New type of PCR primer that offers exquisite discrimination of single nucleotide polymorphisms
Life Sciences : Research Tools
Available for licensing
- Andrew Ellington, Ph.D. , Molecular Biosciences
- Michelle Byrom , University of Texas at Austin
- Yu Sherry Jiang , University of Texas at Austin
- Sanchita Bhadra , Molecular Genetics and Microbiology
Accurate detection of single nucleotide polymorphisms (SNPs) is crucial for diagnosing diseases, assessing disease risk, drug efficacy and side effects, and for disease management.
Real-time polymerase chain reaction (PCR) is currently widely used for rapid and sensitive SNP diagnostics; however, there are at least two drawbacks of this method using conventional PCR primers: first, the presence of impurities or contaminants in prepared samples can lead to non-specific amplification; and second, differences in amplification efficiency from wild-type templates compared to SNP-containing templates are often too small to achieve an ideal level of discrimination.
Thus, there is a need for robust discrimination between SNPs, irrespective of sample origin, condition, preparation, or purity.
The Ellington lab at UT Austin has developed a toolkit of unique primers and primer design rules that distinguish single nucleotide polymorphisms with an exquisite degree of discrimination (i.e., up to 100,000-fold), resulting in little to no false positives.
The rationally designed primers contain unique structural components and a powerful mechanism of action that allow hybridization to matched targets with great efficiency while greatly diminishing binding to non-matching targets. This establishes a large amplification bias in favor of the matched template versus the non-matching template, allowing exquisite allelic distinction in real-time.
Additionally, inexpensive primers for different SNPs can be multiplexed and can lead to definitive identification of each SNP allele, even in parallel.
- Can be used for the identification of SNPs in any context--identifying, for example, disease susceptibility, drug-resistant alleles of infectious organisms, or drug-resistant cancer cells to guide therapy
- Can be used to analyze SNPs from complex mixtures of genetic material
- Can be used in any PCR application to provide a high degree of target discrimination
- Better discrimination than the current competitors on the market
- Digital level of accuracy (up to 100,000-fold discrimination; i.e., yes/no level of accuracy)
- Little to no false positives (i.e., no amplification of unmatched templates, or up to 30 cycles difference between average Cq values for wild-type and SNP containing templates)
- Reduced primer-dimer formation
- The presence of all possible SNPs at one site can be tested simultaneously.
- Primers are relatively cheap and easy to manufacture and do not require specialized nucleotides.
- May be useful for some forensic analyses; e.g., phenotypic prediction and paternity testing
Forensics, diagnostics, SNP analysis
- 1 U.S. patent application filed