Detecting Genetic Differences Using DNA Probes and Enzymes

This 1987 patent describes a method to find tiny differences in DNA sequences by using special DNA pieces (probes) and cutting enzymes, which can help diagnose genetic conditions like sickle cell anemia.

What it covers

This patent outlines a way to spot specific changes in DNA. You start with a DNA sample and a short, custom-made DNA piece called a probe. This probe is designed to match a specific part of the DNA you're interested in, and it's tagged with a label (like a radioactive marker or a fluorescent dye) near where a potential difference might be. The probe is mixed with the DNA sample, and if the DNA sequence matches, the probe sticks to it. Then, a special enzyme that acts like molecular scissors is used. If the DNA has the specific spot the enzyme cuts, it snips the probe. By separating the cut and uncut probes and looking at their labels, scientists can tell if the specific DNA sequence, and thus the restriction site, is present or absent. Claim 1 details this process, including hybridizing, digesting with a restriction endonuclease, separating fragments, and detecting. Claim 2 adds a step using a 'blocking oligomer' to make the test more precise by preventing the probe from sticking to similar but incorrect DNA sequences.

What it doesn't cover

• —Methods that do not use an oligonucleotide probe complementary to the target nucleic acid sequence.
• —Methods that do not involve digesting the hybridized nucleic acid with a restriction enzyme.
• —Methods where the probe is not labeled at the end nearer the restriction site.
• —Techniques that do not separate labeled cleaved fragments from labeled uncleaved fragments.
• —Methods that do not detect the presence or absence of labeled fragments.
• —Detecting nucleic acid sequences without looking for specific restriction sites.

The clever bit

The innovation lies in combining a specifically designed, end-labeled oligonucleotide probe with a restriction enzyme that cuts at a specific site. This allows for a highly sensitive detection system where cutting the probe directly indicates the presence of the target DNA sequence and its associated restriction site, simplifying detection compared to earlier methods.

Why it matters

This patent is foundational for genetic testing and diagnostics. It provided a precise method for identifying genetic variations, which was crucial for understanding and diagnosing inherited diseases. The ability to detect specific DNA sequences and their variations paved the way for personalized medicine and advanced genetic research.

Real-world examples

• 1.Diagnostic tests for sickle cell anemia
• 2.Genetic screening for inherited diseases
• 3.Early DNA sequencing and analysis techniques
• 4.Research tools for molecular biology