How an Enzyme Helps Find Specific DNA in a Sample

This patent describes a method for detecting a specific DNA sequence in a sample by using a labeled DNA probe and an enzyme that cuts the probe, releasing detectable fragments.

What it covers

This patent describes a process for finding a specific target nucleic acid (like DNA) in a sample. First, the sample is mixed with two short DNA pieces, called oligonucleotides. One oligonucleotide binds to a specific part of the target DNA, and a second, labeled oligonucleotide binds to another nearby part of the same target DNA strand (Claim 1a). Then, an enzyme called a template-dependent nucleic acid polymerase, which has a special ability to cut DNA from its 5' end (5' to 3' nuclease activity), is added (Claim 1b). This enzyme cuts the labeled oligonucleotide, releasing its detectable fragments. Finally, these released labeled fragments are detected and/or measured (Claim 1c). For example, this process can be used to detect a specific viral DNA sequence in a patient sample.

What it doesn't cover

• —Does not cover detection methods that do not use a labeled oligonucleotide.
• —Does not cover systems where the nucleic acid polymerase lacks 5' to 3' nuclease activity.
• —Does not cover methods where the first oligonucleotide's 3' end is not upstream of the labeled oligonucleotide's 5' end.
• —Does not cover detection without the release of labeled fragments.
• —Does not cover methods using only a single oligonucleotide probe for detection.
• —Does not cover detection methods that rely solely on nucleic acid polymerization without nuclease activity.

The clever bit

The truly novel aspect was using the nucleic acid polymerase's natural 5' to 3' nuclease activity to cleave a labeled probe *during* the DNA synthesis process itself. This allowed for simultaneous amplification and detection, eliminating the need for separate post-amplification detection steps.

Why it matters

This patent laid the groundwork for a widely used molecular diagnostic technique known as TaqMan, or probe-based real-time PCR. It provided a way to detect and quantify specific DNA or RNA sequences in real-time during a PCR amplification. This innovation significantly sped up and simplified genetic testing, making it a cornerstone for research, medical diagnostics, and forensic science.

Real-world examples

• 1.TaqMan assays for gene expression analysis
• 2.Quantitative PCR (qPCR) diagnostics
• 3.COVID-19 diagnostic tests
• 4.Detection of pathogens in food safety
• 5.Genetic screening for inherited diseases