VALIDATION OF A METHOD FOR AUTOMATED NUCLEOTIDE-SEQUENCE ANALYSIS ANDESTIMATION OF THE LIMITS OF DETECTION OF VARIANT SEQUENCES IN A HETEROGENIC DNA SAMPLE
Dj. Kobelt et al., VALIDATION OF A METHOD FOR AUTOMATED NUCLEOTIDE-SEQUENCE ANALYSIS ANDESTIMATION OF THE LIMITS OF DETECTION OF VARIANT SEQUENCES IN A HETEROGENIC DNA SAMPLE, Analytical letters, 31(1), 1998, pp. 41-54
The assessment of the genetic stability of an expression-system used f
or the production of recombinant drugs and the verification of the nuc
leotide sequence of expression plasmids makes it necessary to estimate
a limit of detection for variant sequences. Therefore, a series of ex
periments was performed in which defined mixtures of two different pla
smid-DNAs were subjected to nucleotide sequence analysis using an auto
matic sequencer (ABI 377) and PCR-based sequencing reactions. A plasmi
d pGR201 (Fig. 1a), encoding recombinant non-glycosylated human prouro
kinase (Saruplase), was spiked in varying ratios (90:10; 80:20; 70:30;
60:40) with a related plasmid, pSJ41 (Fig. 1a), differing in a single
base-substitution and a cluster of multiple mutations, respectively.
Each of these mixtures was sequenced six times in a 50 bp-region cover
ing tile variant sequences in order to estimate a limit of detection f
or point mutations and clusters of multiple mutations. The results are
of general interest since we analysed enough point-mutations as well
as deletion-mutations to allow a statistical assessment. It was shown
that a single point mutation resulting in a base-substitution will be
detected by sequencing in one direction if at least 20 % of the plasmi
ds within the DNA sample are mutated, while the sequencing method is s
ensitive enough to detect a cluster of multiple mutations when at leas
t 10 % of the sequenced plasmid DNA molecules carry the altered sequen
ce. In contrast to other publications the sequencing-procedure present
ed in this paper results in electropherograms with small background si
gnals although only a very simple purification of the sequencing react
ion products was used in order to present a method that is not time-co
nsuming.