Bi-directional dideoxy fingerprinting (Bi-ddF): a rapid method for quantitative detection of mutations in genomic regions of 300-600 bp.
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.
Liu
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Department of Biochemistry and Molecular Biology, Mayo Clinic/Foundation, Rochester, MN 55905, USA.
Feng
J
J
Sommer
S S
SS
D004247:DNA / Q000032:analysis / Q000737:chemistry; D016172:DNA Fingerprinting / Q000379:methods*; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers; D015224:Dideoxynucleosides; D005164:Factor IX / Q000235:genetics; D006801:Humans; D008970:Molecular Weight; D018807:Polymorphism, Single-Stranded Conformational; D012680:Sensitivity and Specificity
D017931:DNA Primers; D015224:Dideoxynucleosides; D005164:Factor IX; D004247:DNA