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Unformatted text preview: same in either direction (“Radar,” “Madam, I’m Adam”). DNA base sequences can be determined by using
DNA polymerase and dideoxynucleotides
Synthesize DNA enzymatically in the presence of a small amount of a labeled
dideoxynucleotide (e.g., ddTTP) and larger amounts of ordinary ATP, GTP, CTP
and TTP, using a primer and one strand of the DNA to be sequenced as a template.
DNA polymerase synthesizes the new strand in the 5’ → 3’ direction. The
dideoxynucleotide interrupts DNA synthesis wherever it is incorporated. This
happens at random points where the corresponding base occurs in the sequence.
In this illustration, each DNA fragment ends in ddT, which is labeled with a red dye. O
O O O P O P O P O-CH2 5’ primer 3’ synthesis
3’ template 5’ O NH O O O ddTTP (continued …) N
O O DNA sequencing can be automated
Repeat the procedure with
ddC, ddA and ddG, using a
different fluorescent dye to
tag the dideoxynucleotide in
each case. Then mix all the
DNA fragments and separate
them according to size by
Electrophoresis of the DNA
fragments gives a “ladder” of
bands, each ending in a
at its 3’ end and tagged with
the corresponding dye. This
procedure has been
automated in machines that
can sequence more than
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This note was uploaded on 02/17/2012 for the course CHEM 212 taught by Professor Staff during the Fall '10 term at Rutgers.
- Fall '10