188
KLAUS DEICHMANN
OH
OH
OH
I
I
I
N
o =
P - 0 -
P--0 -
P - 0 - - - -
1
I
I
OH
OH
OH
H
Fig. 2. A 2' -,3' -dideoxy nucleotide is lacking at its 3' -end the OH-group, which is essential for
chain elongation.
insert
vector
5'
ATGCCGTAATTGGGCATTGACT
3'
ddA C G G C A T T A A C C C G T A A C T G A
3'
5'
21bp
ddA T T A A C C C G T A A C T G A
3'
5'
16bp
ddA A C C C G T A A C T G A
3'
5'
13bp
ddA C C C G A A C T G A
3'
5'
12bp
Fig. 3. The reaction products after a sequencing reaction
Components of dideoxy chain termination sequencing
The components used in dideoxy sequencing are listed below:
• Vectors
Two groups of vectors are used in dideoxy sequencing reactions: singlestranded vectors allowing direct sequencing and double-stranded vectors, which must be denatured prior to the sequencing reaction
(Mead and Kernper 1986).
• M13 derivatives
M13 is a fllamentous, male-specific coliphage (Messing et al. 1977; Wu et
al. 1983; Sambrook et al. 1989) existing in two forms: a double-stranded
replicative form (RF), present during infection of suitable E. coli host
cells and a single-stranded form, which is packed into a protein coat
and extruded out of the cell. The modern sequencing vectors derived