Rapid separation and laser-induced fluorescence detection of mutated DNA by capillary electrophoresis in a self-coating, low-viscosity polymer matrix.
The detection of point and other simple mutations in DNA is important for cancer research and diagnosis and other biological studies. Capillary electrophoresis has been successfully used for separating DNA fragments. However, a low-viscosity polymer sieving buffer for DNA separation with on-line coating has never been reported. In this paper, a new method using capillary electrophoresis with on-line coating and laser-induced fluorescence detection (CE-LIF) for screening for point or simple DNA mutations has been demonstrated. The method uses an on-line dynamic coating technique that increases capillary lifetime and analysis reproducibility, and employs a low-viscosity polymer solution, which allows the user to rinse the capillary rapidly and refill with polymer solution easily. Experiments proved that the additives in the separation buffer for on-line capillary coating do not affect the separation efficiency of the running buffer, and do not interfere with the formation of hydrogen-bonded network between boric acid, mannitol and hydroxypropylmethylcellulose polymers. The stability of the dynamically coated capillary was quantitatively studied; the capillary lifetime was increased 6- to 7-fold compared with that of permanently coated CE columns. Standard DNA fragments containing mutations, with sizes of 209, 219, and 338 bps, were successfully separated and detected with this system, after the mutated DNA fragments were cleaved by CEL-I endonuclease. The technique is very sensitive for the size-separation of low-range, middle-range, and high-range DNA fragments. Results were compared with the HPLC methods developed by Transgenomic, Inc. and were in good agreement. The method should be applicable to mutation detection for all relevant biological and clinical studies. The factors influencing separations and the stability of dynamic capillary coatings are also discussed in the paper.
D001888:Boric Acids; D004247:DNA / Q000032:analysis* / Q000302:isolation & purification; D019075:Electrophoresis, Capillary / Q000379:methods*; D004720:Endonucleases; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D007785:Lactose / Q000031:analogs & derivatives* / Q000737:chemistry; D007834:Lasers; D008353:Mannitol / Q000737:chemistry; D008747:Methylcellulose / Q000031:analogs & derivatives* / Q000737:chemistry; D010078:Oxazines; D017354:Point Mutation; D011108:Polymers / Q000737:chemistry; D015203:Reproducibility of Results; D013499:Surface Properties; D013997:Time Factors
D001888:Boric Acids; D005456:Fluorescent Dyes; D010078:Oxazines; D011108:Polymers; D008353:Mannitol; D008747:Methylcellulose; D004247:DNA; C071191:MK 458; D004720:Endonucleases; C406922:CEL I nuclease; D007785:Lactose; C032688:boric acid