本文關(guān)鍵詞： 毛細管電泳 DNA 基質(zhì)場放大 柱頭場放大 電動 壓力 靈敏度　出處：《華南理工大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

【摘要】： 毛細管電泳技術(shù)具有高效,快速,微量,自動,高通量,清潔無污染等特點,自產(chǎn)生以來便獲得廣泛的關(guān)注和應(yīng)用,但在應(yīng)用過程中,其有限的檢測光學(xué)路程及有限長度所造成的微小進樣量使其靈敏度低,特別是配合常用的UV檢測器時,靈敏度問題更是成為制約其應(yīng)用的一個關(guān)鍵因素。因此,針對其靈敏度問題展開研究已成為一個熱點,其中樣品富集技術(shù)以操作簡單,不需昂貴的儀器設(shè)備,靈敏度高成為研究熱點。而場放大技術(shù)更是以其成熟的理論基礎(chǔ),簡單的操作模式,高的靈敏度效果成為眾多富集技術(shù)中應(yīng)用最多的技術(shù)之一,具有極高的實際應(yīng)用價值。 目前,在CE分析DNA領(lǐng)域,靈敏度問題尤其突出,由于DNA中堿基所形成的雙鍵具有較低的紫外吸收,而常規(guī)的UV檢測器靈敏度低。CE本身又存在有限內(nèi)徑所造成的UV吸收光程短和有限長度造成的進樣量低等固有缺陷。這樣就給CE在核酸分析領(lǐng)域的應(yīng)用造成了極大的困難。導(dǎo)致在DNA CE分析領(lǐng)域,目前大多研究者采用熒光檢測模式來解決靈敏度問題。而熒光檢測器本身價格昂貴,樣品需標(biāo)記熒光引物或嵌合熒光染料,過程繁瑣,費用高,大部分的染料具有一定的毒性(如EB),且對于目前廣泛使用的UV分析圖譜,熒光圖譜必然給數(shù)據(jù)的統(tǒng)一化應(yīng)用帶來困難。因此,熒光方式尚不能大規(guī)模普及應(yīng)用。 所以,本實驗使用場放大技術(shù)提高CGE-UV檢測核酸的靈敏度,使其在簡便的情況下實現(xiàn)對微量核酸的分析,為擴大CE-UV在分子生物學(xué)領(lǐng)域的應(yīng)用奠定基礎(chǔ)。 本論文的研究工作如下： 1.綜述了包括場放大、pH介導(dǎo)的堆積、等速電泳富集、酸堆積、移動化學(xué)反應(yīng)邊界等以電泳為基礎(chǔ)的富集和Sweeping、單滴劑微抽提、SPE等以色譜為基礎(chǔ)的富集,還包括一些根據(jù)新的理論、機制和特殊設(shè)計發(fā)展起來的富集模式,如陰／陽離子選擇性耗盡注射-Sweeping、臨態(tài)捕獲-釋放堆積、膠束坍塌、移動反應(yīng)邊界、甘油-鹽介導(dǎo)的堆積、醋酸纖維素膜包裹的多孔性接合部等新的富集模式。眾多的富集技術(shù)對于克服毛細管的靈敏度問題,促進其在分析化學(xué)領(lǐng)域的應(yīng)用都具有極大的實際價值。 2.采用水柱法場擴大堆積提高CGE-UV分析核酸靈敏度。以已知濃度的DNAMarker為標(biāo)準(zhǔn)樣品,TE緩沖液遞度稀釋,壓力進樣前加一段去離子水柱(0.5psi,20s),在對分離度無明顯影響下,將壓力進樣時間延長至0.5psi,990s,與常規(guī)的壓力進樣(0.5psi,10s)相比,靈敏度提高了94.4倍。與時間延長后的壓力進樣(0.5psi,90s)相比,靈敏度提高8.2倍,達到最低檢測總濃度1ng/μL,檢測限降至80ng/mL(S/N=3),比前人報道的7ng/μL檢測限提高87.5倍。 3.鑒于壓力法場放大盡管極大提高了進樣量,但由于進樣時間的增加及存于管中過長的樣品柱對分離場強的影響,造成了分析時間的增加,且水柱法場放大堆積的靈敏度仍然有限。因此,嘗試采用基質(zhì)場放大進一步提高CE對核酸的檢測靈敏度�；|(zhì)場放大將單個DNA條帶的檢測限降至40ng/mL以下。比壓力進樣的檢測限(0.5psi,90s)和場擴大下壓力進樣(0.5psi,20s水,990s進樣)的靈敏度分別高65倍和8倍。在同一濃度,電動進樣具有比壓力進樣更高的靈敏度。然而,單獨依靠基質(zhì)場放大形式的靈敏度仍然較低(電動進樣時間很難超過30s)。存在高場強下樣品溶液與管口的BGE交界面很容易受到擾動,管口樣品區(qū)帶的擾動、樣品溶液的擾動影響到分離效率,如造成峰展寬、電動時間受限制、結(jié)果的重復(fù)性不好等問題。低離子強度樣品溶液在高場強下的EOF與管內(nèi)BGE EOF的不匹配也會對這種方式的靈敏度效果產(chǎn)生影響。如進樣電流不穩(wěn)定,區(qū)帶變形展寬,基線漂移嚴(yán)重等。因此,實驗嘗試將柱頭場放大和基質(zhì)場放大聯(lián)合使用以克服上述不足。 4.聯(lián)合使用基質(zhì)場放大和柱頭場放大進一步提高CGE-UV分析DNA的靈敏度。方法建立后以PCR后的高鹽DNA樣品驗證方法的可靠性。將DNA樣品稀釋達40萬倍,與普通的電動進樣(10KV,10s)和延長的壓力進樣(0.5psi,90s)相比,對峰形和峰寬無明顯影響下,將電動進樣的時間延至(10KV,420s),離子強度降至(1.5%TE),柱頭場放大靈敏度分別提高了約28和56倍,聯(lián)合使用基質(zhì)場放大和柱頭場放大后,靈敏度分別提高3760和7548倍。DNA的檢測限達0.1ng/mL(S/N=3,CGE-FASI-UV),接近XU Z等報道的0.09ng/mL的DNA檢測限(CGE—tITP—LIF)。在未經(jīng)脫鹽純化下,以建立的方法分析PCR后的DNA產(chǎn)物獲得了極高的靈敏度(分別比普通的壓力進樣和電動進樣提高50477倍和33354倍)。驗證了聯(lián)合使用基質(zhì)場放大和柱頭場放大具有更高的靈敏度效果。同時,為評價建立的場放大技術(shù)的穩(wěn)定性及在DNA定量方面的能力。設(shè)定條件(2%TE為樣品離子強度,0.5psi 20s水柱,10KV,210s進樣),其線性范圍良好(0.4-20ng/mL),相關(guān)系數(shù)達0.9992,峰面積日內(nèi)和日間精密度分別為3.65%、2.83%、1.67%和4.95%、3.06%、5.84%,樣品峰面積和遷移時間的RSD分別為3.86%和0.28%。顯示了良好的定性及定量效果。此方法操作簡單,靈敏度高,也適用于高鹽微量的DNA樣品分析,具有極高的實際應(yīng)用價值,必將促進毛細管電泳在核酸微量分析領(lǐng)域的應(yīng)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.

【學(xué)位授予單位】：華南理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R341

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