Multiplex-PCR consists of multiple primer sets within a single PCR mixture to produce amplicons that are specific to different DNA sequences. By targeting multiple genes at once, additional information may be gained from a single test run that otherwise would require several times the reagents and efforts to perform.
One of the major obstacles that can decrease the assay sensitivity of multiplex-PCR is the accumulation of primer-dimers (PD). A PD consists of primer molecules that hybridize to each other due to strings of complementary bases, particularly at the 3′-ends of the primers. The presence of many primer pairs at very high concentrations in multiplex PCR reactions increases the chances of formation of primer dimers. Once formed, short PD tend to be amplified very efficiently, potentially inhibiting the amplification of the desired DNA sequences by the massive consumption of primers and other reagents. PD formation can be reduced by a combination of different approaches, including special primer design and modification methods, the use of hot start Taq polymerase, PCR additives and optimized PCR cycling conditions.
Various primer design and modification methods have been reported to reduce the PD formation. Brownie et al (Nucleic Acids Res, 25(16): 3235-41, 1997) describe HANDS (Homo-Tag Assisted Non-Dimer System). In HANDS PCR, all target-specific primers contain a common tail sequence at their 5′ ends at low concentration and are mixed with a single tail-specific primer at a higher concentration. After at least two cycles of target specific PCR, the annealing temperature is elevated for the subsequent amplification cycles which are driven entirely by the tail-specific primer. Consequently, the single strands from all PCR products, including desired amplicons and side-products such as PD, have complementary 5′ and 3′ ends leading to the formation of the same stem-loop structures. Due to the high local concentrations of the tail sequences, the stem-loop structures formed in short products, such as PD, are very stable and out-compete the subsequent annealing of the tail-specific primer, resulting in the inhibition of PD amplification. However, with the same tail sequence on each end of all primers, this method requires the targeted amplicons to be long enough to minimize the inhibitory effects of stem loops on the real target products. Depending on the length and the composition of targeted amplicons in a highly multiplexed PCR, the tightness of the stem loop of each amplicon varies, which may lead to significantly imbalanced amplification. Furthermore, the stem loop may not be stable enough to inhibit PD formation between long primers.
U.S. Pat. No. 5,792,607 (Backman et al) and U.S. Patent Application Publication No. 20140329245 disclose a method using endonuclease IV to cleave off the modified non-Extendable 3′ of the primers to activate the primers upon specific primer-template hybridization. Dobosy et al. (BMC Biotechnol. 11: 80, 2011) report a rnase H-dependent PCR (rhpcr) Method using rnase H to cleave off a single RNA base positioned close to the 3′-end of the Blocked primers to activate the primers upon the primer-template specific hybridization. This Method was commercialized recently by IDT (Integrated DNA Technologies, US Patent Application Publication No. 2009/0325169, PCT/US2012/030413). All of these approaches require modified bases in primers and additional enzymes for primer activation, which results in higher cost.
Peleg et al (Appl. Environ. Microbiol., 75: 6393-6398, 2009; WO/2009/004630) report that DNA-RNA chimeric primers in PCR reduces PD formation. Dual Priming Oligonucleotide (DPO) primer (Seegene Technologies) has been reported to reduce PCR PD formation (Chun et al., Nucleic Acids Res. 35(6): e40, 2007). DPO comprises of two separate priming regions (5′-end stabilizer and 3′-end determiner) joined by a polydeoxyinosine linker. Non-specific hybridizations of the primers, such as PD, are reduced at the 3′-end of the DPO primer due to the “bubble”-like structure comprised of the weak hydrogen bonds of the polydeoxyinosine linker. The above RNA bases in the chimeric primers and the polydeoxyinosine linker in the DPO primers significantly increase the complexity and the cost of primer manufacturing.
Scatterfield (J. Mol. Diagn., 16: 163-173, 2013) reports cooperative primers that consist of two DNA sequences linked through a polyethylene glycol linker either 5′ to 5′ or 5′ to 3′. The results indicate that singleplex PCR reactions using cooperative primers greatly reduce primer-primer propagation in the presence of added primer dimers.
Despite these efforts, PD formation remains a big challenge in multiplex PCR. In particular, the multiplex level for target enrichment in next generation sequencing (NGS) applications is extremely high when hundreds of or even thousands of primers are present in the same PCR reaction pool. All of these primers can potentially form primer dimers.