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  • machanism behind the 454 sequencing

    Hi, this confuses me long time. How did 454 machine tell whether it is a A, T, C or G incorporated?

    APS+PPi ATP sulfurylase
    ATP + Luciferin Light + Oxyluciferin luciferase

    There is always one PPi, if add one nt into the DNA strand. Not matter a A, T, C , G is added? How does the machine tell the light strength is from adding an A or C.

    thanks

  • #2
    One uses only one nucleotide at one time and you know the nucleotide you are adding. See: https://www.youtube.com/watch?v=2pzS4JEgKRY (and others)
    Last edited by GenoMax; 02-24-2017, 11:07 AM.

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    • #3
      Originally posted by GenoMax View Post
      One uses only one nucleotide at one time and you know the nucleotide you are adding. See: https://www.youtube.com/watch?v=2pzS4JEgKRY (and others)
      Thanks. BTW, is emPCR also running on the sequencing platform? or There is a special PCR machine for emPCR.

      Thanks,

      Comment


      • #4
        PCR is done on a standard thermocycler. Link for GS FLX Titanium emPCR protocol:

        ftp://ftp.genome.ou.edu/pub/for_broe...il%202009).pdf

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