Multiplex capillary denaturing high-performance liquid chromatography with laser-induced fluorescence detection.
Denaturing high-performance liquid chromatography (DHPLC) is a sensitive, robust, and operationally inexpensive method for the detection of single-base substitutions and small deletions and insertions. To increase sample throughout, we have developed a multiplexing strategy using fluorophores to distinguish different PCR products. The system combines recent advances in the synthesis of monolithic poly(styrene-divinylbenzene) capillary columns with four-color confocal argon ion laser-induced fluorescence detection. Depending on the change in retention caused by the fluorophores, adjustments in the analysis temperature may be required to ensure the maximum mutation detection sensitivity.
Xiao
W
W
Stanford University, Palo Alto, CA, Affymetrix, Santa Clara, CA, USA.
Stern
D
D
Jain
M
M
Huber
C G
CG
Oefner
P J
PJ
D001128:Argon; D020137:Base Pair Mismatch; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D004252:DNA Mutational Analysis / Q000295:instrumentation / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods*; D004867:Equipment Design; D005456:Fluorescent Dyes / Q000032:analysis; D005470:Fluorometry / Q000295:instrumentation*; D020180:Heteroduplex Analysis; D006801:Humans; D007834:Lasers; D008297:Male; D009691:Nucleic Acid Denaturation; D009692:Nucleic Acid Heteroduplexes / Q000032:analysis; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D013696:Temperature; D014998:Y Chromosome / Q000737:chemistry / Q000235:genetics
D005456:Fluorescent Dyes; D009692:Nucleic Acid Heteroduplexes; D001128:Argon