a fluorescent pigment whose fluorescence intensity is increased upon intercalation into a double-stranded nucleic acid;
- and
- (b) measuring the fluorescence intensity of said fluorescent pigment before and after said polymerase chain reaction by means of a device operably linked to a measuring devise, whereby an increase in fluorescence intensity is indicative of the presence of said target nucleic acid.
A method for quantifying a target nucleic acid in a test sample, comprising the steps of:
- (a) amplifying a region of said target nucleic acid in a test reaction by the polymerase chain reaction in the presence of a fluorescent pigment whose fluorescence intensity is increased upon intercalation into a double-stranded nucleic acid;
- (b) monitoring the fluorescence intensity of said fluorescent pigment during said polymerase chain reaction by use of a device operably linked to a container containing said polymerase chain reaction and to a detector suitable for measuring emitted light;
- and
- (c) determining the quantity of said target nucleic acid in said sample.
Dependencies are inferred from the claim text.