基于场放大富集的毛细管凝胶电泳紫外检测核酸灵敏度研究

发布时间：2018-02-05 21:03

本文关键词： 毛细管电泳 DNA 基质场放大柱头场放大电动压力灵敏度　出处：《华南理工大学》2010年硕士论文　论文类型：学位论文

【摘要】： 毛细管电泳技术具有高效,快速,微量,自动,高通量,清洁无污染等特点,自产生以来便获得广泛的关注和应用,但在应用过程中,其有限的检测光学路程及有限长度所造成的微小进样量使其灵敏度低,特别是配合常用的UV检测器时,灵敏度问题更是成为制约其应用的一个关键因素。因此,针对其灵敏度问题展开研究已成为一个热点,其中样品富集技术以操作简单,不需昂贵的仪器设备,灵敏度高成为研究热点。而场放大技术更是以其成熟的理论基础,简单的操作模式,高的灵敏度效果成为众多富集技术中应用最多的技术之一,具有极高的实际应用价值。目前,在CE分析DNA领域,灵敏度问题尤其突出,由于DNA中碱基所形成的双键具有较低的紫外吸收,而常规的UV检测器灵敏度低。CE本身又存在有限内径所造成的UV吸收光程短和有限长度造成的进样量低等固有缺陷。这样就给CE在核酸分析领域的应用造成了极大的困难。导致在DNA CE分析领域,目前大多研究者采用荧光检测模式来解决灵敏度问题。而荧光检测器本身价格昂贵,样品需标记荧光引物或嵌合荧光染料,过程繁琐,费用高,大部分的染料具有一定的毒性(如EB),且对于目前广泛使用的UV分析图谱,荧光图谱必然给数据的统一化应用带来困难。因此,荧光方式尚不能大规模普及应用。所以,本实验使用场放大技术提高CGE-UV检测核酸的灵敏度,使其在简便的情况下实现对微量核酸的分析,为扩大CE-UV在分子生物学领域的应用奠定基础。本论文的研究工作如下： 1.综述了包括场放大、pH介导的堆积、等速电泳富集、酸堆积、移动化学反应边界等以电泳为基础的富集和Sweeping、单滴剂微抽提、SPE等以色谱为基础的富集,还包括一些根据新的理论、机制和特殊设计发展起来的富集模式,如阴／阳离子选择性耗尽注射-Sweeping、临态捕获-释放堆积、胶束坍塌、移动反应边界、甘油-盐介导的堆积、醋酸纤维素膜包裹的多孔性接合部等新的富集模式。众多的富集技术对于克服毛细管的灵敏度问题,促进其在分析化学领域的应用都具有极大的实际价值。 2.采用水柱法场扩大堆积提高CGE-UV分析核酸灵敏度。以已知浓度的DNAMarker为标准样品,TE缓冲液递度稀释,压力进样前加一段去离子水柱(0.5psi,20s),在对分离度无明显影响下,将压力进样时间延长至0.5psi,990s,与常规的压力进样(0.5psi,10s)相比,灵敏度提高了94.4倍。与时间延长后的压力进样(0.5psi,90s)相比,灵敏度提高8.2倍,达到最低检测总浓度1ng/μL,检测限降至80ng/mL(S/N=3),比前人报道的7ng/μL检测限提高87.5倍。 3.鉴于压力法场放大尽管极大提高了进样量,但由于进样时间的增加及存于管中过长的样品柱对分离场强的影响,造成了分析时间的增加,且水柱法场放大堆积的灵敏度仍然有限。因此,尝试采用基质场放大进一步提高CE对核酸的检测灵敏度。基质场放大将单个DNA条带的检测限降至40ng/mL以下。比压力进样的检测限(0.5psi,90s)和场扩大下压力进样(0.5psi,20s水,990s进样)的灵敏度分别高65倍和8倍。在同一浓度,电动进样具有比压力进样更高的灵敏度。然而,单独依靠基质场放大形式的灵敏度仍然较低(电动进样时间很难超过30s)。存在高场强下样品溶液与管口的BGE交界面很容易受到扰动,管口样品区带的扰动、样品溶液的扰动影响到分离效率,如造成峰展宽、电动时间受限制、结果的重复性不好等问题。低离子强度样品溶液在高场强下的EOF与管内BGE EOF的不匹配也会对这种方式的灵敏度效果产生影响。如进样电流不稳定,区带变形展宽,基线漂移严重等。因此,实验尝试将柱头场放大和基质场放大联合使用以克服上述不足。 4.联合使用基质场放大和柱头场放大进一步提高CGE-UV分析DNA的灵敏度。方法建立后以PCR后的高盐DNA样品验证方法的可靠性。将DNA样品稀释达40万倍,与普通的电动进样(10KV,10s)和延长的压力进样(0.5psi,90s)相比,对峰形和峰宽无明显影响下,将电动进样的时间延至(10KV,420s),离子强度降至(1.5%TE),柱头场放大灵敏度分别提高了约28和56倍,联合使用基质场放大和柱头场放大后,灵敏度分别提高3760和7548倍。DNA的检测限达0.1ng/mL(S/N=3,CGE-FASI-UV),接近XU Z等报道的0.09ng/mL的DNA检测限(CGE—tITP—LIF)。在未经脱盐纯化下,以建立的方法分析PCR后的DNA产物获得了极高的灵敏度(分别比普通的压力进样和电动进样提高50477倍和33354倍)。验证了联合使用基质场放大和柱头场放大具有更高的灵敏度效果。同时,为评价建立的场放大技术的稳定性及在DNA定量方面的能力。设定条件(2%TE为样品离子强度,0.5psi 20s水柱,10KV,210s进样),其线性范围良好(0.4-20ng/mL),相关系数达0.9992,峰面积日内和日间精密度分别为3.65%、2.83%、1.67%和4.95%、3.06%、5.84%,样品峰面积和迁移时间的RSD分别为3.86%和0.28%。显示了良好的定性及定量效果。此方法操作简单,灵敏度高,也适用于高盐微量的DNA样品分析,具有极高的实际应用价值,必将促进毛细管电泳在核酸微量分析领域的应用。
[Abstract]:Capillary electrophoresis technique is efficient, rapid, trace, automatic, high throughput, clean and no pollution, since they received extensive attention and application, but in the application process, the small sample size limited detection optical distance and the finite length caused by the low sensitivity, especially with UV detector commonly, sensitivity problem is becoming a key factor in restricting its application. Therefore, research has become a hot spot for its sensitivity, the sample enrichment technology is simple, does not need expensive equipment, high sensitivity has become a research hotspot. The field amplification technology with its mature the theoretical basis, simple mode of operation, the effect of high sensitivity as many enrichment technology in one of the most widely used technology, has very high practical value.
At present, in the CE analysis of the DNA field, the sensitivity problem is particularly prominent absorption due to the formation of DNA base double bonds with low UV, while the conventional UV detector low sensitivity of.CE itself and the defects caused by UV finite diameter and short length optical path caused by the limited amount of sample. This gave low inherent the application of CE in nucleic acid analysis has caused great difficulties. In DNA CE analysis field, most researchers used fluorescence detection model to solve the sensitivity problem. And the fluorescence detector is expensive, sample fluorescence labeled primers or chimeric fluorescent dyes, complicated process and high cost, most of the dye has certain toxicity (such as EB), and the widely used UV analysis of fluorescence spectra, inevitably bring difficulties to the unification of the application data. Therefore, there are still no large-scale popularization and application of fluorescence method.
Therefore, the field amplification technology is used to improve the sensitivity of nucleic acid detection by CGE-UV, so that it can analyze trace nucleic acids in a simple way, and lay a foundation for expanding the application of CE-UV in molecular biology.
The research work of this paper is as follows:
1. reviews including field amplification, accumulation of pH mediated, isotachophoresis preconcentration, acid accumulation, moving chemical reaction boundary on the basis of electrophoresis and enrichment of Sweeping, single drop micro extraction, enrichment by chromatography based SPE, including some according to the new theory, mechanism and special design enrichment mode development together, such as Yin / cation selectiveexhaustive injection of -Sweeping micelle critical state capture release accumulation, collapse, moving reaction boundary, the accumulation of glycerol salt mediated, enrichment model package cellulose acetate membrane porous junction. Many new enrichment techniques to overcome the sensitivity problem of capillary, which has promoted great practical value in the application field of analytical chemistry.
2. by water accumulation increased CGE-UV nucleic acid analysis method to expand the sensitivity. With known concentrations of DNAMarker as the standard sample, TE buffer gradient dilution, pressure sampling before a deionized water (0.5psi, 20s), in has no obvious effect on the separation degree, the sampling time is extended to 0.5psi, 990s, and conventional pressure injection (0.5psi, 10s) compared the sensitivity increased by 94.4 times. With prolonged pressure after injection (0.5psi, 90s) compared to the sensitivity increased by 8.2 times, the minimum detection concentration in 1ng/ L, the detection limit to 80ng/mL (S/N=3), 7ng/ L than previous reports of the detection limit increased by 87.5 times.
3. in view of the pressure field enlarge despite greatly increased the amount of sample, but due to the influence of sample increase and stored in pipe is too long sampling time on the separation of field intensity, resulting in increased analysis time, sensitivity and water field amplified stacking is limited. Therefore, try to use the matrix field to further improve the detection sensitivity of CE amplification the matrix field of nucleic acids. A single DNA band to enlarge the detection limit below 40ng/mL. The detection limit of injection pressure ratio (0.5psi, 90s) and expand the field under pressure injection (0.5psi, 20s water, 990s injection) the sensitivity were 65 times and 8 times. At the same concentration, electric the sample has a higher sensitivity than the pressure sampling. However, relying solely on the sensitivity matrix field amplified form is still low (electric injection time is less than 30s). The presence of BGE sample solution and nozzle under high electric field interface is very susceptible to disturbance, nozzle Disturbed sample zone, sample solution disturbance affect the separation efficiency, such as peak broadening, electric time limit problem of the repeatability of the results is not good. Also the sensitivity effect on the way the influence does not match the low ionic strength of sample solution and EOF tube under high voltage in BGE EOF such as injection current instability, deformation zone broadening, baseline drift is serious. Therefore, the attempt to stigma field amplification and matrix field amplification in combination to overcome these shortcomings.
4. the combination of matrix field amplification and stigma field amplification further improve the sensitivity analysis of CGE-UV DNA method. After the establishment of DNA samples with high salt PCR verification method of reliability. The DNA sample was diluted 400 thousand times, and general electric injection (10KV, 10s) and prolonged pressure injection (0.5psi, 90s) compared to the peak shape and peak width had no significant effect, extended the electrokinetic injection to (10KV, 420s), ionic strength (1.5%TE), stigma to field amplified sensitivity were improved by about 28 and 56 times, the combination of matrix field amplification and stigma field amplification, 3760 and 7548 respectively to improve detection sensitivity.DNA times the limit of 0.1ng/mL (S/N=3, CGE-FASI-UV), DNA XU Z reported the close to detection limit of 0.09ng/mL (CGE tITP LIF). In the desalination and purification, to establish the analysis method of DNA products after PCR was a high sensitivity (respectively than normal pressure sampling and Electric injection increased 50477 times and 33354 times). To verify the combination of matrix field amplification effect and higher sensitivity with stigma field amplification. At the same time, the stability of field amplification technology is established and evaluation ability in quantitative DNA. The conditions set (2%TE for the samples of ionic strength, 0.5psi 10KV, 210s 20s column sample), the good linear range (0.4-20ng/mL), the correlation coefficient was 0.9992, the peak area of the intra day and inter day precision were 3.65%, 2.83%, 1.67% and 4.95%, 3.06%, 5.84%, peak area and migration time were 3.86% and 0.28%. RSD showed good qualitative and quantitative effect of this method. Simple operation, high sensitivity and is suitable for the analysis of trace DNA in high salt samples, has high practical value, will promote the application of capillary electrophoresis in analysis of trace nucleic acid field.

【学位授予单位】：华南理工大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R341

【共引文献】