DNA 恒溫快速擴(kuò)增試劑盒（熒光型） DNA Isothermal Rapid Amplification Kit（Fluorescent Type) Guide Manual

產(chǎn)品參數(shù)Product parameters：

原理概述 Principle overview：

本試劑盒基于一種常溫恒溫核酸快速擴(kuò)增技術(shù)：在常溫恒溫下，重組酶和引物形成蛋白/單鏈核苷酸的復(fù)合體 Rec/ssDNA，在輔助蛋白和單鏈結(jié)合蛋白 SSB 的幫助下，侵入雙鏈 DNA 模板；在侵入位點(diǎn)形成 D-loop區(qū)域，并開(kāi)始對(duì) DNA 雙鏈進(jìn)行掃描；待找到與引物互補(bǔ)的目的區(qū)域后，復(fù)合體Rec/ssDNA 解體的同時(shí)，聚合酶也結(jié)合到引物的 3’末端，開(kāi)始鏈的延伸。本試劑盒在39 ºC 下，依賴(lài)核酸外切酶的作用，加入根據(jù)模板設(shè)計(jì)的特異的分子探針，使用熒光監(jiān)測(cè)設(shè)備能實(shí)現(xiàn)對(duì)目標(biāo)片段擴(kuò)增過(guò)程的實(shí)時(shí)監(jiān)控。

This kit is based on a room temperature and constant temperature nucleic acid rapid amplification technology: at room temperature and constant temperature, the recombinase and primer form the protein/single-stranded nucleotide complex Rec/ssDNA, with the help of auxiliary proteins and single-stranded binding protein SSB , invade the double-stranded DNA template; form a D-loop region at the invasion site, and start scanning the DNA double-strands; after finding the target region complementary to the primer, the Rec/ssDNA complex disintegrates, and the polymerase also binds to The 3' end of the primer initiates chain extension. This kit relies on the action of exonuclease at 39 ºC, adding specific molecular probes designed based on the template, and using fluorescence monitoring equipment to achieve real-time monitoring of the amplification process of the target fragment.

引物設(shè)計(jì) Primer design：

建議使用長(zhǎng)度在 30-35 bp 的引物，引物過(guò)短會(huì)影響擴(kuò)增速度和檢測(cè)靈敏度；引物設(shè)計(jì)避免形成二級(jí)結(jié)構(gòu)而影響擴(kuò)增；擴(kuò)增子長(zhǎng)度建議在 150-300 bp，通常不超過(guò) 500 bp。

It is recommended to use primers with a length of 30-35 bp. Primers that are too short will affect amplification speed and detection sensitivity; primers are designed to avoid the formation of secondary structures that affect amplification; the amplicon length is recommended to be 150-300 bp, usually no more than 500 bp.

熒光探針設(shè)計(jì)Fluorescent probe design：

探針序列不與特異性引物識(shí)別位點(diǎn)重疊，長(zhǎng)度為 46-52 nt，序列避免回文序列、內(nèi)部二級(jí)結(jié)構(gòu)和連續(xù)的重復(fù)堿基。探針共有四個(gè)修飾位點(diǎn)：距離 5’端的≥ 35 nt 的中部位置標(biāo)記一個(gè) dSpacer（四氫呋喃，THF），作為核酸外切酶的識(shí)別位點(diǎn)；THF 位點(diǎn)的上游標(biāo)記一個(gè)熒光基團(tuán)，下游標(biāo)記一個(gè)粹滅基團(tuán)，兩個(gè)基團(tuán)的間距為2-4 nt；THF 距離3’末端≥15 nt，并且 3’末端標(biāo)記一個(gè)修飾基團(tuán)，例如胺基、磷酸基團(tuán)或 C3-Spacer。

The probe sequence does not overlap with the specific primer recognition site, is 46-52 nt in length, and the sequence avoids palindromic sequences, internal secondary structures, and continuous repeated bases. The probe has four modification sites: the middle position ≥ 35 nt from the 5' end is labeled with a dSpacer (tetrahydrofuran, THF) as the recognition site for exonuclease; the upstream of the THF site is labeled with a fluorescent group, and the downstream Label a quenching group, the distance between the two groups is 2-4 nt; THF is ≥15 nt from the 3' end, and the 3' end is labeled with a modifying group, such as an amine group, a phosphate group or a C3-Spacer.

正對(duì)照熒光結(jié)果圖： P：正對(duì)照；N：陰性對(duì)照

產(chǎn)品特點(diǎn)及優(yōu)勢(shì)Product features and advantages：

本試劑盒具有靈敏度高、特異性強(qiáng)、反應(yīng)時(shí)間短（僅需 20 min）等優(yōu)點(diǎn)，反應(yīng)組分為干粉狀態(tài)，操作簡(jiǎn)便，易于保存。可適用于各種品牌的熒光定量 PCR 儀、恒溫?zé)晒鈹U(kuò)增儀器等熒光檢測(cè)設(shè)備。

This kit has the advantages of high sensitivity, strong specificity, and short reaction time (only 20 minutes). The reaction components are in dry powder state, which is easy to operate and easy to store. It can be applied to various brands of fluorescence quantitative PCR instruments, constant temperature fluorescence amplification instruments and other fluorescence detection equipment.

產(chǎn)品用途Application：

適用于實(shí)驗(yàn)室級(jí)別的DNA擴(kuò)增以及其他檢測(cè)用途的DNA擴(kuò)增使用。

Suitable for laboratory-grade DNA amplification and DNA amplification for other detection purposes.

操作步驟 Operation procedure and process：

實(shí)驗(yàn)操作相關(guān)視頻，請(qǐng)微信掃碼聯(lián)系安普未來(lái)生物客服獲取。

For videos related to experimental operations, please scan the QR code on WeChat to contact customer service.

產(chǎn)品線Product Line：

性能對(duì)比數(shù)據(jù)Performance comparison data：

Q：如何避免實(shí)驗(yàn)失敗？

A：使用前請(qǐng)仔細(xì)閱讀說(shuō)明書(shū)，嚴(yán)格按照操作步驟進(jìn)行操作；

Q：實(shí)驗(yàn)不規(guī)范會(huì)影響結(jié)果準(zhǔn)確度么？

A：會(huì)的，樣品處理應(yīng)該嚴(yán)格按照生物安全規(guī)范操作，在進(jìn)行反應(yīng)時(shí)請(qǐng)注意避免核酸污染，并設(shè)置空白對(duì)照；

Q：試劑的存儲(chǔ)條件？

A：儲(chǔ)存條件：儲(chǔ)存溫度 ≤ -20 °C恒溫環(huán)境，避光保存，避免重壓；