Re: DNA sequencing

rmj wrote:

"John Harshman" <jharshman.diespamdie@xxxxxxxxxxx> wrote in message
news:X23Yh.1039$uJ6.981@xxxxxxxxxxxxxxxxxxxxxxxxxxxxx

rmj wrote:

[replacement for a post that disappeared into the ozone]


Does anyone know of any web site that goes into detail regarding the
methodology of sequencing DNA?

No, but I can try explaining a bit.


I can't imagine how the obstacles are overcome. The human chromosome is 3
billion or so base pairs. If one ended up with eight base pair fragments
there would be 4^8 (or almost 17 million) possible sequences; this means
close to 200 positions in the chromosome would have each combination. So
one
would apparently need to work with even longer chains. In addition I
suspect
that splitting the chromosome into smaller parts would be very
unspecific.

I'm not sure what you're getting at here. Sequencing involves two steps,
generally. First amplification (getting more DNA) with PCR or bacterial
cloning. The pieces amplified are usually somewhere between 1000 and
10,000 bases. Then sequencing of smaller pieces; generally 600-800 bases
are about as much as you can get in one go. So you assemble 800-base
"reads" of the 10,000-base fragment until you have complete overlap in
two directions. Then you assemble those fragments into longer sections
by noting the overlaps. Nobody works with anything nearly as short as 8
bp.


In sequencing proteins one works with much smaller molecules with the
additional advantages that there are twenty-two amino acids and one can
create conditions of fairly specific cleaving.

The way in which DNA is sequenced doesn't resemble the way proteins are
sequenced in the slightest.


Perhaps the scientific community is perpetrating a hoax; you know, like
staging the moon landing on a Hollywood set.

Or perhaps you don't understand how DNA sequencing works. It's really
one of the coolest things I know. Essentially what you do is replicate a
piece of DNA many times. The mix of raw materials contains a proportion
of di-deoxynucleotides. When a replicating fragment incorporates a
di-deoxynucleotide, replication stops. So you end up with fragments of
all different lengths from 1 to the length of the piece being
replicated. Now here's the fun part: each di-deoxynucleotide is labeles
with a different color of fluorescent die, one color each for A, C, G,
and T. So if you sort the fragments by size with electrophoresis and
march them past a window in which a laser causes each dye to fluoresce
in turn, the shortest fragment goes past the window first and tells you
what base it ends with, and then the fragment one base longer, and then
one base longer, and so on until you have read the entire sequence. The
limitation on reading is that fragments differ less in mass from their
neighbors as they get longer and longer, and eventually you can't
distinguish adjacent peaks from each other.


An example of how remarkable human ingenuity can be.

Sanger dideoxy sequencing is my all time favorite scientific technique
in terms of sheer coolness.

Here, this seems to do a reasonable job:
http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/sequencing.html




.

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