Use of fluorescent DNA-intercalating dyes in the analysis of DNA via ion-pair reversed-phase denaturing high-performance liquid chromatography.
SYBR Green 1 is an asymmetrical cyanine DNA-binding dye that provides an opportunity for increasing the sensitivity of nucleic acid detection when used in conjunction with gel electrophoresis. In this paper, we summarize the general properties and specific uses of SYBR green 1 in ion-pair reversed-phase denaturing high-performance liquid chromatography (IP DHPLC). We describe several applications for the WAVE DHPLC platform that illustrate the generic potential of such intercalating dyes in mutation detection and gene expression profiling. We show that SYBR Green 1 obviates the need to use end-labeled oligodeoxynucleotides for the sensitive detection of nucleic acids during chromatography. Moreover the incorporation of SYBR Green 1 into samples and elution buffers does not impair resolution and has no significant effect on the retention times of DNA fragments compared with dye-free DHPLC.
Bahrami
Ahmad R
AR
Department of Molecular Biology, The Transgenomic Research Laboratory, Krebs Institute, Firth Court, Western Bank, Sheffield S10 2TN, UK.
Dickman
Mark J
MJ
Matin
Maryam M
MM
Ashby
John R
JR
Brown
Paul E
PE
Conroy
Matthew J
MJ
Fowler
Gregory J S
GJ
Rose
James P
JP
Sheikh
Qaiser I
QI
Yeung
Anthony T
AT
Hornby
David P
DP
D020137:Base Pair Mismatch; D002232:Carbocyanines / Q000737:chemistry; D002454:Cell Differentiation / Q000235:genetics; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004247:DNA / Q000032:analysis* / Q000378:metabolism; D053938:DNA Fragmentation; D018076:DNA, Complementary / Q000032:analysis; D005456:Fluorescent Dyes / Q000737:chemistry*; D020180:Heteroduplex Analysis / Q000379:methods*; D006801:Humans; D007364:Intercalating Agents / Q000737:chemistry*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D002232:Carbocyanines; D018076:DNA, Complementary; D005456:Fluorescent Dyes; D007364:Intercalating Agents; D009838:Oligodeoxyribonucleotides; D004247:DNA