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Anandaraj
I M.Sc Environmental Biotechnology
Bharadhidasan University.
FOR CONTACT: anandaraj.wilson@gmail.com
WHAT IS DNA SEQUENCING ?
PROCEDURE
PRINCIPLE
PROCEDURE
The polymerase does not discriminate between dNTPs and ddNTPs, so the
dideoxynucleotide can be incorporated into the growing chain, but it then blocks
further elongation because it lacks the 3′-hydroxyl group needed to form a
connection with the nucleotide.
(Continue)
This process continue until several hundred nucleotides have been polymerized
before a ddATP is eventually incorporated. The result is therefore a set of new
chains, all of different lengths, but each ending in ddATP.
Now the polyacrylamide gel comes on to play. The family of the presence of
ddNTPAs(ddATP,ddCTP,ddGTP,ddTTP) loaded into four adjacent wells of
the gel.
After electrophoresis, the DNA sequence can be read directly from the positions
of the bands in the gel.
CHAIN T ER MAIN TAIO N M ETHOD
HOW DOES THE DNA TEMPLATE OBTAINED?
Pyrosequencing
Sequencing by hybridization
THERMAL CYCLE SEQUENCING
( Sears et al., 1992)
The discovery of thermo stable DNA polymerases, which led to the
development of PCR has also resulted in new methodologies for chain
termination sequencing.
Thermal cycle sequencing is carried out in a similar way to PCR but just
one primer is used and each reaction mixture includes one of the
ddNTP. Because there is only one primer, only one of the strands of the
starting molecule is copied, and the product accumulates in a linear
fashion, not exponentially as is the case in a real PCR.
In their method, the primer was labeled with one of four different
fluorescent dyes and each was placed in a separate sequencing
reaction with one of the four dideoxynucleotides plus all four
deoxynucleotides.
(CONTINUE)
AUTOMATED DNA SEQUENCING WITH FLUORESCENTLY
LABELED DIDEOXYNUCLEOTIDES
Each dNTP is therefore added separately, one after the other, with a
nucleotidase enzyme also present in the reaction mixture so that if a
dNTP is not incorporated into the polynucleotide then it is rapidly
degraded before the next dNTP is added.
PYROSEQUENCING
The problem with this approach is that the maximum length of the
molecule that can be sequenced is given by the square root of the
number of oligonucleotides in the array.
SEQ UEN CED DY HYBR IDI ZATI ON