Reverse Complement PCR (RC-PCR) is a modification of the polymerase chain reaction (PCR). It is primarily used to generate amplicon libraries for DNA sequencing by Next-generation Sequencing (NGS). This patented and unique technique permits the simultaneous multiplex amplification, adapter ligation and indexing in a single closed tube reaction.
In RC-PCR, no target specific primers are present in the reaction mixture. Instead target specific primers are formed as the reaction proceeds. A typical reaction employing RC-PCR requires four oligonucleotides, that interact with eachother in pairs: one RC-PCR probe, one universal primer (containing functional domains of choice), which hybridize with each other at their 3’ ends. The RC-PCR probe contains the reverse complement sequence of the desired target specific primer sequence. Once RC-PCR probe and universal primer have hybridized, the universal primer can be extended, using the oligonucleotide probe as the template, to yield fully formed, target specific primers, which are then available to amplify the target DNA in subsequent rounds of thermal cycling like in a standard PCR reaction.
RC-PCR kinetics provide higher sensitivity and specificity compared to standard PCR for library prep, as concentrations of primers synthesized during the reaction and the amplicons are more in line. This leads to more balanced reaction components, reducing potential primer dimerization and off-target primer binding.
During the RC-PCR reaction the probe is not consumed; it is available to act as a template for the universal primer to be ‘converted’ into target specific primer during successive PCR cycles. This generation of target specific primer occurs in parallel with standard PCR amplification under standard PCR conditions.
RC-PCR provides the flexibility of attaching sequences or functional domains of choice to either end of any amplicon, using a single target specific probe pair with a whole library of universal primers. This benefit is used to apply sample specific indexes independently to each end of the amplicon construct, so-called Unique Dual Indexes (UDI), allowing effective sample identification in the NGS workflow. Amplified products are pooled for sequening at scale limited only by the number of available indexes. This sequencing strategy, recommended by illumina®, has distinct index sequences for each of the so-called i5 and i7 index reads.
Image rights Illumina, Inc.
RC-PCR also provides the flexibility of obtaining barcode tagged amplicons, by hybridizing universal barcoded primers with RC-PCR probes, which are then available to amplify target DNA in a pooled library. Similar to Unique Dual Indexes, the barcodes are used to demultiplex the library during a Nanopore sequencing run. A dual barcoding strategy, applying inner (PCR) and outer (native) barcodes, is recommended for sequencing of more than 96 samples or sorting libraries into multiple categories, as each barcode can be used to denote a different sample feature.
Image rights Oxford Nanopore Technologies
