Multiplexed DNA sizing by capillary electrophoresis using entangled polymer solutions and diode array detection.
We developed a method for the analysis of multiplexed double-stranded DNA (dsDNA) samples complexed to various intercalating dyes using entangled polymer solution. A commercial single-column capillary electrophoresis (CE) instrument with diode array detection was used for multiplexed detection of DNA samples by addition of intercalating fluorescent molecules. A Phi X174HinfI and a pGEM DNA ladder (1 mg/mL) were used for the electrophoretic separation of dsDNA fragments ranging in size from 24 to 726 and 36 to 2645 bp, respectively. The results suggested that simultaneous electrophoretic separation of different DNA ladders multiplexed with different dyes could be performed in the same capillary yielding fast DNA sizing separations. CE analysis, which is often overpowered by slab gel in sample throughput, could now overcome this disadvantage by allowing multiplexed sample analysis in a fraction of the time needed for slab gel analysis. The separation efficiency of stained DNA molecules with both dyes were dramatically improved with buffers containing a large cation such as tetrapentylammonium ion (Npe(4) (+)) as the only cation in the buffer.
Caruso
Célia Sulzbacher
CS
Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos-SP, Brazil.
Lanças
Fernando Mauro
FM
Carrilho
Emanuel
E
D010584:Bacteriophage phi X 174 / Q000737:chemistry; D001583:Benzoxazoles; D002021:Buffers; D004396:Coloring Agents; D004247:DNA / Q000737:chemistry* / Q000302:isolation & purification*; D004279:DNA, Viral / Q000737:chemistry / Q000302:isolation & purification; D019075:Electrophoresis, Capillary / Q000379:methods*; D007364:Intercalating Agents; D008970:Molecular Weight; D011108:Polymers; D011806:Quinolinium Compounds; D012996:Solutions; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet
D001583:Benzoxazoles; D002021:Buffers; D004396:Coloring Agents; D004279:DNA, Viral; D007364:Intercalating Agents; D011108:Polymers; D011806:Quinolinium Compounds; D012996:Solutions; C075296:1,1'-((4,4,7,7-tetramethyl)-4,7-diazaundecamethylene)bis-4-(3-methyl-2,3-dihydro(benzo-1,3-oxazole)-2-methylidene)quinolinium; D004247:DNA