machanism behind the 454 sequencing

[SDPA_Pet]

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

[GenoMax]

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)

[SDPA_Pet]

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,

[nucacidhunter]

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