荧光标记DNA片段高效液相色谱质谱分析及荧光特性研究
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摘要
荧光标记DNA片段包括荧光标记寡核苷酸探针和荧光标记长片段双涟DNA,在许多前沿生物化学检测分析技术中都具有举足轻重的作用,高新技术和广泛领域的应用对荧光标记DNA片段及其标准提出了更高的要求,如何研制高品质、满足计量需求的荧光标记DNA片段标准成为关注的重点。其中分离纯化制备、定性定量分析等关键研制技术研究是需要突破的方向。本研究建立的反相离子对色谱法及液质联用分析方法适用于高纯度荧光标记DNA片段标准的分离纯化制备、准确定性定量分析,为该标准的定值研究提供了方法基础。
本研究通过考察并优化色谱和质谱条件,建立荧光标记DNA片段的反相离子对高效液相色谱法以及液质联用分析方法。离子对试剂醋酸三乙胺浓度为0.01mol/L,pH=7的条件下,不同长度荧光标记寡核苷酸的分离度最佳。该条件适用于荧光标记寡核苷酸的液相质谱联用定性定量分析,避免了钠、钾离子的干扰。该分离条件转移至制备液相可进行荧光标记寡核苷酸的纯化与制备。荧光标记长片段双链DNA的分离纯化条件为更高浓度醋酸三乙胺(0.05-0.10 mol/L)。本研究还对荧光标记寡核苷酸在反相离子对色谱中的保留机理进行分析,与非标记寡核苷酸的保留进行对比,阐述了不同长度荧光标记寡核苷酸与非标记寡核苷酸保留时间相反的原因。本研究建立的方法不仅可用于荧光标记DNA片段的纯化制备,分离定性定量分析,而且可以广泛应用于其他核酸生物技术研究领域。
采用本研究建立的分离纯化方法制取的荧光标记DNA片段满足了荧光特性分析对高纯度的要求。通过初步研究荧光标记DNA片段的荧光特性,分析荧光激发光谱、发射光谱和荧光量子产率等参数,结果表明标记不同长度寡核苷酸会对荧光染料的荧光特性造成影响,在定性定量分析时应避免以长度不一致的荧光标记寡核苷酸作为分析用标准或参比。本研究为荧光标记类生物相关标准物质的研发提供了研究思路及技术,初步的研究结果也为准确的荧光定量分析方法研究提供了技术支持。
Fluorescent dye-labeled DNA fragments, including fluorescent labeled oligonucleotides and labeled long double-strand DNA (dsDNA), play an important role in many cutting-edge biological technologies. The analytical method of fluorescent labeled DNA fragments is established by ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) and HPLC coupled to negative-ion electrospray ionization mass spectrometry (ESI-MS) in this work. The results show that the best resolution of different length fluorescent labeled oligonucleotides is observed under the HPLC condition of 0.01 mol/L triethylamine-acetic acid (TEAA), pH 7.0, while the optimal condition for labeled long dsDNA is 0.05-0.10 mol/L TEAA. Another study shows that the retention behavior of fluorescent labeled oligonucleotides is significantly different from that of unlabeled isomers in terms of the retention order. The retention time of labeled oligonucleotides is reduced with the increase of the length of the oligonucleotides.
The low concentration of TEAA as mobile phase (0.01mol/L) enables this IP-RP-HPLC method to be used in ESI-MS. Thus, it is available for the fluorescent labeled oligonucleotides to be detected easily by LC-MS. In order to obtain the high sensitivity, the parameters of LC-ESI-MS are improved. Moreover, this approach is able to avoid the impact of Na+ and K+ on the detection of molecular weight of oligonucleotides effectively.
The fluorescent properties of fluorescent labeled DNA fragments, which are purified by IP-RP-HPLC method, are investigated preliminarily. Interating sphere technique is used to determine fluorescence quantum yields, emission spectra and excitation spectra. The results indicate that the effect of different length of labeled oligonuleotides on fluorescent properties must not be overlooked. It is very useful for the accurate quantitative analysis of fluorescent labeled DNA fragments.
本研究通过考察并优化色谱和质谱条件,建立荧光标记DNA片段的反相离子对高效液相色谱法以及液质联用分析方法。离子对试剂醋酸三乙胺浓度为0.01mol/L,pH=7的条件下,不同长度荧光标记寡核苷酸的分离度最佳。该条件适用于荧光标记寡核苷酸的液相质谱联用定性定量分析,避免了钠、钾离子的干扰。该分离条件转移至制备液相可进行荧光标记寡核苷酸的纯化与制备。荧光标记长片段双链DNA的分离纯化条件为更高浓度醋酸三乙胺(0.05-0.10 mol/L)。本研究还对荧光标记寡核苷酸在反相离子对色谱中的保留机理进行分析,与非标记寡核苷酸的保留进行对比,阐述了不同长度荧光标记寡核苷酸与非标记寡核苷酸保留时间相反的原因。本研究建立的方法不仅可用于荧光标记DNA片段的纯化制备,分离定性定量分析,而且可以广泛应用于其他核酸生物技术研究领域。
采用本研究建立的分离纯化方法制取的荧光标记DNA片段满足了荧光特性分析对高纯度的要求。通过初步研究荧光标记DNA片段的荧光特性,分析荧光激发光谱、发射光谱和荧光量子产率等参数,结果表明标记不同长度寡核苷酸会对荧光染料的荧光特性造成影响,在定性定量分析时应避免以长度不一致的荧光标记寡核苷酸作为分析用标准或参比。本研究为荧光标记类生物相关标准物质的研发提供了研究思路及技术,初步的研究结果也为准确的荧光定量分析方法研究提供了技术支持。
Fluorescent dye-labeled DNA fragments, including fluorescent labeled oligonucleotides and labeled long double-strand DNA (dsDNA), play an important role in many cutting-edge biological technologies. The analytical method of fluorescent labeled DNA fragments is established by ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) and HPLC coupled to negative-ion electrospray ionization mass spectrometry (ESI-MS) in this work. The results show that the best resolution of different length fluorescent labeled oligonucleotides is observed under the HPLC condition of 0.01 mol/L triethylamine-acetic acid (TEAA), pH 7.0, while the optimal condition for labeled long dsDNA is 0.05-0.10 mol/L TEAA. Another study shows that the retention behavior of fluorescent labeled oligonucleotides is significantly different from that of unlabeled isomers in terms of the retention order. The retention time of labeled oligonucleotides is reduced with the increase of the length of the oligonucleotides.
The low concentration of TEAA as mobile phase (0.01mol/L) enables this IP-RP-HPLC method to be used in ESI-MS. Thus, it is available for the fluorescent labeled oligonucleotides to be detected easily by LC-MS. In order to obtain the high sensitivity, the parameters of LC-ESI-MS are improved. Moreover, this approach is able to avoid the impact of Na+ and K+ on the detection of molecular weight of oligonucleotides effectively.
The fluorescent properties of fluorescent labeled DNA fragments, which are purified by IP-RP-HPLC method, are investigated preliminarily. Interating sphere technique is used to determine fluorescence quantum yields, emission spectra and excitation spectra. The results indicate that the effect of different length of labeled oligonuleotides on fluorescent properties must not be overlooked. It is very useful for the accurate quantitative analysis of fluorescent labeled DNA fragments.
引文
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2 Watson JCrick F. Genetic implications of the structure of DNA[J]. Nature[J],1953,171964-967
3 Jackson D, Symons RBerg P. Biochemical method for inserting new genetic information into DNA of SV40:circular SV40 DNA molecules containing lambda phage genes and the galactose operon of Escherichia coli[J]. Proc. Natl. Acad. Sci. USA,1972,69 2904-2909
4 Maxam AGilbert W. A new method for sequencing DNA[J]. Proceedings of the National Academy of Sciences,1977,74 (2):560-564
5 Sanger F, Nicklen SCoulson A. DNA sequencing with chain-terminating inhibitors[J]. Proceedings of the National Academy of Sciences,1977,74 (12):5463-5467
6 Mullis K, Faloona F, Scharf S, Saiki R, Horn GErlich H. Specific enzymatic amplification of DNA in vitro [J]. Quant. Biol,1986,51 263-273
7 Cello J, Paul AWimmer E. Chemical synthesis of poliovirus cDNA:generation of infectious virus in the absence of natural template[J]. Science,2002,297 (5583):1016-1018
8 Edwards MGibbs R. Multiplex PCR:advantages, development, and applications[J]. Genome Research,1994,3 (4):S65-S75
9 Dovichi N. DNA sequencing by capillary electrophoresis[J]. Electrophoresis,1997,18 (12-13): 2393-2399
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11 Pastinen T, Raitio M, Lindroos K, Tainola P, Peltonen LSyv nen A. A system for specific, high-throughput genotyping by allele-specific primer extension on microarrays[J]. Genome Research,2000,10(7):1031-1042
12 Gewirtz A. Oligonucleotide therapeutics:a step forward[J]. Journal of Clinical Oncology,2000, 18(9):1809-1811
13 Moreira BG, You Y, Behlke MAOwczarzy R. Effects of fluorescent dyes, quenchers, and dangling ends on DNA duplex stability[J]. Biochemical and Biophysical Research Communications,2005, 327473-484
14 Lowry M, Fakayode SO, Geng ML, Baker GA, Wang L, McCarroll ME, Patonay GWarner IM. Molecular Fluorescence, Phosphorescence, and Chemiluminescence Spectrometry [J]. Analytical Chemistry,2008,80 (12):4551-4574
15 许金钩,王尊本.荧光分析法(第三版)[M].科学出版社，2006,3-63
16黄晓峰,张远强,张英起.荧光探针技术[M].人民军医出版社，2004,2
17 王彦广,刘洋.化学标记与探针技术在分子生物学中的应用[M].化学工业出版社,2007,26-32
18 Mackay I, Arden KNitsche A. Real-time PCR in virology[J]. Nucleic Acids Research,2002,30 (6): 1292-1305
19 Tyagi SKramer F. Molecular beacons:probes that fluoresce upon hybridization[J]. Nature biotechnology,1996,14 (3):303-308
20 Wittwer C, Ririe K, Andrew R, David D, Gundry RBalis U. The LightCycler:a microvolume multisample fluorimeter with rapid temperature control[J]. Biotechniques,1997,22 (1):176-181
21 Fauth CSpeicher M. Classifying by colors:FISH-based genome analysis[J]. Cytogenetic and Genome Research,2000,93 (1-2):1-10
22 J SH J. Next-generation DNA sequencing[J]. Nat Biotechnol,2008,26 (10):1135-1145
23 Van Orden A, Cai H, Goodwin PKeller R. Efficient detection of single DNA fragments in flowing sample streams by two-photon fluorescence excitation[J]. Anal. Chem,1999,71 (11):2108-2116
24 Blow N. PCR's next frontier[J]. Nature Methods,2007,4(10):869-874
25鞠熀先,邱宗荫,丁世家.生物分析化学[M].科学出版社，2007,36-52
26 Gilar MBouvier E. Purification of crude DNA oligonucleotides by solid-phase extraction and reversed-phase high-performance liquid chromatography[J]. Journal of Chromatography A,2000, 890(1):167-177
27 Fearon K, Stults J, Bergot B, Christensen LRaible A. Investigation of the'n-1'impurity in phosphorothioate oligodeoxynucleotides synthesized by the solid-phase{beta}-cyanoethyl phosphoramidite method using stepwise sulfurization[J]. Nucleic Acids Research,1995,23 (14): 2754-2761
28 Frank R, Muller DWolff C. Identification and suppression of secondary structures formed from deoxy-oligonucleotides during electrophoresis in denaturing polyacrylamide-gels[J]. Nucleic Acids Research,1981,9(19):4967-4979
29 Fountain KJ, Gilar M, Budman YGebler JC. Purification of dye-labeled oligonucleotides by ion-pair reversed-phase high-performance liquid chromatography[J]. Journal of Chromatography B, 2003,78361-72
30 McKeon JKhaledi M. Evaluation of liposomal delivery of antisense oligonucleotide by capillary electrophoresis with laser-induced fluorescence detection[J]. Journal of Chromatography A,2003, 1004 (1-2):39-46
31 Warren WVella G. Principles and methods for the analysis and purification of synthetic deoxyribonucleotides by high-performance liquid chromatography[J]. Molecular biotechnology, 1995,4(2):179-199
32 CG HA K. Analysis of nucleic acids by capillary ion-pair reversed-phase HPLC coupled to negative-ion electrospray ionization mass spectrometry[J]. Anal. Chem,1999,71 (17):3730-3739
33邹汉法.离子对高效液相色谱法[M].河南科学技术出版社，1994,6-7
34 Huber C, Oefner PBonn G. High-resolution liquid chromatography of oligonucleotides on nonporous alkylated styrene-divinylbenzene copolymers[J]. Analytical biochemistry,1993,212 (2): 351-358
35邹汉法,张玉奎卢佩章.反相离子对色谱法及其应用[J].色谱，1992,10(006)：329-333
36 Ikuta S, Chattopadhyaya RDickerson R. Reverse-phase polystyrene column for purification and analysis of DNA oligomers[J]. Analytical Chemistry,1984,56 (12):2253
37 Crowther J, Jones RHartwick R. High-performance liquid chromatography of the oligonucleotides[J]. Journal of Chromatography,1981,217479-490
38 Gilar M, Fountain K, Budman Y, Neue U, Yardley K, Rainville P, Russell I, Reb JGebler J. Ion-pair reversed-phase high-performance liquid chromatography analysis of oligonucleotides:: Retention prediction[J]. Journal of Chromatography A,2002,958 (1-2):167-182
39 Gelhaus SLaCourse W. Separation of modified 2'-deoxyoligonucleotides using ion-pairing reversed-phase HPLC[J]. Journal of Chromatography B,2005,820 (2):157-163
40骆雪芳,尹小英,付静静胡育筑.柱温和流速对反相离子对色谱法分忻寡核苷酸的影响[J].分析化学，2008,36(5)：627-631
41 骆雪芳,陈蓉,付静静胡育筑.反相离子对色谱法分析3’,5’反向寡核苷酸[J].色谱，2007,25 (6)：814-819
42 Gilar M. Analysis and purification of synthetic oligonucleotides by reversed-phase high-performance liquid chromatography with photodiode array and mass spectrometry detection[J]. Analytical biochemistry,2001,298 196-206
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