Food Chemistry

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 termi-
nation 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 single-
stranded 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 ineach case. The primer

serves as a defined starting position and also as an

initiator for the start of the synthesis of the com-

plementary 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 de-

termined 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 dideoxynu-

cleotides can also be radioactively labelled

(e. g., with^32 P). In this case, four separate DNA

syntheses are also required.

(1.90)