Detection of mutations by automated fluorescence/RNA-based dideoxy fingerprinting (ARddF)

Abstract

Dideoxy ®ngerprinting (ddF) is a hybrid technique which combines aspects of single strand conformational polymorphism (SSCP) and dideoxy sequencing to detect the presence of single base changes in a de®ned fragment of nucleic acid. ddF is no more technically demanding than SSCP, yet it is more sensitive in detecting point mutations. We describe here the adaptation of conventional ddF to an automated sequencing system usinḡ uorescent Cy5 labeled primers. We show that automated RNA-based ddF (ARddF) has several advantages over conventional radioisotope-based ddF, including: (1) analysis of larger nucleic acid fragments (up to 10 3 bp), due to the ability to continuously analyse and compile sequencing information; (2) greater reliability for distinguishing mutant sequences from wild type sequences (particularly when the mutation leads to gain or loss of a dideoxy termination segment); (3) the use of¯uorescent labeled primers, making ARddF less hazardous than methods requiring radionucleotides. The use of ARddF in conjunction with new methods for isolating RNA from a small number of cells facilitates mutational analysis of small tissue biopsies and other limited samples, and will allow more widespread application of mutational screening in the setting of clinical diagnostic laboratories.

Rapid separation and laser‐induced fluorescence detection of mutated DNA by capillary electrophoresis in a self‐coating, low‐viscosity polymer matrix

“…Point mutations in DNA can be detected by several methods [39][40][41][42][43][44][45][46][47]. Slab gel electrophoresis is the most commonly used technique.…”

Nucleic Acid Analysis

“…The authors analyzed lung cancer tissue specimens (196). Martincic and Whitlock compared SSCP and dideoxy-fingerprinting with sequencing for detection of mutations in exons 5-8 of the p53 tumor suppressor gene (197). An automated fluorescence-based PCR-SSCP capillary electrophoresis system was developed and applied to the analysis of p53 gene mutations (198).…”

Multiplex Detection of DNA Mutations by the Fluorescence Fingerprint Spectrum Technique

“…[8][9][10] By comparing dideoxy fingerprint patterns of samples with the established standard patterns, DNA mutations can be intuitively and sensitively determined. The dideoxy fingerprint method, which combines the dideoxy sequencing reaction and single-strand conformation polymorphism (SSCP) analysis, has been developed to identify DNA mutations.…”

“…The dideoxy fingerprint method, which combines the dideoxy sequencing reaction and single-strand conformation polymorphism (SSCP) analysis, has been developed to identify DNA mutations. [8][9][10] By comparing dideoxy fingerprint patterns of samples with the established standard patterns, DNA mutations can be intuitively and sensitively determined. Nevertheless, this method is also based on electrophoresis separa-tion and is difficult to simultaneously detect mutations on multiple exons or genes.…”