How to Make Many Copies of a Specific DNA Segment

This patent describes a process for detecting a specific nucleic acid sequence by repeatedly copying it. First, a sample containing nucleic acids is treated with two short DNA pieces called oligonucleotide primers, one for each strand of the target sequence, under conditions where they attach and an enzyme builds new complementary strands (Claim 1a). Next, the sample is heated to separate these newly formed strands from their templates (Claim 1b). The primers and enzyme then build new strands again using the separated strands as templates (Claim 1c). Repeating these steps (Claim 2) exponentially increases the amount of the specific nucleic acid. Finally, a labeled probe is added to bind to the amplified sequence, allowing its detection (Claim 1d, 1e). For example, this process could be used to find a specific viral DNA sequence in a patient sample.

The clever bit

The truly novel aspect was the idea of using a pair of primers and repeated cycles of denaturation, annealing, and extension to exponentially amplify a specific DNA segment, with each newly synthesized strand serving as a template for subsequent rounds.

What it does not cover

• ×Does not cover methods that amplify DNA without using two oligonucleotide primers, one for each strand of the target sequence.
• ×Does not cover methods that amplify DNA without repeating the denaturation and extension steps at least once.
• ×Does not cover methods for detecting a nucleic acid sequence that do not involve adding a labeled oligonucleotide probe and determining its hybridization.
• ×Does not cover amplification processes where the primers do not create extension products that can themselves serve as templates for the other primer.
• ×Does not cover methods that use enzymes not listed or functionally equivalent to those described in Claim 15 for polymerization.