1344490 九、發明說明： 【發明所屬之技術領域】 本發明係關於使用聚合酶鏈反應放大核酸之方法。 【先前技術】 美國專利5,512,462是關於一種長片段去氧核醣核酸 (DNA)進行放大反應的方法之專利，於反應中添加兩種 聚合酶，其一為3,至5,端外切酶，另一為去氧核醣核酸 聚合酶’且亦添加甘油與二曱基亞砜以降低去氧核醣核酸 之解鏈溫度（melting temperature)，進一步增加聚合酶鍵反 應之專一性。 歐洲專利公開申請案U19719 A1係關於去氧核醣核酸 的合成與放大之方法。該案中揭示利用兩種經修飾的埶穩 定性之酵素進行聚合酶鏈反應，其—具有3，端核酸外切酶 之活性而無去氧核醣核酸聚合酶之活一 力 昇有去氧核 醣：酸聚合酶之活性進行聚合酶鏈反應。於該案發明所用 之聚合酶鏈反應緩衝溶液中，添加甜菜鹼加⑻加)、二曱 基亞砜、甘油或海满糖（trehal〇se)等試劑，用以降：去 氧核醣核酸之解鏈溫度，進一步增加聚合酶鏈反糞— 秀囫寻利6，150,094 (inert c f t ^ ……卞❼肛井沽化表面 ace) ”生物分子間的非共價之影響的方 包括偶級離子的滲透物（zwitted()nie。 甜菜鹼（betaine)等。 ）的添加’如 1344490 美國專利6,399,304,主要係利用熱循環合成核酸的方 …其中使用至少兩種對雙去氧核苦酸具有不同活性之酵 2 ’在熱循環升高溫度反應中，至少有—熱穩定酵素比另 一熱穩定酵素較晚地受到活化。 ^美國專利公開申請案030049614與040072213中，揭 :―種放大、合成與突變核酸之方法，係利用熱穩定且不 具有权正功能（non_pr〇〇freading)之聚合酶及另一具有校正 功能^聚合SI，並使用可抑制不欲之核酸或其衍生物=入 至去氧核醣核酸聚合物之因子，如聚合酶加強因子 (polymerase enhancing fact〇rs )。此外在該等前案揭示之 方法中，亦可於放大特長片段之核酸時，添加二甲基亞砜。 歐洲專利2〇〇4〇39947係利用突變之去氧核畴核酸聚 合酶增加其反轉錄之活性。 上述文獻資料中教示的方法，具有共同的技術上不利 點。這些先前技術往往在進行聚合酶鏈反應放大時，需要 繁覆多次的反應條件測試，因此在得到既具高專一性且言 產量的聚合酶鏈反應放大產物時，已然耗費了許多時間與 材料。特別是使用基因體核酸作為模版時，經常會遇到無 法成功以聚合酶鏈反應放大產物的情況，而1 一 下 1¾重要 的原因在於巨大基因體核酸分子的二級構造會干擾弓丨子 (primer)與模版核酸序列片段的黏合（annealing)。 目前市售的聚合酶鏈反應條件最適化測試試劑，仍# 測試各種的離子濃度與種類及引子黏合溫度等， …、法提 供快速且有效率之方法，能夠在最少一次實驗即能達到測 6 1344490 試出最佳的聚合酶鏈反應放大條件並得到產物。 【發明内容】 本發明方法之技術特點’主要在於在一般聚合酶鏈反 應緩衝系統中，加入一種欲放大之模版核酸序列片段，再 個別加入三種不同之添加劑：二曱基亞砜、海藻糖（trehalose) 與甜菜鹼（betaine)，並配合預先選定的溫度區間設定，即 了在單一聚合轉鍵反應貫驗中決定同時能達到高專一性與 高產量目的之最佳化條件。 在一方面，本發明提供一種決定聚合酶鏈反應最佳化 條件的方法，其包括下列步驟： (a) 提供三個或更多的聚合酶鏈反應單元群組； (b) 在相異群組的聚合酶鏈反應單元内分別單獨加入二 曱基亞砜、海藻糖及甜菜鹼； (c) 將步驟（b)所得到之每個群組中的個別聚合酶鏈反應 單元，分別置於預先選定溫度區間内之複數溫度下進行聚 合酶鏈反應； (d) 判讀聚合酶鏈反應結果；及 (e) 決定聚合酶鏈反應最佳化條件。 在本發明方法之較佳事實中，該核酸序列片段係天然 核酸序列片段或人工合成核酸序列片段。特別較佳地，該 天然核酸序列片段係來自生物檢體、生物組織或生物細 胞。更較佳地，該生物檢體係指全血，血清，尿液，唾液， 腦脊髓液，精液，淚水，喉嚨棉拭，直腸棉拭或排洩物。 1344490 麻在本發明決定聚合酶鏈反應最佳化條件的方法之較佳 ^中’二甲基亞硕在個別聚合酶鏈反應單元中提供的最 個=範圍為1至1〇%，特別較佳地，該二甲基亞硬在 個別聚合酶鏈反應單元中提供的最終濃度為4%。 在本發明決定聚合酶鏈反應最佳化條件的方法之較佳 爭：r中’海藻糖在個別聚合 度為〇 6m。 K口^反應早4提供的最終濃 =本發明決定聚合酶鏈反應最佳化條件的方法之較佳 ^中’甜菜驗在個„合酶鏈反應單 度範圍為〇 5至？ ς λ/ί时 捉仏的成終濃 合酶鏈反料：L 別較佳地，該甜菜驗在個別聚 鏈反應早70中提供的最終濃度為2Μ。 事每t本Γ月決定聚合酶鍵反應最佳化條件的方法之較佳 Λ 個別聚合酶鏈反應單元内提供 終道声氣/1。/ 丫 &亞石風的最 最終二為二海糖的最終濃度為。·6Μ;且甜菜驗的 事實i = 酶鏈反應最佳化條件的方法之較佳 水合轉前先進行熱起始反應。 。括於添加 名务明、、Jr a '、疋聚合酶鏈反應最佳化條件的太土士 先選定溫度區間可為一般聚合…預 可能發生黏合的Λ 心中引子與核酸模版 的m度範圍。在本發明方法 — 該原始設計弓丨早阳 較L事貫中’ 別較佳地，該預I 溫度區間係為4 〇至7 〇°C，特 65 1待測=選定引子最佳點合溫度區間係為49至 式時，該預先選定引子最佳點合溫度區間传 1344490 為 4 9 至 6 1 °C。 在本發明決定聚合酶鏈反應最佳化條件的 預先選定溫度區間内可選擇多種不同溫度進行聚合酶鍵反 應，以反應結果決定最佳化條件。在本發明方法之較佳事 實中’該複數溫度係指四種溫度。 在另-方面，本發明提供一種在生物檢體中偵測核酸 序列片段的方法，其包括下列步驟： U)使用本發明決定聚合酶鏈反應最佳化條件的方法， 決定該核酸序列片段之聚合酶鏈反應最佳化條件； (b) 以該最佳化條件對該生物檢體進行聚合酶鏈反應； (c) 判讀聚合酶鏈反應結果；及 (d) 決定該生物檢體中是否存在該核酸序列片段。 本發明在生物檢體中偵測核酸序列片段的方法之較佳 事貝中，違生物檢體係指全血’血清，尿液，唾液，腦脊 链液’精液’淚水，喉嚨棉拭，直腸棉拭或排洩物。 在另一方面，本發明提供一放大具有核酸多型性片段 之方法’其包括下列步驟： (a) 使用本發明決定聚合酶鍵反應最佳化條件的方 法’決定該核酸序列片段之聚合酶鏈反應最佳化條件； (b) 以該最佳化條件就對應於該核酸序列片段之複數異 源性核酸進行聚合酶鏈反應； (c) 分析聚合酶鏈反應結果；及 (d) 决疋s玄核酸序列片段是否存在單—核苦酸多型性。 本發明放大具有核酸多型啦片段之方法之較佳事實 M449〇 ’該核酸序列片段係來自生物檢體、生物組織或生物細 $。本發明放大具有核酸多型性片段之方法之另—較佳事 、中’該生物檢體係指全血’血清，尿液，唾液，腦脊趙 液，精液，淚水，喉嚨棉拭，直腸棉拭或排洩物。 在另一方面，本發明提供一種製造核酸序列片段之方 去’其包括下列步驟： U)使用本發明決定聚合酶鏈反應最佳化條件的方 去’決定該核酸序列片段之聚合酶鏈反應最佳化條件； (b) 以該最佳化條件使用該核酸序列X段作為模版進行 聚合酶鏈反應；及 (c) 分離聚合酶鏈反應得到的核酸序列片段。 在本發明製造核酸序列片段的方法之較佳事實中，該 核峻序列片段係天然核酸序列片段或人工合成核酸序列 4^ 〇 在本發明製造核酸序列片段的方法之較佳事實中，該 天然核酸序列片段係來自生物檢體、生物組織或生物細 跑；在本發明製造核酸序列片段之方法之另一較佳事實 中，遠生物檢體係指全血，血清，尿液，每液，腦脊趙液， 和液，淚水，喉嚨棉栻’直腸棉拭或排洩物。 在另一方面’本發明提供一種決定聚合酶鏈反應最佳 化條件的套組，其包括： 適量的二甲基亞砜、海藻糖及甜菜鹼。 較佳地’本發明決定聚合酶鏈反應最佳化條件的套組 進而含有用於實施本發明決定聚合酶鏈反應最佳化條件的 10 1344490 方法的操作指南。 較佳地’本發明決定聚合酶鏈反應最佳化條件的套組 所提供的二曱基亞砜，在個別聚合酶鏈反應單元中的最終 /辰度範圍為1至1 〇 %，特別較佳地，該二甲基亞艰在個 別聚合酶鏈反應單元中提供的最終濃度為4 %。^ 較佳地，本發明決定聚合酶鏈反應最佳化條件的套組 所提供的海藻糖，在個別聚合酶鏈反應單元中的最終濃度 為 0 · 6 Μ。 較佳地’本發明決定聚合酶鏈反應最佳化條件的套組 所提供的甜菜鹼，在個別聚合酶鏈反應單元中的最终濃度 範圍為0 . 5至3，5 Μ，特別較佳地，該甜菜鹼在個別聚合 酶鍵反應單元中提供的最終濃度為2Μβ 較佳地’本發明決定聚合酶鏈反應最佳化條件的套 組，在個別聚合酶鏈反應單元中提供二甲基亞砜的最終濃 度為4 % 海藻糖的最終濃度為〇 6 Μ ;且甜菜鹼的最終 濃度為2 Μ。 在另一方面’本發明提供一種在生物檢體中偵測核酸 序列片段的套組，其包括： 適量的二曱基亞碱、海藻糖及甜菜鹼。 較佳地’本發明在生物檢體中偵測核酸序列片段的套 組進而含有用於實施本發明在生物檢體中偵測核酸序列片 段的方法的操作指南。 較佳地’本發明在生物檢體中偵測核酸序列片段的套 組所提供的二甲基亞砜，在個別聚合酶鏈反應單元中的最 丄 M4490 碾在 、冬♦度範圍為1至；！ 〇 % ’特別較佳地，該二甲基亞 個別聚合酶鏈反應單元中提供的最終濃度為4 %。 車X佳地，本發明在生物檢體中偵測核酸序列片段的套 纽所提供的海藻糖，在個別聚合酶鏈反應單元中的最終濃 度為0 . 6 Μ。1344490 IX. Description of the Invention: [Technical Field to Which the Invention Is Ascribed] The present invention relates to a method of amplifying a nucleic acid using a polymerase chain reaction. [Prior Art] U.S. Patent No. 5,512,462 is a patent for a method for amplifying a long-segment deoxyribonucleic acid (DNA) by adding two polymerases, one of which is 3, to 5, an exonuclease, and the other. One is a deoxyribonucleic acid polymerase' and glycerol and dimercaptosulfoxide are also added to lower the melting temperature of the deoxyribonucleic acid, further increasing the specificity of the polymerase bond reaction. European Patent Application Laid-Open No. U19719 A1 is a method for the synthesis and amplification of deoxyribonucleic acid. In this case, a polymerase chain reaction using two modified hydrazine-stabilized enzymes is disclosed, which has the activity of a 3'-end exonuclease without the activity of a deoxyribonucleic acid polymerase. : Activity of acid polymerase for polymerase chain reaction. In the polymerase chain reaction buffer solution used in the invention, a reagent such as betaine plus (8) plus), dimercaptosulfoxide, glycerin or trehal〇se is added to reduce the solution of deoxyribonucleic acid. Chain temperature, further increase the polymerase chain anti-dew - Xiu Yu Xian Li 6,150,094 (inert cft ^ ... 卞❼ anal well 沽 surface ace) "The non-covalent effect of biomolecules including the even ion Permeate (zwitted () nie. betaine, etc.) is added as '1344490 US Patent 6,399,304, mainly using thermocycling to synthesize nucleic acids...in which at least two have different activities on di-deoxynucleotide In the heat cycle elevated temperature reaction, at least - the thermostable enzyme is activated later than the other heat stable enzyme. ^ US Patent Application Nos. 030049614 and 040072213, the disclosure of: "magnification, synthesis and The method of mutating a nucleic acid utilizes a polymerase which is thermally stable and has no positive function (non_pr〇〇freading) and another polymer having a correction function, and uses a nucleic acid which inhibits unwanted or its derivative = A factor of a deoxyribonucleic acid polymer, such as a polymerase enhancing fact rs. In addition, in the methods disclosed in the above, dimethyl sulfoxide may also be added when amplifying a nucleic acid of a very long fragment. European Patent 2〇〇4〇39947 uses mutated deoxyribonucleic acid nucleic acid polymerase to increase its reverse transcription activity. The methods taught in the above literature have common technical disadvantages. These prior techniques often involve polymerases. When the chain reaction is amplified, it is necessary to test a plurality of reaction conditions repeatedly, so that it takes a lot of time and materials to obtain a polymerase chain reaction amplification product which is highly specific and yields, especially using genomic nucleic acid as a nucleic acid. When stenciling, it is often the case that the product cannot be successfully amplified by polymerase chain reaction, and the important reason is that the secondary structure of the large genomic nucleic acid molecule interferes with the primer and the template nucleic acid sequence fragment. Adaling. Currently commercially available polymerase chain reaction conditions optimize test reagents, still #test various ion concentrations With the kind and the bonding temperature of the primer, etc., the method provides a fast and efficient method, and can achieve the best polymerase chain reaction amplification condition and obtain the product by measuring 6 1344490 in at least one experiment. The technical features of the method of the invention are mainly to add a template nucleic acid sequence fragment to be amplified in a general polymerase chain reaction buffer system, and then add three different additives: dimercaptosulfoxide, trehalose and betaine. (betaine), combined with pre-selected temperature interval settings, is the optimal condition for achieving high specificity and high yield in a single polymerization transfer reaction. In one aspect, the invention provides a method of determining conditions for optimizing polymerase chain reaction, comprising the steps of: (a) providing three or more polymerase chain reaction unit groups; (b) in a different population Each group of polymerase chain reaction units is separately added with dimethyl sulfoxide, trehalose and betaine; (c) each individual polymerase chain reaction unit in each group obtained in step (b) is placed separately The polymerase chain reaction is carried out at a complex temperature in a pre-selected temperature range; (d) the polymerase chain reaction results are interpreted; and (e) the polymerase chain reaction optimization conditions are determined. In a preferred aspect of the method of the invention, the nucleic acid sequence fragment is a fragment of a native nucleic acid sequence or a fragment of a synthetic nucleic acid sequence. Particularly preferably, the natural nucleic acid sequence fragment is derived from a biological specimen, biological tissue or biological cell. More preferably, the biopsy system refers to whole blood, serum, urine, saliva, cerebrospinal fluid, semen, tears, throat swabs, rectal swabs or excretions. 1344490 Ma in the preferred method of determining the conditions for optimizing the polymerase chain reaction of the present invention, the most = range of 1 -1% in the individual polymerase chain reaction units, especially in the range of 1 to 1% Preferably, the dimethyl subhard provides a final concentration of 4% in individual polymerase chain reaction units. In the method of the present invention for determining the conditions for optimizing the polymerase chain reaction, it is preferred that r in the individual degree of polymerization is 〇 6 m. K port ^ reaction early 4 provides the final concentration = the preferred method for determining the polymerase chain reaction optimization conditions in the present invention, the 'beet test in a single enzyme chain reaction single degree range is 〇 5 to ? ς λ / ί 仏 仏 仏 浓 浓 浓 : : : : : : : : : : : : : : : : L L L L L L L L L L L L L L L L L L L 较佳 较佳 较佳 较佳 较佳 较佳 较佳 较佳 较佳 较佳The best method for optimizing the conditions Λ The final concentration of the final gas in the individual polymerase chain reaction unit /1. / 丫 & 亚 亚 风 的 的 的 为 最终 最终 最终 最终 最终 且 甜菜 甜菜 甜菜 甜菜 甜菜 甜菜The fact that i = the optimal conditions for the enzyme chain reaction is preferred to carry out the hot start reaction before the hydration. It is included in the optimization conditions of the addition of the name, the Jr a ', and the polymerase chain reaction. The temperature range selected by Tusi can be the general polymerization... the range of the m-degree of the primer and the nucleic acid template in the pre-probable bonding. In the method of the present invention - the original design is better than the L in the morning. The pre-I temperature range is 4 〇 to 7 〇 ° C, and the special 65 1 is to be tested = the best point for the selected primer When the degree interval is 49 to the formula, the optimal spot temperature range of the pre-selected primer is 1344490, which is 4 9 to 61 ° C. It can be selected in the preselected temperature range of the present invention to determine the optimal conditions for the polymerase chain reaction. The polymerase bond reaction is carried out at a plurality of different temperatures, and the optimization result is determined by the reaction result. In the preferred fact of the method of the present invention, the plural temperature means four temperatures. In another aspect, the present invention provides a biological sample. A method for detecting a fragment of a nucleic acid sequence, comprising the steps of: U) determining a polymerase chain reaction optimization condition of the nucleic acid sequence fragment using the method for determining a polymerase chain reaction optimization condition of the present invention; (b) The optimized condition conditions the polymerase chain reaction of the biological sample; (c) interpreting the result of the polymerase chain reaction; and (d) determining whether the nucleic acid sequence fragment is present in the biological sample. The present invention is in a biological sample. In the preferred method of detecting nucleic acid sequence fragments, the biological test system refers to whole blood 'serum, urine, saliva, cerebrospinal fluid 'sperm' tears, throat cotton swabs, rectal cotton swabs or In another aspect, the invention provides a method of amplifying a fragment having a nucleic acid polymorphism comprising the steps of: (a) determining a fragment of the nucleic acid sequence using the method of the invention for determining a condition for optimizing a polymerase bond reaction. Optimizing the polymerase chain reaction conditions; (b) performing polymerase chain reaction on the plurality of heterologous nucleic acids corresponding to the nucleic acid sequence fragment under the optimized conditions; (c) analyzing the results of the polymerase chain reaction; d) determining whether a singular nucleic acid sequence fragment has a mono-nucleic acid polymorphism. The present invention amplifies the fact that a nucleic acid polymorphic fragment is preferred. M449〇' The nucleic acid sequence fragment is derived from a biological specimen or biological tissue. Or biological fine $. The present invention amplifies a method having a nucleic acid polymorphic fragment, preferably, the biological test system refers to whole blood 'serum, urine, saliva, cerebrospinal fluid, semen, tears, throat Cotton swab, rectal cotton swab or excrement. In another aspect, the invention provides a method of making a fragment of a nucleic acid sequence comprising the steps of: U) determining the polymerase chain reaction of the nucleic acid sequence fragment using the method of determining the optimal conditions for polymerase chain reaction according to the invention. Optimizing conditions; (b) using the X segment of the nucleic acid sequence as a template for polymerase chain reaction; and (c) isolating a fragment of the nucleic acid sequence obtained by polymerase chain reaction. In a preferred aspect of the method of making a nucleic acid sequence fragment of the present invention, the nuclear sequence fragment is a natural nucleic acid sequence fragment or a synthetic nucleic acid sequence which is preferred in the method of making a nucleic acid sequence fragment of the present invention, the natural The nucleic acid sequence fragment is derived from a biological specimen, biological tissue or biological run; in another preferred fact of the method for producing a nucleic acid sequence fragment of the present invention, the far biopsy system refers to whole blood, serum, urine, fluid, brain Ridge Zhao fluid, and liquid, tears, throat, cotton axillary 'rectal cotton swab or excrement. In another aspect, the invention provides a kit for determining conditions for optimizing polymerase chain reaction, comprising: an appropriate amount of dimethyl sulfoxide, trehalose, and betaine. Preferably, the kit of the present invention which determines the conditions for optimizing the polymerase chain reaction further contains instructions for the practice of the 10 1344490 method for determining the conditions for optimizing the polymerase chain reaction of the present invention. Preferably, the dimercaptosulfoxide provided by the kit for determining the conditions for optimizing the polymerase chain reaction of the present invention has a final/length in the individual polymerase chain reaction units ranging from 1 to 1%, especially Preferably, the dimethyl sulfoxide provides a final concentration of 4% in individual polymerase chain reaction units. Preferably, the present invention determines the trehalose provided by the kit for optimizing the conditions of the polymerase chain reaction, and the final concentration in the individual polymerase chain reaction units is 0 · 6 Μ. Preferably, the betaine provided by the kit for determining the conditions for optimizing the polymerase chain reaction of the present invention has a final concentration in the individual polymerase chain reaction units ranging from 0.5 to 3,5 Torr, particularly preferably. The final concentration of the betaine provided in the individual polymerase bond reaction unit is 2Μβ. Preferably, the present invention determines a kit for optimizing the conditions of the polymerase chain reaction, providing a dimethyl group in an individual polymerase chain reaction unit. The final concentration of sulfone is 4%. The final concentration of trehalose is 〇6 Μ; and the final concentration of betaine is 2 Μ. In another aspect, the invention provides a kit for detecting fragments of a nucleic acid sequence in a biological sample, comprising: an appropriate amount of a dimercapto base, trehalose, and betaine. Preferably, the kit of the present invention for detecting a fragment of a nucleic acid sequence in a biological sample further comprises an operation guide for carrying out the method of the present invention for detecting a fragment of a nucleic acid sequence in a biological specimen. Preferably, the dimethyl sulfoxide provided by the kit for detecting a fragment of a nucleic acid sequence in a biological sample is smashed in the last M4490 of the individual polymerase chain reaction unit, and the winter range is 1 to ;! 〇 % ' particularly preferably, the final concentration provided in the dimethyl sub-polymerase chain reaction unit is 4%. The trehalose provided by the kit for detecting a fragment of a nucleic acid sequence in a biological specimen of the present invention has a final concentration of 0.6 Μ in an individual polymerase chain reaction unit.
較佳地，本發明在生物檢體中偵測核酸序列片段的套 組所提供的甜菜鹼，在個別聚合酶鏈反應單元中的最終濃 又範圍為〇 ’ 5至3 . 5 Μ ’特別較佳地，該甜菜鹼在個別聚 合酶鏈反應單元中提供的最終濃度為2μ。 較佳地本發明在生物檢體中·<(貞測核酸序列片段的套 組’在個別聚合酶鏈反應單元中提供二甲基亞硬的最終濃 度為4% ;海藻糖的最終濃度為〇 6Μ :且甜菜鹼的最終 ;辰度為2 Μ。 在另一方面，本發明提供一放大具有核酸多型性片段 的套組，其包括： 適里的二甲基亞砜'海藻糖及甜菜鹼。 較佳地，本發明係一放大具有核酸多型性片段的套組 進而含有用於實施本發明放大具有核酸多型性片段的方法 的操作指南。 較佳地，本發明係一放大具有核酸多型性片段的套組 所提供的二甲基亞磾，在個別聚合酶鏈反應單元中的最終 農度範圍為1 1 1 Q %，特別較佳地，該二甲基亞硬在個 別聚合酶鏈反應單元中提供的最終濃度為4 %。 較佳地，本發明係一放大具有核酸多型性片段的套組 12 1344490 所提供的海藻糖，在個別肜人& „ 丄 以聚合酶鏈反應早兀中的最终濃度 為 0 . 6 Μ。 較佳地，本發明係1大具有«多型性片段的b 所提供的甜菜鹼’在個别聚合酶鏈反應單元中的最終濃度 範圍為Q·5至3.5M1難㈣，㈣㈣在個別聚合 酶鏈反應單元中提供的最终濃度為2M。 。 較佳地，本發明係—放大具有核酸多型性片段的套 植’在個別聚合酶鍵反應單元中提供二甲基亞硬的最終濃 度為4% ;海薄糖的最終濃度為〇 6Μ;且甜菜驗的最终 濃度為2 Μ。 在另一方面，本發明提供一種製造核酸序列片段的套 組，其包括： 適量的二甲基亞砜、海藻糖及甜菜鹼。 較佳地’本發明製造核酸序列月段的套組進而含有用 於實施本發明製造核酸序列片段的方法的操作指南。 較佳地’本發明製造核酸序列月段的套組所提供的二 曱基亞颯’在個別聚合酶鏈反應單元中的最終濃度範圍為 1至1 〇 % ’特別較佳地，該二甲基亞砜在個別聚合酶鍵 反應單元中提供的最終濃度為4 %。 較佳地’本發明製造核酸序列片段的套組所提供的海 蒸糖，在個別聚合酶鏈反應單元中的最終濃度為〇 6 μ。 較佳地，本發明製造核酸序列月段的套組所提供的封 菜鹼，在個別聚合酶鏈反應單元中的最終濃度範圍為〇 5至3 · 5 Μ ’特別較佳地，該甜菜驗在個別聚合酶鍵反應 13 1344490 單元中提供的最終濃度為2M。 較佳地’本發明製造核酸序列片段的套組，在個別聚 合酶鏈反應單元中提供二甲基亞砜的最終濃度為4%;海 藻糖的最終濃度為0.6M;且甜菜鹼的最終濃度為2m。 本發明說明書中所使用之名詞「聚合酶鏈反應單元」， 係指能夠進行聚合酶鏈反應之最小單位，通常含有聚合聘 鏈反應所需之材料 '試劑及物質等’但不含二甲基亞碼、 海藻糖及甜菜驗三者任一。在本發明較佳之具體事實中， 一個聚合酶鏈反應單元可含有適量之核酸模版序列、最终 濃度為0.2 μΜ之個別引子、最終濃度為〇 . 4 mM之去氧 核酸單體、適量之離子濃度與適量之聚合酶。 本發明說明書所使用之名詞「核酸序列片段」，係由 多個核皆酸為單元體接成之巨分子。包含核醣核酸或去氧 核膽核酸’為單股或雙股或經過化學修飾之片段。 本發明說明書所使用之名詞「熱起始反應」，係指在 混合適量之核酸序列片段 '引子對、去氧核酸單體與適量 之離子濃度後，在添加聚合酶前先利用高溫來降低混合物 中引子非專一性地結合至模版序列的情形，從而減少發生 非專一性地放大反應’且亦避免引子二聚體之形成。 本發明說明書所使用之名詞「異源性核酸」，係指屬 同種（species)生物間不同個體來源之核酸。 本發明說明書所使用之名詞「套組」，係包含被劃分 的多個區隔（compartment)或容器（container)，在該多個區 隔或容器中至少分別獨立包含適量的二甲基亞砜、海藻糖 14 丄j 及甜菜鹼。在本發明的套組 ^ 而分別獨立包含進行本發明㊃㈣隔或容11亦可進 核酸序列片段、引子對、去：需之試劑及材料，例如 之級衝溶液等。在本發明 ^" .,t , , #組中另包含操作指南，該择 作札南係指用來實施本發明方 术 ,D ^ 赞月方去之說明。操作指南可以被 印在適虽的基質上，例如：套 ^ 、'之包裝上，或是可被電腦 β買取之格式，例如：磁片等. 乃寻’或是網路上可被使用者讀取 之方式等。 Λ ^ 【實施方式】 鲁 本發明其他的特徵及優點將可明顯見於下列較佳具體 事實及申請專利範圍。 實例 下列實施例用於示範說明本發明。這些實施例不以任 何方式意欲限制本發明之範圍，但用於指示如何實施本發 明的材料及方法。 聚合醢鉍及應、 · 1 ·樣品來源：從來自台大醫院檢體抽取之DNA。 2 ‘基因放大標的：分別以人類基因體之過氧化小體 增生活化受體（peroxisome-proliferator activated receptor, PPAR)、人類基因體之衍脂蛋白E ( Apolipoprotein E,Preferably, the betaine provided by the kit for detecting a fragment of a nucleic acid sequence in a biological sample has a final concentration in the individual polymerase chain reaction unit ranging from 〇' 5 to 3.5 Μ ' Preferably, the betaine is provided in a final concentration of 2 [mu] in individual polymerase chain reaction units. Preferably, the present invention provides a final concentration of 4% dimethyl subhard in the individual polymerase chain reaction units in a biosample (> (measurement of a set of nucleic acid sequence fragments); the final concentration of trehalose is 〇6Μ: and the final of betaine; the degree is 2 Μ. In another aspect, the invention provides a kit for amplifying a polymorphic fragment having a nucleic acid comprising: a suitable dimethyl sulfoxide trehalose and Preferably, the present invention is a method of amplifying a kit having a nucleic acid polymorphic fragment and further comprising a method for carrying out the method of amplifying a nucleic acid polymorphic fragment of the present invention. Preferably, the present invention is amplifying. The dimethyl hydrazine provided by the kit having the polymorphic fragment of nucleic acid has a final agronomic range in individual polymerase chain reaction units of 11 1 Q%, and particularly preferably, the dimethyl sub-hard The final concentration provided in the individual polymerase chain reaction units is 4%. Preferably, the present invention amplifies the trehalose provided by the kit 12 1344490 having the polymorphic fragment of the nucleic acid, in the individual deaf & Polymerase chain reaction Preferably, the present invention is a large concentration of Q. 5 to 3.5 M1 in a single polymerase chain reaction unit. (d), (iv) (iv) The final concentration provided in the individual polymerase chain reaction unit is 2M. Preferably, the present invention is to amplify a cassette having a nucleic acid polymorphic fragment to provide a dimethyl group in an individual polymerase bond reaction unit. The final concentration of the subhard is 4%; the final concentration of the sea sugar is 〇6Μ; and the final concentration of the beet is 2 Μ. In another aspect, the invention provides a kit for making a fragment of a nucleic acid sequence, comprising: Dimethyl sulfoxide, trehalose, and betaine. Preferably, the kit for making a nucleic acid sequence of the present invention, in turn, contains instructions for performing the methods of the present invention for making nucleic acid sequence fragments. Preferably, the present invention The kit for making the nucleic acid sequence of the month provides a final concentration of dimercaptopurine in the individual polymerase chain reaction units ranging from 1 to 1%%. Particularly preferably, the dimethyl sulfoxide is polymerized individually. Enzyme bond reaction unit The final concentration is provided at 4%. Preferably, the set of nucleic acid sequence fragments of the invention provides a sea evaporated sugar having a final concentration of 〇6 μ in individual polymerase chain reaction units. Preferably, the invention The kit for the manufacture of the nucleic acid sequence of the monthly phase provides a final concentration in the individual polymerase chain reaction units ranging from 〇5 to 3·5 Μ. Particularly preferably, the beet is tested in individual polymerase bond reactions. 13 1344490 The final concentration provided in the unit is 2 M. Preferably, the set of nucleic acid sequence fragments of the present invention provides a final concentration of 4% dimethyl sulfoxide in individual polymerase chain reaction units; the final of trehalose The concentration is 0.6 M; and the final concentration of betaine is 2 m. The term "polymerase chain reaction unit" as used in the specification refers to the smallest unit capable of performing polymerase chain reaction, usually containing the polymerization chain reaction required. The material 'reagents and substances, etc.' but does not contain any of the dimethyl subcode, trehalose and beet test. In a preferred embodiment of the present invention, a polymerase chain reaction unit may contain an appropriate amount of a nucleic acid template sequence, a final primer concentration of 0.2 μΜ, a final concentration of 0.4 mM of deoxyribonucleic acid monomer, and an appropriate amount of ion concentration. With the right amount of polymerase. The term "nucleic acid sequence fragment" as used in the specification of the present invention is a macromolecule in which a plurality of nuclear acid is a unit body. The ribonucleic acid or deoxyribonucleic acid' is a single or double strand or chemically modified fragment. The term "hot initiation reaction" as used in the specification of the present invention refers to the use of high temperature to reduce the mixture before adding a polymerase after mixing an appropriate amount of the nucleic acid sequence fragment 'primer pair, deoxyribonucleic acid monomer and an appropriate amount of ion concentration. The middle primer is non-specifically bound to the template sequence, thereby reducing the occurrence of non-specific amplification reactions and also avoiding the formation of primer dimers. The term "heterologous nucleic acid" as used in the specification of the present invention refers to a nucleic acid of a different individual origin between the species. The term "set" as used in the specification of the present invention includes a plurality of compartments or containers, and at least each of the plurality of compartments or containers independently contains an appropriate amount of dimethyl sulfoxide. , trehalose 14 丄 j and betaine. In the kit of the present invention, each of the four (four) compartments or the volume 11 of the present invention may be independently included, and the nucleic acid sequence fragment, the primer pair, the reagent and the material to be used, for example, a graded solution, and the like may be included. In the ^".,t, ,# group of the present invention, an operation guide is additionally included, and the selection is used to carry out the method of the present invention, and D ^Zhayuefang goes to the description. The operating instructions can be printed on a suitable substrate, such as a package, a 'package', or a format that can be purchased by the computer beta, such as a magnetic disk, etc. It can be read by the user or read on the Internet. Take the way and so on. Λ ^ [Embodiment] Other features and advantages of the present invention will be apparent from the following specific details and claims. EXAMPLES The following examples are intended to illustrate the invention. The examples are not intended to limit the scope of the invention in any way, but are intended to indicate how to practice the materials and methods of the invention. Polymerization and application, · 1 · Sample source: DNA extracted from samples from National Taiwan University Hospital. 2 ‘Gene amplification: Peroxisome-proliferator activated receptor (PPAR), human genomic lipoprotein E (Apolipoprotein E, respectively)
ApoE )與人類基因體之低密度脂蛋白（iip〇pr〇tein Hpase， LPL)為放大之標的基因。以上的序列來自the European 15 1344490ApoE) and human genomic low-density lipoprotein (iip〇pr〇tein Hpase, LPL) are the amplified genes. The above sequence is from the European 15 1344490
Bioinformatics Institute所公布的基因序列 3，步驟： 3 · 1 ·聚合酶鏈反應之混合溶液製備： 於每一聚合酶鏈反應管中添加最終濃度為0.4 mM去Bioinformatics Institute published gene sequence 3, steps: 3 · 1 · Polymerase chain reaction mixed solution preparation: Add a final concentration of 0.4 mM to each polymerase chain reaction tube
氧核苷酸單體（deoxynucleotide triphosphate,dNTP )、2 mM 氣化鎂（MgCl2) 、0.2 μΜ之各個引子與25ng 去氧核醣 核酸（DNA )模版，另外再分別添加二曱基亞砜（dimethyl sulphoxide，DMSO )、海藻糖（trehalose )與甜菜鹼（betaine ) 之添加劑使其最終濃度分別為4 % ' 〇 · 6 Μ與2 Μ於不同 之聚合酶鏈反應管中。 於聚合酶鏈反應管所添加之混令溶液體葙(μί ) 對照組 4%二甲·*亞磾 〇·6Μ海藻糖 2Μ甜菜鹼 去氧核醣核酸 2 2 2 2 去氧核苷酸單體 (dNTP,l〇 mM) 1 1 1 1 氣化鎂 (50 mM) 1 1 1 1 二次水 12.5 11.5 2.5 海藻糖（1·2Μ) 12.5 二甲基亞（4%) 1 甜菜鹼（5Μ) 10 引子（ΙΟμΜ ) 0.5 0.5 0.5 0.5 引子（I ΟμΜ ) 0.5 0.5 _----- _ -- 0.5 0.5 總體積 17.5 一 17.5 17.5 17.5 3 . 2 .聚合酶鏈反應之緩衝溶液製備：使其最終濃 16 1344490 度含有20 mM Tris ' 50 mM氣化鉀（KCl )與2 mM氣化 鎂（MgCl2)，且PH為8.4以及2.5U之Taq聚合酶’總 體積為7.5pL。 3 . 3 ·熱起始反應：將含有聚合酶鏈反應混合溶液 之聚合酶鏈反應管置於聚合酶鏈反應儀（RoboCycler®Deoxynucleotide triphosphate (dNTP), 2 mM magnesium hydride (MgCl2), 0.2 μΜ each primer and 25 ng DNA template, and dimethyl sulphoxide , DMSO), trehalose and betaine additives to a final concentration of 4% '〇·6 Μ and 2 Μ in different polymerase chain reaction tubes. Mixture solution added to the polymerase chain reaction tube μ(μί) Control group 4% dimethyl·*Aaquinium·6Μ Trehalose 2Μ Betaine deoxyribonucleic acid 2 2 2 2 Deoxynucleotide monomer (dNTP, l〇mM) 1 1 1 1 Magnesium sulfide (50 mM) 1 1 1 1 Secondary water 12.5 11.5 2.5 Trehalose (1·2Μ) 12.5 Dimethyl (4%) 1 Betaine (5Μ) 10 primer (ΙΟμΜ) 0.5 0.5 0.5 0.5 primer (I ΟμΜ ) 0.5 0.5 _----- _ -- 0.5 0.5 total volume 17.5 -17.5 17.5 17.5 3 . 2 . Polymerase chain reaction buffer solution preparation: make it final Concentrated 16 1344490 degrees contains 20 mM Tris '50 mM potassium carbonate (KCl) and 2 mM magnesium sulfide (MgCl2), and the total volume of Taq polymerase' at a pH of 8.4 and 2.5 U is 7.5 pL. 3. 3 · Thermal initiation reaction: Place the polymerase chain reaction tube containing the polymerase chain reaction mixture solution in the polymerase chain reaction apparatus (RoboCycler®
Gradient 96，Stratagen，La Jolla，CA)中，以 95。(：加熱 5 分鐘。 3.4.聚合酶鏈反應放大： 將最終 2.5U 之 Taq 聚合酶（Cat No. 11615-010, Invitr〇gen )、緩衝溶液（含有Taq聚合酶）與二次水加入 上述步驟3 . 2之聚合酶鏈反應管後，使其總反應體積為 2 5 mL ^之後於聚合酶反應儀中’進行3 〇次熱循環，條件 如下：951加熱30秒（使DNA變性之反應），之後於不同之 反應管中各自設定溫度為61、58、55與52 〇C或58、55、 52與49°C加熱45秒後（依照引子對之最適黏合溫度上下各 取至少一個適當溫度，進行黏合反應），再以721加熱i 分鐘（延長反應）。最後’再以7 21：加熱7分鐘，使延長反 應完全。 4 · 使用之引子序列 • 模版 引子名稱 序列（5 ‘ 一 3 ’） PPARy PPARG2IN45 5’-CTCCAAGTCATCCACGTTTTCCC (SE〇 ID ΝΟ:1) PPARG2IN5 5’-CATCATCCCACCCTCTTTCATAG (SEQ ID NO:2) 17 1344490 PPARa PPARAIN56 5’-CTGCTCATGCCTGTGTTTCCCC (SE〇 ID NO:3) PPARAIN6 5’-CTTCGCTGTCCTTTGCTTAACTGTG (SE〇 ID NO:4) ApoE ApoE(298-3 1 8) 5’-GCGCTGATGGACGAGACCATG (SEQ ID NO:5) ApoE(1020-997) 5，-CGGCGTTCAGTGATTGTCGCTGGG (SEQ ID NO:6) LPL LPL(607-627) 5，-GAGTTTAACTACCCTCTGGAC (SEQ ID NO:7) LPL(717-700) 5，-GCCAGTAATTCTGTTGACTTTC (SEQ ID NO:8)Gradient 96, Stratagen, La Jolla, CA), 95. (: heating for 5 minutes. 3.4. Polymerase chain reaction amplification: Add the final 2.5U Taq polymerase (Cat No. 11615-010, Invitr〇gen), buffer solution (containing Taq polymerase) and secondary water to the above steps. After the polymerase chain reaction tube of 3.2, the total reaction volume was 25 mL ^, and then 3 cycles of thermal cycling in the polymerase reactor under the following conditions: 951 heating for 30 seconds (reaction of DNA denaturation) Then, after setting the temperature in each of the different reaction tubes to 61, 58, 55, and 52 〇C or 58, 55, 52 and 49 ° C for 45 seconds (according to the optimal adhesion temperature of the primer, take at least one suitable temperature , carry out the bonding reaction), and then heat 721 for 1 minute (prolong the reaction). Finally '7 7: heating for 7 minutes to extend the reaction completely. 4 · Use primer sequence • Template primer name sequence (5 ' one 3 ' PPARy PPARG2IN45 5'-CTCCAAGTCATCCACGTTTTCCC (SE〇ID ΝΟ:1) PPARG2IN5 5'-CATCATCCCACCCTCTTTCATAG (SEQ ID NO:2) 17 1344490 PPARa PPARAIN56 5'-CTGCTCATGCCTGTGTTTCCCC (SE〇ID NO:3) PPARAIN6 5'-CTTC GCTGTCCTTTGCTTAACTGTG (SE〇ID NO: 4) ApoE ApoE (298-3 1 8) 5'-GCGCTGATGGACGAGACCATG (SEQ ID NO: 5) ApoE (1020-997) 5, -CGGCGTTCAGTGATTGTCGCTGGG (SEQ ID NO: 6) LPL LPL (607 -627) 5,-GAGTTTAACTACCCTCTGGAC (SEQ ID NO: 7) LPL (717-700) 5, -GCCAGTAATTCTGTTGACTTTC (SEQ ID NO: 8)
5 ·結果： 以ΡΡΑΙΙγ、PPARa、ApoE與LPL作為放大標的，同 時測試不同溫度與不同種類之添加劑進行聚合酶鍵反應之 放大，再將聚合酶鏈反應產物進行電泳分析，結果分別如 第一圖、第二圖、第三圖與第四圊所示，箭號係為產物出 現之位置。其中ΡΡΑΙΙγ最佳放大條件為：黏合溫度52。(：， 添加終濃度4°/◦二曱基亞颯、或0.6Μ海藻糖或2Μ甜菜鹼； PPARa最佳放大條件為：黏合溫度52°C，添力口終濃度0.6Μ 海藻糖或2M甜菜驗；ApoE最佳放大條件為：黏合溫度6 1 °C， 添加終濃度2M甜菜鹼；LPL最佳放大條件為：黏合 溫度4 9 °C，不加任何添加物。 18 1344490 根據本發明可作之不同修正及變化對於熟習該項技術 者而s均顯然不會偏離本發明的範圍與精神。雖然本發明 已敘述特疋的較佳具體事實，必須瞭解的是本發明不應被 不當地限制於該等特定具體事實上。事實i ,在實施本發 明之已述模式方面，熟習該項技術者而言_而易知之 不同修正亦被涵蓋於下列申請專利範圍之内。 [ 圖式簡 單說明】 第 一圖： 以聚合酶鏈反應放大 ΡΡΑΙΙγ之電泳分析 Μ ：DNA 分子重量標示。 1 ： 6「C， 無添加物之對照組。 2 ： 58〇C， 無添加物之對照組。 3 ： 5 5°C， 無添加物之對照組。 4 ： 52〇C， 無添加物之對照組。 5 ： 61°C > 添加最終濃度為4 % 二曱基亞碾。 6 : 58〇C ' 添加最終濃度為4 % 二甲基亞砜。 7 ： 55。。， 添加最終濃度為4 % 二曱基亞砜。 8 ： 52〇C * 添加最終濃度為4% 二曱基亞碾。 9 ： 61〇C， 添加最終濃度為0.6M海藻糖。 10 ：58〇C ’添加最終濃度為〇_6M海藻糖。 11 ：55〇C ’添加最終濃度為〇. < 5Μ海藻糖。 12 ：52〇C ’添加最終濃度為〇.6M海藻糖。 13 ：61°C ’添加最終濃度為2M甜菜鹼。 14 ：58〇C ’添加最終濃度為2M甜菜鹼。 19 1344490 1 5 : 5 5 °C，添加最終濃度為2M甜菜鹼。 16 : 52°C，添加最終濃度為2M甜菜鹼。 第二圖：以聚合酶鏈反應放大PPARa之電泳分析圖。 M : DNA分子重量標示。 1 : 61°C，無添加物之對照組。 2 : 58°C，無添加物之對照組。 3 : 5 5 °C，無添加物之對照組。 4 : 52°C，無添加物之對照組。 5 : 6 1°C，添加最終濃度為4%二曱基亞砜。 6 : 58°C，添加最終濃度為4%二甲基亞颯。 7 : 55°C，添加最終濃度為4%二曱基亞颯。 8 : 52°C，添加最終濃度為4%二曱基亞砜。 9 : 61°C，添加最終濃度為0.6M海藻糖。 10 : 58°C，添加最終濃度為0.6M海藻糖。 11 : 55°C，添加最終濃度為0.6M海藻糖。 12 : 52°C，添加最終濃度為0.6M海藻糖。 1 3 : 6 1°C，添加最終濃度為2M甜菜鹼。 1 4 : 58°C，添加最終濃度為2M甜菜鹼。 1 5 : 5 5 °C，添加最終濃度為2M甜菜鹼。 16 : 52°C，添加最終濃度為2M甜菜鹼。 第三圖：以聚合酶鏈反應放大ApoE之電泳分析圖。 M : DNA分子重量標示。 1 : 61°C，無添加物之對照組。 2 : 58°C，無添加物之對照組。 20 1344490 3 : 55°C，無添加物之對照組。 4 : 5 2 °C，無添加物之對照組。 5 : 6 1 °C，添加最終濃度為4%二曱基亞砜。 6 : 58°C，添加最終濃度為4%二曱基亞砜。 7 : 55°C，添加最終濃度為4%二曱基亞砜。 8 : 52°C，添加最終濃度為4%二曱基亞砜。 9 : 6 1°C，添加最終濃度為0.6M海藻糖。 10 : 58°C，添加最終濃度為0.6M海藻糖。 1 1 : 55°C，添加最終濃度為0.6M海藻糖。 12 : 52°C，添加最終濃度為0.6M海藻糖。 1 3 : 6 1°C，添加最終濃度為2M甜菜鹼。 14 : 58°C，添加最終濃度為2M甜菜鹼。 1 5 : 5 5°C，添加最終濃度為2M甜菜鹼。 1 6 : 52°C，添加最終濃度為2M甜菜鹼。 第四圖：以聚合酶鏈反應放大L P L之電泳分析圖。 M : DNA分子重量標示。 1 : 58°C，無添加物之對照組。 2 : 55°C，無添加物之對照組。 3 : 52°C，無添加物之對照組。 4 : 49°C，無添加物之對照組。 5 : 58°C，添加最終濃度為4%二曱基亞砜。 6 : 55°C，添加最終濃度為4%二曱基亞砜。 7 : 52°C，添加最終濃度為4%二曱基亞颯。 8 : 49°C，添加最終濃度為4%二曱基亞砜。 21 1344490 9 : 58°C，添加最終濃度為0.6M海藻糖。 10 : 55°C，添加最終濃度為0.6M海藻糖。 1 1 : 52°C，添加最終濃度為0.6M海藻糖。 12 : 49°C，添加最終濃度為0.6M海藻糖。 13 : 58°C，添加最終濃度為2M甜菜鹼。 14 : 55°C，添加最終濃度為2M甜菜鹼。 15 : 52°C，添加最終濃度為2M甜菜鹼。 16 : 49°C，添加最終濃度為2M甜菜鹼。 【主要元件符號說明】 225 ·Results: ΡΡΑΙΙγ, PPARa, ApoE and LPL were used as amplification targets. At the same time, the amplification of polymerase bond reactions of different temperatures and different kinds of additives was tested, and the polymerase chain reaction products were analyzed by electrophoresis. The results are shown in the first figure. The second, third, and fourth arrows show the position where the product appears. The optimum amplification condition of ΡΡΑΙΙγ is: bonding temperature 52. (:, adding a final concentration of 4 ° / ◦ 曱 飒 飒, or 0.6 Μ trehalose or 2 Μ betaine; PPARa optimal amplification conditions: bonding temperature 52 ° C, Timing mouth final concentration 0.6 Μ trehalose or 2M Beet test; ApoE optimal amplification condition is: bonding temperature 6 1 °C, adding final concentration 2M betaine; LPL optimal amplification condition is: bonding temperature 4 9 °C, without any additives. 18 1344490 According to the invention The various modifications and variations are obvious to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has described the preferred specific facts of the invention, it must be understood that the invention should not be unduly Limitations to such specific specific facts. Facts i, in the implementation of the described modes of the invention, those skilled in the art are aware of the various modifications that are also within the scope of the following claims. Description: Figure 1: Electrophoresis analysis of ΡΡΑΙΙγ by polymerase chain reaction Μ: DNA molecular weight labeling 1 : 6 "C, no additive control group 2 : 58 〇 C, no additive control group. 3 : 5 5 ° C, no additive control group 4 : 52 〇 C, no additive control group 5 : 61 ° C > Add final concentration of 4 % bismuth yam. 6 : 58 〇 C ' Add The final concentration is 4% dimethyl sulfoxide. 7 : 55., adding a final concentration of 4% dimercaptosulfoxide. 8 : 52 〇 C * Adding a final concentration of 4% dimercapto ruthenium 9: 61 〇C, add a final concentration of 0.6M trehalose. 10:58〇C 'Add the final concentration to 〇_6M trehalose. 11 :55〇C 'Add the final concentration to 〇. < 5 Μ trehalose. 12 :52〇 C 'Add a final concentration of 〇.6M trehalose. 13 : 61 ° C 'Add a final concentration of 2 M betaine. 14 : 58 〇 C 'Add a final concentration of 2 M betaine. 19 1344490 1 5 : 5 5 °C, Add a final concentration of 2M betaine. 16 : 52 ° C, add a final concentration of 2M betaine. Figure 2: Electrophoretic analysis of PPARa amplified by polymerase chain reaction. M : DNA molecular weight indication. 1 : 61 ° C Control group without additives 2: 58 ° C, no additive control group 3 : 5 5 ° C, no additive control group 4 : 52 C, no additive control group 5: 6 1 ° C, adding a final concentration of 4% dimethyl sulfoxide. 6 : 58 ° C, adding a final concentration of 4% dimethyl hydrazine. 7 : 55 ° C, a final concentration of 4% dimercaptopurine was added. 8 : 52 ° C, a final concentration of 4% dimercaptosulfoxide was added. 9 : 61 ° C, adding a final concentration of 0.6 M trehalose. 10: 58 ° C, a final concentration of 0.6 M trehalose was added. 11 : 55 ° C, adding a final concentration of 0.6 M trehalose. 12: 52 ° C, adding a final concentration of 0.6 M trehalose. 1 3 : 6 1 ° C, adding a final concentration of 2M betaine. 1 4 : 58 ° C, adding a final concentration of 2M betaine. 1 5 : 5 5 ° C, adding a final concentration of 2M betaine. 16 : 52 ° C, adding a final concentration of 2M betaine. Figure 3: Electrophoretic analysis of ApoE amplified by polymerase chain reaction. M : DNA molecule weight labeling. 1 : 61 ° C, no additive control group. 2: 58 ° C, no additive control group. 20 1344490 3 : 55 ° C, no additive control group. 4 : 5 2 ° C, no additive control group. 5 : 6 1 ° C, adding a final concentration of 4% dimethyl sulfoxide. 6 : 58 ° C, a final concentration of 4% dimercaptosulfoxide was added. 7 : 55 ° C, a final concentration of 4% dimercaptosulfoxide was added. 8 : 52 ° C, a final concentration of 4% dimercaptosulfoxide was added. 9 : 6 1 ° C, adding a final concentration of 0.6 M trehalose. 10: 58 ° C, a final concentration of 0.6 M trehalose was added. 1 1 : 55 ° C, adding a final concentration of 0.6 M trehalose. 12: 52 ° C, adding a final concentration of 0.6 M trehalose. 1 3 : 6 1 ° C, adding a final concentration of 2M betaine. 14 : 58 ° C, adding a final concentration of 2M betaine. 1 5 : 5 5 ° C, adding a final concentration of 2M betaine. 1 6 : 52 ° C, adding a final concentration of 2M betaine. Figure 4: Electrophoresis analysis of L P L amplified by polymerase chain reaction. M : DNA molecule weight labeling. 1 : 58 ° C, no additive control group. 2: 55 ° C, no additive control group. 3: 52 ° C, no additive control group. 4: 49 ° C, no additive control group. 5: 58 ° C, a final concentration of 4% dimercaptosulfoxide was added. 6 : 55 ° C, a final concentration of 4% dimercaptosulfoxide was added. 7 : 52 ° C, adding a final concentration of 4% dimercaptopurine. 8 : 49 ° C, a final concentration of 4% dimercaptosulfoxide was added. 21 1344490 9 : 58 ° C, adding a final concentration of 0.6 M trehalose. 10: 55 ° C, adding a final concentration of 0.6 M trehalose. 1 1 : 52 ° C, adding a final concentration of 0.6 M trehalose. 12: 49 ° C, adding a final concentration of 0.6 M trehalose. 13 : 58 ° C, adding a final concentration of 2M betaine. 14 : 55 ° C, adding a final concentration of 2M betaine. 15 : 52 ° C, adding a final concentration of 2M betaine. 16 : 49 ° C, adding a final concentration of 2M betaine. [Main component symbol description] 22