DNA SEQUENCING

 process of determining the precise order of nucleotides within a DNA molecule.

 It includes any method or technology that is used to determine the order of the four bases—
adenine, guanine, cytosine, and thymine—in a strand of DNA.

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/D/DNAsequencing.html
The dideoxy method gets its name from the critical role played
by synthetic nucleotides that lack the -OH at the 3′ carbon atom (red arrow). A
dideoxynucleotide (dideoxythymidine triphosphate — ddTTP — is the one shown
here) can be added to the growing DNA strand but when it is,chain elongation
stops because there is no 3′ -OH for the next nucleotide to be attached to. For this
reason, the dideoxy method is also called thechain termination method
https://www.youtube.com/watch?v=bEFLBf5WEtc

https://www.youtube.com/watch?v=tiG-rxkhlqg maxam gilbert

the above structure in the image shows a normal dntp which is actually a dttp bcoz it contains thymine
as the nitrogen base. now y does its name begin with deoxy ... it is bcoz a normal ribose sugar contains
two hydroxyl groups but in a dntp the robise lacks one of the two hydroxyl groups at the 2nd carbon.

now notice the below structure. it is exactly same with only one difference i.e. at the 2nd carbon of
ribose .. the hydroxyl group is missing. so since this molecule lacks both the hydroxyl gps it is termed as
dideoxythymine triphosphate. and for this reason sanger method is also knwn as dideoxy method.

now lemme explain y is this method also called chain termination method. while dna synthesis is going
on and the dna strand is growing, continuously a nucleotide is attached at the hydroxyl gp at the 2nd
carbon of the previous nucleotide . the two oh grps combine and a water molecule is released which is
termed as dehydration synthesis. and so the furthewr nucleotides are attached . but if a ddntp (
dideoxyntp) gets attached in the chain.. it wud terminate the reaction there as it lacks the one n only
hydroxyl grp req. by the new incoming dntp.

lets talk abt just one of the four tt . in this several reactions r going on and strands of dna are getting
synthesised complementary to the template strand that we hv added. but it contains some amount of
dideoxynucleotides also. lets say dideoxythymine triphosphate. now there wud be a possibility wen this
dideoxythymine does not get attached in the chain and a full complete comple strand is synthesised. but
there can b also a possibility that this dideoxynucleo gets attached in the chain terminating it there and
cutting the strand short.

now we can reconstruct the seq..

since the fragment which has travelled farthest lies in lane T so the first nucleotide is T. Then next wud
be G then A then T and then T. since the fragments are present in all the lanes at last.. this shows that
one possibility where the dideoxynucleo did not get attached. these are the complete sequences.

maxam gilbert

we take a ds dna which is our sample.

then we separate the strands as we did in the sanger method and we use the single strand.

then to this strand we add a radiolabelled phosphate which is 32p as 5' p

now we take this strand in ample amounts in 4 diff test tubes, hence we create 4 diff set ups

we label the first one as G 2nd as A+G 3rd as T+c and 4th as C