2009_Book_FoodChemistry

1.4 Proteins 47
Therefore, the primary sequence of a protein can
be derived from the nucleotide (base) sequence.
For the sequencing of DNA, the method of
choice is the dideoxy process (chain termination
process) introduced by Fred Sanger
in 1975. The principle is based on the specific
termination of the enzymatic synthesis of a
DNA strand by means of DNA polymerase
by using a 2 ′ ,3 ′ -dideoxynucleotide, i. e., to
prevent polymerization with the formation
of the 3 ′ → 5 ′ -phosphodiester at the position
of the base in question. For instance, if the
2 ′ ,3 ′ -dideoxynucleotide of guanine is used,
the biosynthesis is stopped at guanine in each
case. To detect all the guanine residues, only
about 0.5 mol% of the dideoxynucleotide in
question (based on the 2-deoxynucleotide) is
used. In this way, DNA fragments of varying
length are obtained which all have the same
5 ′ -terminal and thus mark the position of the
base.
The starting material is a hybrid of the singlestranded
DNA to be sequenced and a primer
consisting of about 20 nucleotides. This is
lengthened with the help of DNA polymerase
and a mixture of the 4 nucleotides and one
2 ′ ,3 ′ -dideoxynucleotide in each case. The primer
serves as a defined starting position and also as an
initiator for the start of the synthesis of the complementary
DNA strand. The DNA fragments of
different length obtained in four experiments are
then separated electrophoretically according to
molecular size. For detection, either the primer
can be labelled with four different fluorescent
dyes (TAG) or the four dideoxynucleotides are
labelled with different fluorescent dyes. In the
former case, 4 series of experiments are carried
out with differently labelled primers and one
of the 4 dideoxynucleotides in each case. The
charges are combined and electrophoretically
separated together. The primary sequence is determined
from the signals measured at different
wave lengths (Fig. 1.13). When 4 differently
labelled dideoxynucleotides are used, the primer
is not labelled. Alternatively, the dideoxynucleotides
can also be radioactively labelled
(e. g., with 32 P). In this case, four separate DNA
syntheses are also required.
(1.90)