Base sequence- and T m-dependent DNA oligomer separation by open tubular capillary columns carrying complementary DNA oligomers as probes.
DNA chips prepared on a flat glass surface have unavoidable drawbacks when used for quantitative analysis. In an attempt to overcome this problem, we constructed an HPLC-type system suitable for quantitative analysis that enables base sequence- and T (m)-dependent DNA oligomer separation in a flow system. A small open tubular capillary column (300-mm x 100-microm I.D.) was used. The DNA oligomers used as probes had an amino group at the 5'-end and were immobilized on the inner silica surface of the capillary column which had been sequentially treated with 3-aminopropyltriethoxysilane, butyltrimethoxysilane, and disuccinimidylglutarate. Using the combination of probe-immobilized column placed in a column oven equipped with temperature gradient function, a nano-flow-controllable pump, a small sample-loading injector, and a capillary-fitted UV detector, we succeeded in separating complementary and non-complementary DNA oligomers in specific and quantitative modes. We also designed a temperature gradient strategy for efficient separation of target DNA oligomers in DNA mixture samples. Using a column carrying two different probes with similar T (m) values, their complementary target DNA oligomers were also separated and detected. The developed DNA open tubular capillary column system investigated in the present study could be further improved as an alternative tool to DNA chips to be used for the quantitative analysis of DNA or mRNA samples.
Devarayapalli
Kamakshaiah Charyulu
KC
Institute of Advanced Energy, Kyoto University, Gokasho, Uji, Kyoto, Japan.
Pack
Seung Pil
SP
Kamisetty
Nagendra Kumar
NK
Nonogawa
Mitsuru
M
Watanabe
Seiya
S
Kodaki
Tsutomu
T
Makino
Keisuke
K
D001483:Base Sequence; D015374:Biosensing Techniques; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification*; D015342:DNA Probes / Q000032:analysis* / Q000737:chemistry; D011437:Propylamines; D012333:RNA, Messenger / Q000032:analysis / Q000737:chemistry; D012821:Silanes / Q000737:chemistry; D013388:Succinimides / Q000737:chemistry; D013499:Surface Properties; D013696:Temperature; D013997:Time Factors
D015342:DNA Probes; D011437:Propylamines; D012333:RNA, Messenger; D012821:Silanes; D013388:Succinimides; C096464:disuccinimidyl glutarate; D004247:DNA; C477625:amino-propyl-triethoxysilane; C071717:trimethoxysilane