dideoxy DNA Sequencing

1
dideoxy DNA Sequencing
polymerases use a free 3' hydroxyl to add a new
dNTP to a growing DNA chain, releasing 2
phosphates dideoxynucleotides end DNA synthesis
no 3' hydroxyl for the next dNTP to
bind often run 4 reactions, one each with
ddA, ddC, ddG, and ddT to stop randomly at
each base label each ddNTP with 35S or
fluorescence
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dideoxy DNA Sequencing
combining dideoxy nucleotides with PCR, can
sequence small amounts of DNA-- linear
amplification of pieces instead of
exponential newer method- use different
fluorescent colors representing the different
base can have all 4 ddNTPs in one lane- gives
more sequences per gel, with 1 sequence per
lane
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Genomic Sequencing
Fragment the human genome using X rays-- clone
large fragments into artificial
chromosomes order the artificial chromosomes
and subclone into cosmids order cosmids along
the artificial chromosomes sequence DNA from
cosmids in a random fashion-- shotgun
sequencing put lots of overlapping fragments
into a computer to sequence a cosmid use
overlapping cosmid sequences to map artificial
chromosomes use overlapping artificial
chromosomes to generate a complete sequence
build up overlapping short sequences