Nucleic acid amplification techniques are powerful tools in making more copies of a nucleic acid that is few in number. Amplification techniques have widespread application including, for example, diagnostics, drug development, forensic investigations, environmental analysis, and food testing.
There are a number of well known methods for amplifying nucleic acid sequences in vitro including, for example, polymerase chain reaction (PCR), ligase chain reaction (LCR), replicase-mediated amplification, strand-displacement amplification (SDA), “rolling circle” types of amplification, and various nucleic acid sequence-based amplification (NASBA) and transcription-medicated amplification (TMA) methods. These methods use different techniques to make amplified sequences, which usually are detected by using a variety of methods. PCR amplification uses a DNA polymerase, oligonucleotide primers, and thermal cycling to synthesize multiple copies of both strands of a double-stranded DNA (dsDNA) or dsDNA made from a cDNA (see, e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159 to Mullis et al.). LCR amplification uses an excess of two complementary pairs of single-stranded probes that hybridize to contiguous target sequences and are ligated to form fused probes complementary to the original target, which allows the fused probes to serve as a template for further fusions in multiple cycles of hybridization, ligation, and denaturation (see, e.g., U.S. Pat. No. 5,516,663 and EP 0320308 B1 to Backman et al.). Replicase-mediated amplification uses a self-replicating RNA sequence attached to the analyte sequence and a replicase, such as Qβ-replicase, to synthesize copies of the self-replicating sequence specific for the chosen replicase, such as a Qβ viral sequence (see, e.g., U.S. Pat. No. 4,786,600 to Kramer et al.). The amplified sequence is detected as a substitute or reporter molecule for the analyte sequence. SDA amplification uses a primer that contains a recognition site for a restriction endonuclease which allows the endonuclease to nick one strand of a hemimodified dsDNA that includes the target sequence, followed by a series of primer extension and strand displacement steps (see, e.g., U.S. Pat. No. 5,422,252A to Walker et al., and U.S. Pat. No. 5,547,861 to Nadeau et al.). “Rolling circle” types of amplification rely on a circular or concatenated nucleic acid structure that serves as a template used to enzymatically replicate multiple single-stranded copies from the template (see, e.g., U.S. Pat. No. 5,714,320 to Kool, and U.S. Pat. No. 5,834,252 to Stemmer et al.). TMA amplification refers to methods that amplify a sequence by producing multiple transcripts from a nucleic acid template. Such methods generally use one or more oligonucleotides, of which one provides a promoter sequence, and enzymes with RNA polymerase and DNA polymerase activities to make a functional promoter sequence near the target sequence and then transcribe the target sequence from the promoter (see, e.g., U.S. Pat. Nos. 5,399,491 and 5,554,516 to Kacian et al., U.S. Pat. No. 5,437,990 to Burg et al., WO 88/010315 to Gingeras et al., U.S. Pat. No. 5,130,238 to Malek et al., U.S. Pat. Nos. 4,868,105 and 5,124,246 to Urdea et al., and US 2006-0046265 A1 to Becker et al.). Nucleic acid amplification methods may amplify a specific target sequence (e.g., a gene sequence), a group of related target sequences, or a surrogate sequence, which may be referred to as a tag or reporter sequence that is amplified and detected in place of the analyte sequence. The surrogate sequence is only amplified if the analyte target sequence is present at some point during the reaction.
The above described technologies often require the use of two or more primers for target amplification. Design and optimization of the primers for each target is required. This is a costly, time-consuming and often challenging and difficult process, especially in multiplex assays for amplifying two or more different target sequences where cross-reaction of primer pairs could be a serious challenge to reliability of the assays. It is also extremely difficult (if not impossible) to design and optimize two primers for amplification of small targets (such as microRNAs, which contains only 18 to 25 nucleotides).
The present invention describes amplification methods using a single composite primer which overcomes the difficulties described above.