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Archived Comments for: An improved 2b-RAD approach (I2b-RAD) offering genotyping tested by a rice (Oryza sativa L.) F2 population

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  1. How the PCR amplification performed?

    Ren-Gang Zhang, Beijing University of Chemical Technology

    20 January 2015

    I am a reader. The first paragraph at Page 10 of 13 showed that the non-phosphorylated adapters were used. Thus, after ligation of digested DNA and adaptors, there should be two nicks on one DNA ligation product (Figure below). Between the steps of ligation and PCR amplification, there was no step to repair the nicks (see the 2b-RAD protocol of the paper). So comes the question, how the primers that annealed to the bottom adaptor extended (Figure below), as the PCR condition was: 98℃ for 30 s followed by 12 cycles of 98℃ for 30 s, 65℃ for 30 s, 72℃ for 30 s with a final Taq extension step at 72℃ for 5 min? 



                                                        ↓ Ligate

                       5'_________________________ _______3' (space indicates a nick)

                       3'________ ________________________5'

                                                        ↓ Denature & Anneal & Extend

       primer 5'--------------->?

                       3'________ ________________________5'



    Competing interests

    None declared