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Supplementary file 1: RC-PCR, an innovative and effective method for multiplexing forensically relevant SNP marker systems

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posted on 05.08.2021, 12:43 by Magdalena M. Bus, Erik de Jong, Jonathan L. King, Walter van der Vliet, Joop Theelen, Bruce Budowle

Supplementary file 1. The graphical presentation of the RC-PCR technology. The (universal) index primer contains a unique dual index i7/i5 (single indexing also possible), sequence adapter, and universal tail. The RC probe includes an extension blocker with a universal sequence and the reverse complement of the SNP target-specific region (F/R). The indexing and multiplex PCR amplification are performed at the same time in one closed tube. The RC-PCR consists of 2 major steps. 1a – 1c) The universal tail sequences of UIPs hybridize, and the target specific index primers are generated by the Taq polymerase that copies the sequence of the RC probe. In each PCR cycle, new target specific index primers are generated. 2a – 2d) The PCR amplification of SNP-specific amplicons. DNA samples are now tagged with a sample-specific index and Illumina sequence adapter. Now samples can be pooled, purified, and sequenced.


This work was funded in part by Award No. 2020-DQ-BX-0005, awarded by the National Institute of Justice (NIJ), Office of Justice Programs, U.S. Department of Justice. The opinions, findings, and conclusions expressed within this publication are those of the authors and do not necessarily reflect those of the U.S. Department of Justice.