201239090 - 六、發明說明： 【發明所屬之技術領域】 本發明係關於一種可提升核酸擴增反應效果之方法，更詳而言之，尤 指一種應用於聚合酶連鎖反應(Polymerase Chain Reaction，PCR)之反應 裝置’透過控制試劑内緩衝液之熱對流速度來增加單次熱對流循環時間， 俾以有效提升核酸擴增反應之效果。 【先前技術】 按’進行遺傳學、分子生物學等基因研究或動植物疫病之檢測時，需 先以擴增核酸的手段(例如聚合酶連鎖反應’ Polymerase Chain Reaction， PCR)，將少量的核酸樣本在短時間内複製擴增到可以被偵測到的量。上述 核酸擴增產物可進一步經由雜合作用（Hybridization)而使目標核酸片段與 帶有螢光、放射物質或呈色酵素的核酸探針(pr〇de)連結，產生螢光、放射 影像或呈色反應。 上述之聚合酶連鎖反應中，其過程需要進行三個主驟，包括變性反應、 黏合反應、延伸反應，而其分別需要不同之反應溫度。而現今商業化之pcR 擴增技術所需樣本包含欲擴增之模板DNA、與模板DNA各股上特定序列互 補之寡核苷酸引子對 '熱安定DNA聚合酶，以及去氡核苷三磷酸(dNTP)，201239090 - VI. Description of the Invention: [Technical Field] The present invention relates to a method for enhancing the effect of a nucleic acid amplification reaction, and more particularly, to a polymerase chain reaction (PCR) The reaction device 'increased the single heat convection cycle time by controlling the heat convection velocity of the buffer in the reagent to effectively enhance the effect of the nucleic acid amplification reaction. [Prior Art] When performing genetic research such as genetics, molecular biology, or animal and plant disease, a small amount of nucleic acid sample is first obtained by means of amplifying nucleic acid (for example, polymerase chain reaction, Polymerase Chain Reaction, PCR). The amplification is amplified in a short time to an amount that can be detected. The nucleic acid amplification product may further bind the target nucleic acid fragment to a nucleic acid probe (pr〇de) with fluorescent, radioactive or chromogenic enzymes via hybridization to generate fluorescence, radiographic or Color reaction. In the above-mentioned polymerase chain reaction, the process requires three main steps, including a denaturation reaction, a binding reaction, and an extension reaction, which respectively require different reaction temperatures. The samples required for the current commercial PCR amplification technology contain the template DNA to be amplified, the oligonucleotide primer pair complementary to the specific sequence on each strand of the template DNA, 'thermal stability DNA polymerase, and depurinated nucleoside triphosphate ( dNTP),
Ik後藉由反覆加熱與冷卻樣本，使樣本於不同溫度間進行循環，藉以擴增 模板DNA核酸序列之特定部份。 於上述變性反應中，將樣本加熱至高溫，俾使雙股之模板DNA分離成 單股DNA，而黏合反應中，將樣本冷卻至較低溫度，俾使引子與變性反應 所形成之單股DNA結合，以形成dNA與引子之複合物；而延伸（聚合)反應 201239090 中，將樣本維持於適當溫度，藉由DNA聚合酶之作用，使DNA與引子之複 合物中之引子得以延伸，進而產生與模板DNA各股互補之新單股口问厶；藉 由將每次組成之循環可複製兩份引子結合位置間之DNA序列。 習用之PCR反應裝置’樣本試管之溫度係以熱傳導方式進行控制，例 如將PCR樣本試管與具高導熱性之固體金屬塊接觸，以透過加熱、冷卻來 改變其概度，以達到試管樣本之需求溫度，然而，由於該樣本試管於加熱 時，大都係將樣本試管以垂直角度進行承放，並將具有導熱性之金屬塊置 放於試管末端處，藉由試f内產生溫度差而使勒部之緩衝液產生熱對流 循％現象’細’賊擴增反應之效果會受職衝祕職速度所影響， 當熱對流速度職時’會導致單次熱對频環_小於單次核_增反應 時間’而使雜_增反應未完全，反之，#熱騎速度珊時，會導致 增加核酸擴增反應所需耗用之時間；而_緩衝液之流動速率會受到多重 因素所影響，例如緩衝液之組成，以及試管頂端與底端之溫度差，因此為 了改變緩誠之流動辭’目前㈣上之方式係透過於緩衝液崎加物質 以改變其黏度，亦或控制試管頂端與底端之溫度差，雖透過此方式確可使 得熱對流速率產纽變，但由於其相#不^控制，使得麟流速率往往無 法有效掌握。 【發明内容】 綜合上開先前技術的缺點，大致上包括上述"pcR反應裝置，由於 核^擴增反應之效果會受到緩衝液熱雌速度所影響，而為了改變緩衝液 之机動速率’目刖習用上之方式係透過於緩衝液内添加物質以改變其黏 度’亦或控制試管頂端與底端之溫度差，但由於其相當不紐制，使得熱 201239090 本發明提出一種生化 對流速率往往無法有效掌握；而鑑於解決上述缺點， 反應之熱對流速度控制裝置及其方法。 ------ 祝/乃问，進而 改變其流動速度糾間，俾使魏衝液之料騎流循環時_於/大於該 本發明係為一種生化反應裝置之熱對流速度控制裝置及其方法勺 括：至少一試管，係活動固定於一座體上，該試管内可供填充至少一生化 反應之緩衝液卜加熱源，係'設於該試f底部或側面處，以供進行加熱； 以及-流速赃裝置，係供控制緩衝液於試管内之熱對流流動方向，進而 生化反應之單次核酸擴增反應時間。 上述生化反應之方法，係包含以下步驟： a) 將至少一生化反應之緩衝液填充於一試管内； b) 將試管活動固定於一座體上； c) 利用一加熱源對試管底部或側面進行加熱； d)藉外界S氣對該試管之頂端進行降溫，並使該緩賊產生熱對流循 句依照緩衝液所需之單次核酸擴增反應時間，調整試管之傾斜角度， 動方向’進而改變其流動速度與時間。 係在於藉由流速調整裝置以控制試管内緩衝液進 以控制緩衝液之流動方向 本發明之主要目的，係在 灯…、對咖之视動騎，變其流_颇時間，俾使該緩舰之單次熱 對桃循¥時間等於/大於該生化反應之單次核麵增反應時間，俾以提升核After Ik, the sample is circulated at different temperatures by repeatedly heating and cooling the sample to amplify a specific portion of the template DNA nucleic acid sequence. In the above denaturation reaction, the sample is heated to a high temperature, and the double-stranded template DNA is separated into single-stranded DNA, and in the bonding reaction, the sample is cooled to a lower temperature, and the single-stranded DNA formed by the primer and the denaturation reaction is formed. Binding to form a complex of dNA and primer; and in extension (polymerization) reaction 201239090, the sample is maintained at an appropriate temperature, and the primer in the complex of DNA and primer is extended by the action of DNA polymerase, thereby generating A new single-stranded sputum complementary to each strand of the template DNA; the DNA sequence between the two primer-binding positions can be replicated by cycling each composition. Conventional PCR reaction device 'The temperature of the sample tube is controlled by heat conduction. For example, the PCR sample tube is contacted with a solid metal block with high thermal conductivity to change its profile by heating and cooling to meet the needs of the test tube sample. Temperature, however, since the sample tube is heated, most of the sample tubes are placed at a vertical angle, and a thermally conductive metal block is placed at the end of the tube, and the temperature difference is caused by the test f. Buffers generate heat convection according to the phenomenon of 'fine' thief amplification reaction will be affected by the speed of the job, when the heat convection speed job 'will lead to a single heat to frequency ring _ less than a single core _ Increasing the reaction time' makes the hetero-increase reaction incomplete. Conversely, when the thermal riding speed is slow, it will lead to an increase in the time required for the nucleic acid amplification reaction; and the flow rate of the buffer is affected by multiple factors. For example, the composition of the buffer, and the temperature difference between the top and bottom of the tube, so in order to change the flow of the word of truth, the current (four) method is to change the buffer through the substance Degree, or that control the temperature difference between the top and bottom of the tube, although obtained via this method can determine the thermal convection speed becomes Zealand production, but because of its relative # ^ not control the flow rate such that the Lin often can not effectively control. SUMMARY OF THE INVENTION The above disadvantages of the prior art are generally included in the above-mentioned "pcR reaction device, since the effect of the nuclear amplification reaction is affected by the hot female speed of the buffer, and in order to change the mobility rate of the buffer The method used in the past is to change the viscosity by adding substances in the buffer, or to control the temperature difference between the top end and the bottom end of the test tube, but because of its relatively unrefined structure, the heat 201239090 proposes that a biochemical convection rate is often impossible. Effectively grasping; and in view of solving the above disadvantages, the reaction heat convection speed control device and method thereof. ------ Zhu / Nai asked, and then changed its flow speed correction, so that the flow of Wei Chong liquid ride cycle _ / / greater than the present invention is a biochemical reaction device thermal convection speed control device and The method includes: at least one test tube is fixed on a body, and the test tube is filled with a buffer source for at least one biochemical reaction, and is disposed at the bottom or side of the test f for heating; And a flow rate 赃 device is used to control the flow direction of the heat convection in the test tube, and then the single nucleic acid amplification reaction time of the biochemical reaction. The above biochemical reaction method comprises the steps of: a) filling at least one biochemical reaction buffer in a test tube; b) fixing the test tube to the body; c) using a heating source to test the bottom or side of the test tube Heating; d) cooling the top end of the test tube by external S gas, and causing the thief to generate a heat convection according to the single nucleic acid amplification reaction time required by the buffer, adjusting the tilt angle of the test tube, and moving in the direction Change its flow rate and time. The main purpose of the present invention is to control the flow direction of the buffer by the flow rate adjusting device to control the flow of the buffer in the test tube, and to light the flow of the light, and to change the flow. The single heat of the ship on the peach cycle time is equal to / greater than the single nuclear surface increase reaction time of the biochemical reaction,
酸擴增反應之效果。 f實施方式:JThe effect of the acid amplification reaction. f implementation: J
為便於朗树日胳上猶明内容 201239090 體實施例表達。實關巾純;ρ同物件係按適於制之_、尺寸 '變形 S或位移量而描繪，而非按實際元件的比例予以繪製，合先敘明。且以下 的說明中，類似的元件是以相同的編號來表示。 本發明係為一種生化反應之熱對流速度控制裝置及其方法，而圖式中 各組件及設置方式僅為便於說明本發明而已，本發明主要技術内容可實現 於各種型態之反應裝置’並不限於圖例所示，合先敘明；如第五圖所示， 為使本發明可達到生化反應所需之熱對流速度控制之目的，與本發明主要 技術内容相關之組件包括一試管彳0、一加熱源2〇，以及_流速調整裝置3〇。 該試管10供進行核酸萃取，其内部填充有至少一生化反應之緩衝液H 以及至少一螢光反應物質’該緩衝液糾可為一聚合酶連鎖反應之緩衝液， 該緩衝液及螢光反應物質可視各式定量分析方法而有所不同，例如可為 DNA染劑(DNA Intercalate ’其又可為ErBr, SyBr Green…·等）、濁度反應 (Turbidity ’其又可為magnesjum PyiOph〇sphate)、榮光染色法(F丨⑽啡從巾 dye ’其又可為FAM,Cy3，Cy5)、可見光偵測或呈色反應劑等。 5亥加熱源20係設於該試管1〇底部或側面處，該加熱源2〇係可為一導熱 片，並以一熱源提供導熱之效果，亦或其它一般用於生化反應所使用之加 熱源’僅要能達到試管1〇加熱之相同目的，皆能作為加熱源2〇之實現方式； 該加熱源20可透過一驅動裝置21控制其接觸於試管1〇 ,以使試管1〇内之緩 衝液11(如第七圖所示)呈現核酸恆溫擴增反應之需求溫度，其中，該加熱源 20加熱使試管1〇底部升溫至緩衝液中dna裂解反應所需之溫度，而以目前 核酸擴增反應係以攝氏90〜99度為最佳，該試管1〇頂端之溫度則需降溫至 低於引子黏合反應所需之溫度；透過試管50内兩端之緩衝液糾溫度形成溫 6 201239090 度差，以產生熱對流現象。 該流速調整裝置30係用 。藉由控繼衝液11机動方向，以改變其流動速度與時間，俾使該緩衝 液11之單次熱對顏環_等於/大_生化絲之單次碰擴增反應時 間’俾以提升核酸擴增反應之效果。 明暸上述反應裝置之主要組成及原職，町係針對本發明之動作及 原理作一詳細說明： 如第四圖至第六圖所示，本發明之操作方法係包含以下步驟： a) 將至少-生化反應之緩衝液Ή填充於一試管1〇内； b) 將試管1〇活動固定於—座體4〇上； c) 利用一加熱源20對試管1〇底部或側面進行加熱； d) 藉外界空氣對該試管1Q之頂端進行降溫，並使該緩衝液ή產生熱 對流循環； e) 依照緩衝液11所需之單次核酸擴增反應時間，調整試管1〇之傾斜 角度’以控制緩衝液11之流動方向，進而改變其流動速度與時間。 如第五圖至第七圖所示，係為本發明反應裝置以—種實施態樣之動作 示意圖，該座體40係設於一機台50上，其中，該座體4〇係可供試管1〇活動 承放，而該機台50係可為一平板，該流速調整裝置3〇係連接於機台5〇，用 以控制該機台50及座體40之傾斜角度；該流速調整裝置30係可如圖所示， 透過連桿方式帶動機台5G調整角度，亦或可以其它任何可用以調整機台5〇 角度之結構實現之，例如以齒輪馬達帶動方式。 藉由驅動裝置21控制加熱源20接觸於試管1〇，以進行加熱，當蜮管扣 201239090 底部升溫時，緩衝液11中之DNA即會產生裂解，而緩衝液11流動至試管10 頂端以產生降溫時，緩衝液11中之DNA即會開始複製，藉此，緩衝液11即 可於升溫、降溫之循環中進行PCR增生反應。 藉由上述結構之設置，使得其可依照緩衝液扣所需之熱對流速度，以 藉由座體40帶動試管1〇進行傾斜角度之調整，來改變試管1〇垂直方向及水 平方向之比例，進而使得緩衝液11之流向產生改變；如第一圖至第三圖所 不，係分別為試管10呈垂直、水平，及傾斜方向承放之使用狀態示意圖； 當試管10承放之方向愈趨近於垂直方向時，緩衝液仞之流動速度會愈快， 反之，當試管10承放之方向愈趨近於水平方向時，緩衝液杓之流動速度會 愈慢，而本發明經過實驗操作，試管10承放角度以與鉛直線方向呈大於0。 並小於45之交角為宜，其中，以目前核酸擴增反應的緩衝液彳彳所需熱對流 之適當速度’試管10承放角度尤以呈大於〇。並小於15。之傾斜角度為最佳。 藉由其傾斜角度，使得緩衝液扣於進行熱對流時，因往上升流動之緩 衝液11會增加與試管1〇管賴之細面積，因此她於錄1()呈垂直方向 承放，確可親緩歸11之熱職鄕速度與時間，藉此可令緩衝液”之 單次熱對賴環時間等於/大闕纽反應之單:欠核酸綱聽_，因此 可避免緩驗11 SJ單次熱對賴環速度職，導致單次鱗祕環時間小 於緩衝液11中DNA進行單次核酸擴增反應時間，而影響核酸擴增反應之結 果，由此可見，本發明確可有效提升核酸擴增反應之效果。 本發明之其它實施例巾’為使試管侧定於_傾斜角度，該流速調整 裝置30亦可為設於座體4〇上供承放試管1〇之一承載座(圖未示），且該承載 座可於座體40上進行角度難’亦或可直接於座體4Q上設—斜楔面，以供 201239090 試管10傾斜承放；除上述之方式外，亦可_手持—供鱗試管1岭試管 夾，並以手動操作方式令試管職生傾斜，亦可達到上述之相同目的。 雖本發明是以數個最佳實施例作，但精於此技藝者能在不脫離本 發明精神與範_下作各種不同形式的改變。以上所舉實施例僅用以說明本 發明而已’非用以限制本發明之顧。舉凡不違本發明精神所從事的種種 修改或變化，俱屬本發明申請專利範圍。 【圖式簡單說明】 第-圖係為生化反應中樣本試管呈垂直狀態之熱對流循環示意圖。 第-圖係為生化反應中樣本試管呈水平狀態之熱對流循環示意圖。 第三圖係為生化反應中樣本試管呈傾斜狀態之熱對流循環示意圖。 第四圖係為本發明之操作流程示意圖。 第五圖係為本發明試管角度調整前之使用狀態示意圖。 第六圖係為本發明試管角度調整後之使用狀態示意圖。 第七圖係為本發明試管呈傾斜角度之動作示意圖。 【主要元件符號說明】 絲10 座體40 緩衝液11 機台5〇 加熱源20 驅動裝置21 流速調整裝置30In order to facilitate the sacred content of the tree, 201239090 body expression. The actual closing towel is pure; the ρ-like object is drawn according to the suitable size, the deformation 'S or the displacement amount, and is not drawn according to the proportion of the actual components. In the following description, like elements are denoted by the same reference numerals. The present invention is a thermal convection speed control device for biochemical reactions and a method thereof, and the components and arrangement modes in the drawings are only for convenience of description of the present invention, and the main technical contents of the present invention can be realized in various types of reaction devices' Not limited to the legends, as described in the fifth figure; as shown in the fifth figure, in order to achieve the purpose of the heat convection speed control required for the biochemical reaction, the components related to the main technical contents of the present invention include a test tube 彳0. , a heating source 2 〇, and a _ flow rate adjusting device 3 〇. The test tube 10 is used for nucleic acid extraction, and is filled with at least one biochemical reaction buffer H and at least one fluorescent reaction substance. The buffer solution can be a polymerase chain reaction buffer, the buffer and the fluorescent reaction. The substance may vary depending on various quantitative analysis methods, such as DNA dye (DNA Intercalate 'which may be ErBr, SyBr Green, etc.), turbidity reaction (Turbidity 'which may be magnesjum PyiOph〇sphate) Glory staining method (F丨(10) brown from towel dye 'which can be FAM, Cy3, Cy5), visible light detection or coloring reagent. The 5H heating source 20 is disposed at the bottom or side of the test tube 1 , and the heating source 2 can be a heat conductive sheet, and provides heat conduction effect by a heat source, or other heating generally used for biochemical reactions. The source 'can only achieve the same purpose as the heating of the test tube, and can be used as the heating source 2〇; the heating source 20 can be controlled by a driving device 21 to contact the test tube 1 to make the test tube 1 The buffer 11 (as shown in the seventh figure) exhibits a required temperature for the constant temperature amplification reaction of the nucleic acid, wherein the heating source 20 is heated to raise the temperature of the bottom of the tube 1 to the temperature required for the DNA cleavage reaction in the buffer, and the current nucleic acid The amplification reaction is preferably 90 to 99 degrees Celsius, and the temperature of the top end of the test tube needs to be lowered to a temperature lower than the temperature required for the adhesion reaction of the primer; the temperature of the buffer at both ends of the test tube 50 is corrected to form a temperature of 6 201239090 The difference is to produce a heat convection phenomenon. This flow rate adjusting device 30 is used. By controlling the direction of movement of the liquid 11 to change its flow speed and time, the single heat of the buffer 11 is equal to / large _ biochemical filament single-shot amplification reaction time '俾 to enhance nucleic acid The effect of the amplification reaction. The main components and original functions of the above reaction apparatus are clarified, and the operation and principle of the present invention are described in detail. As shown in the fourth to sixth figures, the operation method of the present invention comprises the following steps: a) At least - The biochemical reaction buffer is filled in a test tube; b) the test tube 1〇 is fixed on the body 4〇; c) the bottom or side of the test tube 1 is heated by a heating source 20; d) The outside air cools the top end of the test tube 1Q, and the buffer ή generates a thermal convection cycle; e) adjusts the tilt angle of the test tube 1 according to the single nucleic acid amplification reaction time required for the buffer 11 to control the buffer The flow direction of the liquid 11, which in turn changes its flow rate and time. As shown in FIG. 5 to FIG. 7 , it is a schematic diagram of the action of the reaction device of the present invention. The seat body 40 is disposed on a machine 50 , wherein the seat body 4 is available. The test tube 1 is erected, and the machine 50 is a flat plate. The flow rate adjusting device 3 is connected to the machine 5 〇 to control the tilt angle of the machine 50 and the seat 40; The device 30 can be adjusted with the motive table 5G through the connecting rod as shown in the figure, or can be realized by any other structure that can adjust the angle of the machine 5, for example, by a gear motor. The heating source 20 is controlled to be in contact with the test tube 1 by the driving device 21 for heating. When the bottom of the tube buckle 201239090 is heated, the DNA in the buffer 11 is cracked, and the buffer 11 flows to the top of the test tube 10 to generate When the temperature is lowered, the DNA in the buffer 11 starts to be replicated, whereby the buffer 11 can perform a PCR proliferation reaction in a cycle of temperature rise and temperature decrease. By the arrangement of the above structure, according to the heat convection speed required by the buffer buckle, the inclination angle of the test tube 1 is adjusted by the seat 40 to change the ratio of the vertical direction and the horizontal direction of the test tube 1 . Further, the flow direction of the buffer 11 is changed; as shown in the first to third figures, the test tube 10 is vertically, horizontally, and tilted, and the direction of use is shown; When the vertical direction is near, the flow rate of the buffer liquid will be faster. On the contrary, when the direction of the test tube 10 is closer to the horizontal direction, the flow speed of the buffer liquid will be slower, and the present invention is experimentally operated. The test tube 10 is placed at an angle greater than zero in the direction of the lead. Preferably, the angle of intersection is less than 45, wherein the buffer tube of the current nucleic acid amplification reaction, the appropriate speed of heat convection required for the test tube 10, is particularly greater than 〇. And less than 15. The tilt angle is optimal. By the angle of inclination, when the buffer is buckled for thermal convection, the buffer 11 which rises in the upward direction will increase the area which is close to the tube 1 of the test tube. Therefore, she is placed vertically in the recording 1 (). You can slow down the speed and time of the hot job, so that the single heat of the buffer can be equal to the single reaction of the big ring: the nucleic acid class is _, so you can avoid the slow test 11 SJ A single heat is applied to the temperature of the Lai ring, resulting in a single scaly ring time less than the time in which the DNA in the buffer 11 is subjected to a single nucleic acid amplification reaction, thereby affecting the result of the nucleic acid amplification reaction, and thus it can be seen that the present invention can effectively improve The effect of the nucleic acid amplification reaction. According to another embodiment of the present invention, in order to set the tube side to the slant angle, the flow rate adjusting device 30 may also be provided on the base 4 to support one of the test tubes 1 . (not shown), and the carrier can be angled on the body 40 or can be directly placed on the body 4Q - a wedge surface for the 201239090 test tube 10 to be tilted; in addition to the above, Can also be _ hand-held - scale test tube 1 ridge test tube clamp, and manually operated The same is the same as the above-described purpose of the present invention. While the invention has been described in terms of several preferred embodiments, various modifications can be made without departing from the spirit and scope of the invention. The above-mentioned embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Any modifications or variations that do not depart from the spirit of the present invention belong to the scope of the present invention. The first-graph is a schematic diagram of the thermal convection cycle in which the sample tube is in a vertical state in the biochemical reaction. The first-graph is a schematic diagram of the thermal convection cycle in which the sample tube is in a horizontal state in the biochemical reaction. The third picture shows the sample tube in the biochemical reaction. Schematic diagram of the thermal convection cycle of the state. The fourth diagram is a schematic diagram of the operation flow of the present invention. The fifth diagram is a schematic diagram of the use state before the adjustment of the test tube angle of the present invention. The seventh figure is a schematic diagram of the operation of the test tube at an oblique angle. [Main component symbol description] Silk 10 Seat 40 Buffer 11 Machine 5加热 Heating source 20 Driving device 21 Flow rate adjusting device 30