Human Molecular Genetics, Vol 5, 107-114, Copyright © 1996 by Oxford University Press
Q Liu, J Feng and SS Sommer
There is a great need for rapid screening methods that detect essentially
all mutations. Dideoxy fingerprinting (ddF) is a highly sensitive screening
method that is performed by electrophoresing one lane of a Sanger dideoxy
termination reaction through a nondenaturing gel. Mutations may produce an
extra segment or eliminate a segment from the termination products
(informative dideoxy component). In addition, mutations can be detected by
the altered mobility of one or more termination segments (informative SSCP
component). To screen larger segments with virtually 100% sensitivity,
bi-directional ddF (Bi-ddF) was developed. Bi-ddF is a 'second generation
ddF' in which the dideoxy termination reaction is performed simultaneously
with two opposing primers. Bi-ddF has two important advantages over ddF:
(i) the dideoxy component can detect 10 of the 12 types of possible
single-base substitutions; and (ii) the SSCP component is enhanced because
alterations of mobility can be detected in either the downstream or
upstream direction. As a result, Bi-ddF can screen larger regions of
genomic DNA with virtually 100% sensitivity. Bi-ddF detected 100% of 28
single-base substitutions in a 494 bp segment containing exons B and C of
the human factor IX gene and 100% of 42 single-base substitutions and one
microdeletion present in a 577 bp region containing exon H. In a blinded
analysis in which 39 wildtype samples were randomly mixed with 51 mutant
samples, all mutations were detected with no false positives. Bi-ddF
requires essentially the same effort as ddF, yet twofold more DNA sequence
can be screened reliably per unit effort.
ARTICLES
Bi-directional dideoxy fingerprinting (Bi-ddF): a rapid method for quantitative detection of mutations in genomic regions of 300-600 bp
Department of Biochemistry and Molecular Biology, Mayo Clinic/Foundation, Rochester, MN 55905, USA.
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