Parameters affecting the sensitivities of dideoxy fingerprinting and SSCP.
The goals of the present experiments are (1) to improve dideoxy fingerprinting (ddF) and (2) to utilize ddF as a tool to evaluate the relative merits of different conditions for single-strand conformation polymorphism (SSCP). ddF is performed by electrophoresing one dideoxy termination reaction through a nondenaturing gel. The ddF pattern can be divided into a "dideoxy component" and an "SSCP component". If dideoxy CTP (ddCTP) is utilized for the termination reaction of ddF, the dideoxy component is abnormal when an extra segment is produced by a sequence change that creates an extra C or when a segment is eliminated by a change of C to another base. All subsequent segments produced by the termination reaction constitute the SSCP component that contains the mutation in a nested series of ddCTP termination products. The SSCP component is informative if abnormal mobility is detected in one or more of the segments. Herein, we utilize 84 different single-base changes in the human factor IX gene to examine the effects of gel matrix, temperature, and different primers on the sensitivity of ddF. The effects of glycerol and cross-linker ratio were examined on fewer mutations. The following conclusions emerge: 1. The sensitivity of the dideoxy component is invariant, but the sensitivity of the SSCP component can vary greatly with gel matrix, temperature, segment size, and sequence context. 2. For a given segment containing a mutation, it is likely that a mobility shift will be seen under some conditions but not under other conditions. By examining the mobility of the SSCP component in > 2200 segments, it was found that some conditions are statistically more likely to result in altered mobility, thereby increasing the average sensitivity of mutation detection by ddF or conventional SSCP. 3. GeneAmp and MDE gels are superior to polyacrylamide gels and electrophoresis at 8 degrees C is superior to electrophoresis at 23 degrees C. GeneAmp at 8 degrees C provided the highest SSCP component efficiency of all conditions tested; all 84 hemizygotes and 40 heterozygotes were detected readily by ddF under these conditions. 4. The segments that terminate near the mutation site are likely to show an abnormal mobility on polyacrylamide gels at 23 degrees C. 5. The likelihood of mobility shifts decreases with segment size, but sequence context can have a major effect on SSCP component efficiency.(ABSTRACT TRUNCATED AT 400 WORDS)
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Department of Biochemistry and Molecular Biology, Mayo Clinic/ Foundation, Rochester, Minnesota 55905, USA.
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D001483:Base Sequence; D004247:DNA; D016172:DNA Fingerprinting / Q000379:methods*; D003845:Deoxycytosine Nucleotides; D054306:Dideoxynucleotides; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005164:Factor IX / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D018807:Polymorphism, Single-Stranded Conformational; D013696:Temperature
D003845:Deoxycytosine Nucleotides; D054306:Dideoxynucleotides; C037406:2',3'-dideoxycytidine 5'-triphosphate; D005164:Factor IX; D004247:DNA