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Detection and Cloning of Spliced Transcripts by RT-PCR
采用RT-PCR进行剪切体的检测和克隆   

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Abstract

Using a Reverse Transcriptase-PCR approach spliced transcripts can be converted to cDNA, amplified and cloned into an expression plasmid. Sequencing of the obtained cDNA allows identification of the splicing events that generated the detected RNA (Grewe et al., 2012).

Keywords: Pre-mRNA(前体mRNA), Splicing(拼接), Transcription(转录), RT-PCR(RT-PCR), Retrovirus(逆转录病毒)

Materials and Reagents

I.   RT-PCR

  1. QuantiTect Probe RT-PCR Kit (QIAGEN, catalog number: 204443 )
  2. Primers (sense and antisense) (Biomers)
  3. Isolated RNA
  4. PCR tubes (Starlab)
  5. Nuclease-free water (B Braun)

II.  Cloning

  1. Agarose (Roth, catalog number: 2267.4 )
  2. Ethidium bromide (AppliChem)
  3. DNA ladders (1 kb and 100 bp) (Life Technologies, InvitrogenTM)
  4. Geneclean III Kit (Qbiogene, catalog number: 1001-600 )
  5. Example of an acceptor plasmid: pcDNA3.1 (Life Technologies, InvitrogenTM, catalog number: V790-20 )
  6. Restriction endonucleases with reaction buffers (New England Biolabs)
  7. Antarctic phosphatase with reaction buffer (New England Biolabs, catalog number: M0289L )
  8. TaKaRa-DNA-Ligation-Kit version 2.1 (TaKaRa, catalog number: 6022 )
  9. Ultracompetent XL2-blue bacteria (Stratagene, catalog number: 200150 )
  10. Carbenicillin (AppliChem, catalog number: A1491 )
  11. Bacteria cell culture tubes (Sarstedt)
  12. Plasmid preparation Kits (QIAGEN, catalog number: 27104 , 12362 )
  13. Tris-HCl (pH 7.8)
  14. Sodium acetate
  15. EDTA
  16. NaCl
  17. KCl
  18. MgCl2
  19. MgSO4
  20. LB medium (see Recipes)
  21. TAE buffer (see Recipes)
  22. 6x loading dye (see Recipes)
  23. SOC medium (see Recipes)
  24. LB agar (see Recipes)

Equipment

  1. PCR cycler (Mastercycler gradient 5331, Eppendorf, catalog number: 5331000.010 )
  2. Microwave oven (Alaska, model: M 1001 )
  3. Gel electrophoresis chamber (Perfect Blue electrophoresis system 40-1214, PeqLab) and power supply (Power supply E844, Consort)
  4. UV light box (UV-Transilluminator) (Vilber)
  5. Incubators (Thermomixer comfort, Eppendorf, catalog number: 5355000.011 ; 37 °C shaker for bacterial cell cultures Novotron, Infors HT)
  6. Scalpel (B Braun)
  7. Scalpel (B Braun)
  8. Bacteria cell culture tubes (Sarstedt)

Procedure

Depending on the experiment isolation of cytoplasmic or total RNA should be done. In general, cytoplasmic RNA is enriched in spliced RNA lacking all introns whereas total RNA contains unspliced, partially-spliced and fully-spliced RNA. A protocol for the isolation of cytoplasmic RNA from mammalian cells can be obtained from the first part of the bio-protocol "Packaging of retroviral RNA into viral particles analyzed by quantitative Reverse Transcriptase-PCR". Sense and antisense primers should hybridize upstream of the splice donor and downstream of the splice acceptor sequence, respectively (Figure 1A). It is also possible that one primer overlaps the exon-exon junction ensuring perfect hybridization to spliced RNA only. For subsequent cloning, both primers should contain recognition sites for restriction endonucleases (cloning via restriction enzymes) or sequences identical to the acceptor plasmid (cloning via homologous sequences) at their 5' ends. A cDNA containing the whole open reading frame (ORF) of a gene can be generated when sense and antisense primers hybridize to the beginning of the ORF sequence or to the 5' untranslated region and to the end of the ORF sequence or the 3' untranslated region, respectively (Figure 1B).

  1. Set up RT-PCR reaction with the QuantiTect Probe RT-PCR Kit (QIAGEN) as follows:
    PCR mix (2x, contains Taq, dNTPs and buffer)
    10 μl
    Reverse Transcriptase enzyme
    0.2 μl
    Primer sense (10 μM)
    0.4 μl
    Primer antisense (10 μM)
    0.4 μl
    Isolated RNA
    0.5 to 1 μg template RNA in up to 5 μl
    Add nuclease-free water to 20 μl.

    Also prepare a control without addition of the Reverse Transcriptase (RT) in order to control for (genomic or plasmid) DNA contamination.
    When the cDNA obtained in the RT-PCR will be inserted in an acceptor plasmid via restriction enzyme digestion it must carry the desired restriction enzyme recognition sites which can be added to the 5' ends of the primers. Be aware that approximately 4 additional random nucleotides should be added upstream of the recognition sites in the primers to allow efficient cleavage to take place.
  2. RT-PCR reaction:
    Note: The QuantiTect Probe RT-PCR Kit (Qiagen) allows the RT reaction and the PCR to be performed in one run without interruption.
    RT step at 50 °C for 30 min followed by 15 min of 95 °C. Subsequently, amplify the cDNA by 30 PCR cycles starting at 95 °C for 30 sec, 50 to 60 °C depending on the melting temperature of the primers for 30 sec and 72 °C for 30 sec per 500 bp of amplicon length. It is possible to add a final extension step at 72 °C for up to 7 min to allow the DNA polymerase to elongate remaining single stranded DNA. Keep the PCR at 4 °C until agarose gel electrophoresis.
    Short elongation steps are necessary to preferentially detect spliced versus unspliced/partially-spliced transcripts. Sometimes it may be necessary to perform a nested-PCR to preamplify the cDNA. Perform the first RT-PCR with primers surrounding the sequence that is amplified in the second PCR. Primers for the second PCR must bind the fragment amplified in the first RT-PCR. One microliter directly from the first RT-PCR (without any purification) should be sufficient as template for the second PCR. However, the first amplicon can also be purified by a PCR clean-up kit and up to 200 ng of DNA per 20 μl reaction can be used as template for the second PCR which can be performed with the QuantiTect Probe RT-PCR Kit omitting the RT step and starting with 15 min at 95 °C.
  3. Prepare 1.5 % agarose gels in TAE buffer containing ethidium bromide (see recipes).
  4. Add 4 μl of 6x loading dye to the 20 μl PCR reaction and transfer the solution to the agarose gel.
  5. Agarose gel electrophoresis is done for approximately 30 min at 100 to 120 V (5 to 6 V per cm distance between electrodes). Analyze DNA ladder(s) in parallel to be able to determine the DNA fragment sizes. Please note that Ethidium bromide molecules are positively charged and move in the opposite direction in the agarose gel as the DNA molecules. Analyze the gel when the loading dye band reaches approximately the middle of the agarose gel (see Figure 1D).
  6. Document the result under a UV lamp gel documentation system. The intercalated Ethidium bromide allows detection of the amplified cDNA fragments (Figure 1D). Since UV light can damage the DNA, reduce the exposure time to a minimum.
  7. Cut out small gel pieces containing the desired DNA fragments at the UV table with a clean scalpel. Please wear laboratory safety glasses and reduce the exposure time to the UV light to a minimum.
  8. Extract the DNA from the gel pieces with the Geneclean III Kit. Elute the DNA from the glass beads with 10 μl of nuclease-free water.
  9. Prepare a restriction enzyme digestion of the isolated cDNA (the entire 10 μl) and of approximately 3 μg acceptor plasmid DNA in parallel:
    DNA solution
    10 μl
    Restriction buffer (10x)
    2 μl
    Bovine serum albumin solution (10x)
    2 μl
    Restriction enzyme(s) (20 U/μl)
    1 μl
    Add water to 20 μl

    Incubate for 1 to 3 h at 37 °C. When more than one restriction enzyme is used or when restriction enzymes are used that have star activity please increase the reaction volume to 30 μl. The enzyme(s) should not constitute more than 10 % (v/v) of the reaction volume. Star activity can also be reduced by decreasing the digestion time. However, complete digestion is critical for successful ligation and transformation. Extended incubation times up to overnight should only be performed with restriction enzymes without star activity or in increased reaction volumes (up to 50 μl).
    When compatible cohesive ends are generated during digestion (e.g. by using just one restriction enzyme) the linearized acceptor plasmid must be dephosphorylated to prevent self-ligation. After restriction enzyme digestion add Antarctic phosphatase together with its buffer directly to the reaction mix. After 30 min at 37 °C the enzyme should be inactivated by incubation for 5 min at 65 °C followed by agarose gel electrophoresis.
    For transient expression of genes in mammalian cells the plasmid pcDNA3.1 (Life Technologies, InvitrogenTM) (http://tools.invitrogen.com/content/sfs/vectors/pcdna3.1+.pdf) can be used as an acceptor plasmid. Due to the immediate early Cytomegalovirus (CMV) enhancer/promoter and the Bovine Growth Hormone (BGH) polyadenylation sequence it allows high expression levels. The cDNA from the RT-PCR must be inserted into the multiple cloning site. In addition, an efficient Kozak sequence (A/GCCATGG) should be added surrounding the start codon of the open reading frame which can be implemented into the sense primer during RT-PCR.
  10. Repeat steps 3 to 8 with the digested DNA. Cut out small gel slices containing only the linearized acceptor plasmid. Contamination with still circular plasmid DNA will lead to efficient re-transformation of the original plasmid and strongly reduce the amount of bacterial colonies carrying the insert-containing plasmid.
  11. Mix 2 μl of the digested and purified insert, 3 μl of the digested and purified acceptor plasmid DNA and 5 μl of solution I of the Takara Ligation Kit. Incubate at 16 °C for overnight. Alternatively, the DNA concentration can be measured and a vector: Insert DNA ratio of 0.03 pmol: 0.03 - 0.3 pmol can be used for ligation (manufacturer's instructions from the TaKaRa-DNA-Ligation-Kit version 2.1).
    A detailed protocol for transformation of DNA and subsequent amplification of plasmids in bacteria can be found in the bio-protocol "Standard DNA Cloning" (He, 2011b) and "Plasmid DNA Extraction from E. coli Using Alkaline Lysis Method" (He, 2011a) (please generate hyperlink).
    In brief, the following steps have to be performed:
  12. Transform ultracompetent XL-2 bacteria with 1.5 μl of the ligation reaction by heat shock.
  13. Let transformed bacteria grow for 1 h at 37 °C in SOC medium without antibiotics.
  14. Cultivate bacteria on LB agar plates containing an appropriate antibiotic at 30 to 37 °C overnight.
  15. Pick single colonies and let them growth overnight at 30 to 37 °C in 3 to 6 ml LB medium containing an appropriate antibiotic.
  16. Extract plasmid DNA from 1.5 to 3 ml of the bacterial culture. Keep the rest of the cultures at 4 °C.
  17. Digest the plasmid DNA with appropriate restriction enzymes to identify bacteria colonies containing successfully generated plasmids. In addition, sequence those plasmids that show fitting restriction enzyme digestion patterns. This sequencing reaction allows identifying the splicing event that actually generated the detected and cloned RNA.
  18. Prepare Midi, Maxi or Giga plasmid preparations from the identified bacteria colonies harboring the successfully generated plasmid. Endotoxin-free plasmid preparation kits avoid the presence of lipopolysaccharide in the plasmid DNA solution that will be used for transfections or to treat animals.


    Figure 1. Principle of the RT-PCR approach for the detection and cloning of spliced RNA. A. Sense and antisense primers (horizontal arrows) hybridize adjacent to the exon-exon boundary generated by fusion of splice donor 1 (SD1) and splice acceptor 1 (SA1) sequences during splicing. Restriction enzyme recognition sites (RE1 and RE2, vertical arrows) are present in the plasmid DNA and in the RT-PCR-amplified DNA generated from the partially-spliced RNA which can be used to insert the PCR fragment into the plasmid. B. Sense and antisense primers (horizontal arrows) contain restriction enzyme recognition sites (RE1 and RE2, vertical arrows) at their 5' ends. They hybridize in the 5' and 3' untranslated regions (UTR). Amplification of the fully-spliced RNA by RT-PCR allows insertion of the DNA into a plasmid (e.g. pcDNA3.1) using restriction enzymes 1 and 2. C. Depicted is a plasmid used to transfect HEK293T cells and its transcripts. RNAs generated by splicing between SD1 and SA5 or after an additional splicing event between SD4 and SA7 were reinserted into VHgenomic generating the plasmids VHenv or VHnef, respectively (Grewe et al., 2012). D. Shown is the agarose gel electrophoresis result obtained after RT-PCR amplification of spliced RNA from RNA isolated from cells transfected with the plasmid VHgenomic using primer set 1 containing forward primer and antisense primer 1 or primer set 2 containing forward primer and antisense primer 2 (see horizontal arrows in C) (Grewe et al., 2012). SD1-SA5 or SD1-SA5, SD4-SA7 indicates the splicing events detected after sequencing of the cDNA.
    UTR, untranslated region; RE, restriction enzyme recognition site; white bars, introns; dark grey bars, open reading frame; polyA, polyadenylation signal; SD, splice donor; SA, splice acceptor; horizontal arrows, primers; vertical arrows, location of restriction enzyme recognition sites; black dot, RNA 5' CAP; black oval; 3' polyA tail; LTR, lentiviral long terminal repeats; CMV, cytomegalovirus immediate early enhancer/promoter; GFP, green fluorescence protein.

Recipes

  1. 1x TAE buffer
    40 mM Tris-HCl (pH 7.8)
    5 mM Sodium acetate
    1 mM EDTA
  2. Agarose
    1.5 % (w/v) agarose in 1x TAE buffer
    Cook for 1 min in microwave oven and shake carefully
    Add 0.7 μg/ml Ethidium bromide [e.g. 7 µl of 1 % ethidium bromide solution (10 mg/ml) per 100 ml] after the solution is cooled down but still liquid and shake carefully.
  3. 6x loading dye
    30 mM Tris-HCl (pH 7.6)
    1 mM EDTA
    35 % (v/v) glycerine
    0.35 % (w/v) bromphenol blue
  4. SOC medium
    2 % (w/v) tryptone
    0.5 % (w/v) yeast extract
    20 mM glucose
    10 mM NaCl
    2.5 mM KCl
    10 mM MgCl2
    10 mM MgSO4
    Sterilize by autoclaving.
  5. LB agar
    1.5 % (w/v) Bacto-Agar
    In LB-Medium
    Sterilize by autoclaving.
  6. LB medium
    1 % (w/v) tryptone
    0.5 % (w/v) yeast extract
    171 mM NaCl
    pH 7.4
    Sterilize by autoclaving.

Acknowledgments

The protocol was adapted from our paper Grewe et al. (2012). This work was funded by a grant from the German Research Foundation (DFG) to Klaus Überla (Ue45/11-1). Bianca Hoffmann is and Bastian Grewe was supported by a fellowship from the DFG graduate school (GRK 1045). Beside Bianca Hoffmann and Bastian Grewe, Katrin Ehrhardt, Maik Blissenbach, Sabine Brandt, Klaus Überla, Alexander Stang, Thomas Grunwald, Klaus Sure, and Bettina Tippler were part of the team which established the methods described.

References

  1. Grewe, B., Ehrhardt, K., Hoffmann, B., Blissenbach, M., Brandt, S., and Uberla, K. (2012). The HIV-1 Rev protein enhances encapsidation of unspliced and spliced, RRE-containing lentiviral vector RNA. PloS One 7(11), e48688.
  2. He F. (2011a). Plasmid DNA extraction from E. coli using alkaline lysis method. Bio-protocol 1(3): e30. 
  3. He F. (2011b). Standard DNA cloning. Bio-protocol 1(7): e52.

简介

使用逆转录酶-PCR方法,可以将剪接的转录物转化为cDNA,扩增并克隆到表达质粒中。 获得的cDNA的测序允许鉴定产生检测的RNA的剪接事件(Grewe等,2012)。

关键字:前体mRNA, 拼接, 转录, RT-PCR, 逆转录病毒

材料和试剂

I.   RT-PCR

  1. QuantiTect Probe RT-PCR Kit(QIAGEN,目录号:204443)
  2. 引物(有义和反义)(Biomers)
  3. 分离的RNA
  4. PCR管(Starlab)
  5. 无核酸酶水(B Braun)

II。  克隆

  1. 琼脂糖(Roth,目录号:2267.4)
  2. 溴化乙锭(AppliChem)
  3. DNA梯(1kb和100bp)(Life Technologies,Invitrogen TM)
  4. Geneclean III试剂盒(Qbiogene,目录号:1001-600)
  5. 受体质粒的实例:pcDNA3.1(Life Technologies,Invitrogen TM ,目录号:V790-20)
  6. 具有反应缓冲液的限制性内切酶(New England Biolabs)
  7. 具有反应缓冲液的南极磷酸酶(New England Biolabs,目录号:M0289L)
  8. TaKaRa-DNA-Ligation-Kit version 2.1(TaKaRa,目录号:6022)
  9. Ultracompetent XL2-blue bacteria(Stratagene,目录号:200150)
  10. 羧苄青霉素(AppliChem,目录号:A1491)
  11. 细菌培养管(Sarstedt)
  12. 质粒制备试剂盒(QIAGEN,目录号:27104,12362)
  13. Tris-HCl(pH7.8)
  14. 醋酸钠
  15. EDTA
  16. NaCl
  17. KCl
  18. MgCl 2
  19. MgSO 4 4 /
  20. LB介质(见配方)
  21. TAE缓冲区(请参阅配方)
  22. 6x loading染料(见配方)
  23. SOC介质(参见配方)
  24. LB琼脂(见配方)

设备

  1. PCR循环仪(Mastercycler gradient 5331,Eppendorf,目录号:5331000.010)
  2. 微波炉(阿拉斯加,型号:M 1001)
  3. 凝胶电泳室(Perfect Blue电泳系统40-1214,PeqLab)和电源(电源E844,Consort)
  4. UV光盒(UV-透射器)(Vilber)
  5. 孵育器(Thermomixer comfort,Eppendorf,目录号:5355000.011;用于细菌细胞培养物的Novotron,Infors HT的37℃振荡器)
  6. Scalpel(B Braun)
  7. Scalpel(B Braun)
  8. 细菌培养管(Sarstedt)

程序

根据实验,应该进行细胞质或总RNA的分离。一般来说,细胞质RNA富含缺少所有内含子的剪接的RNA,而总RNA含有未剪接的,部分剪接的和完全剪接的RNA。从哺乳动物细胞分离细胞质RNA的方案可以从生物协议的第一部分"通过定量逆转录酶-PCR分析将逆转录病毒RNA包装到病毒颗粒中"。有义和反义引物应分别在剪接供体的上游和剪接受体序列的下游杂交(图1A)。也有可能一个引物与外显子 - 外显子连接重叠,确保仅与剪接的RNA完全杂交。对于随后的克隆,两个引物应在其5'端含有限制性内切核酸酶的识别位点(通过限制性酶克隆)或与受体质粒相同的序列(通过同源序列克隆)。当有义和反义引物与ORF序列的起始或5'非翻译区和ORF序列的末端或3'非翻译区杂交时,可产生含有基因的完整开放阅读框(ORF)的cDNA区域(图1B)。

  1. 使用QuantiTect Probe RT-PCR Kit(QIAGEN)设置RT-PCR反应,如下:
    PCR混合物(2x,含有Taq,dNTP和缓冲液)
    10微升
    反转录酶
    0.2μl
    引物检测(10μM)
    0.4μl
    引物反义(10μM)
    0.4μl
    分离的RNA
    0.5至1μg模板RNA至多5μl
    加入无核酸酶水至20μl
    还要制备一个没有加入逆转录酶(RT)的对照,以控制(基因组或质粒)DNA污染。
    当在RT-PCR中获得的cDNA通过限制性酶消化插入受体质粒时,它必须携带所需的限制酶识别位点,其可以加到引物的5'末端。注意,应在引物的识别位点上游添加大约4个额外的随机核苷酸,以允许有效裂解发生。
  2. RT-PCR反应:
    注意:QuantiTect Probe RT-PCR试剂盒(Qiagen)允许RT反应和PCR一次运行而不中断。
    RT步骤在50℃30分钟,然后15分钟95℃。随后,通过从95℃30秒,50至60℃开始的30个PCR循环扩增cDNA,取决于引物的解链温度30秒和72℃30秒/500bp扩增子长度。可以在72℃下添加最终延伸步骤长达7分钟,以允许DNA聚合酶延长剩余的单链DNA。保持PCR在4°C,直到琼脂糖凝胶电泳 短伸长步骤对于优先检测剪接的与未剪接的/部分剪接的转录物是必要的。有时可能需要进行巢式PCR以预扩增cDNA。使用在第二PCR中扩增的序列周围的引物进行第一次RT-PCR。第二次PCR的引物必须结合在第一次RT-PCR中扩增的片段。直接来自第一次RT-PCR(无任何纯化)的1微升应足够作为第二次PCR的模板。然而,第一扩增子也可以通过PCR纯化试剂盒纯化,并且每20μl反应高达200ng的DNA可以用作第二PCR的模板,其可以用QuantiTect Probe RT-PCR试剂盒进行,省略RT步骤,并在95℃下开始15分钟
  3. 在含有溴化乙锭的TAE缓冲液中制备1.5%琼脂糖凝胶(见配方)
  4. 加入4μl的6x负载染料到20μlPCR反应,并将溶液转移到琼脂糖凝胶
  5. 在100至120V(5至6V/cm电极间距离)下进行琼脂糖凝胶电泳约30分钟。平行分析DNA梯度以能够确定DNA片段大小。请注意,溴化乙锭分子带正电荷,并在琼脂糖凝胶中作为DNA分子以相反的方向移动。当加载染料带达到琼脂糖凝胶的中间时分析凝胶(见图1D)
  6. 将结果记录在UV灯凝胶文档系统下。插入的溴化乙锭允许检测扩增的cDNA片段(图1D)。由于紫外光可以损害DNA,将曝光时间降至最低。
  7. 用干净的手术刀在UV台上切出含有所需DNA片段的小凝胶片。请佩戴实验室安全眼镜,并将对紫外线的暴露时间降至最低
  8. 用Geneclean III试剂盒从凝胶片中提取DNA。 用10μl无核酸酶的水从玻璃珠中洗脱DNA
  9. 准备限制酶消化分离的cDNA(全部10μl)和约3μg受体质粒DNA平行进行:
    DNA溶液
    10微升
    限制缓冲区(10x)
    2微升
    牛血清白蛋白溶液(10x)
    2微升
    限制酶(20U /μl)
    1微升
    加水至20μl

    在37°C孵育1至3小时。当使用多于一种限制酶时或当使用具有星号活性的限制性酶时,请将反应体积增加至30μl。酶不应构成超过反应体积的10%(v/v)。星形活性也可以通过减少消化时间来减少。然而,完全消化对于成功连接和转化是关键的。延长孵育时间直至过夜只能使用没有星号活性或增加反应体积(高达50微升)的限制性酶。
    当在消化期间(例如通过仅使用一种限制性酶)产生相容的粘性末端时,线性化的受体质粒必须去磷酸化以防止自连接。限制酶消化后,将南极磷酸酶及其缓冲液直接加入到反应混合物中。在37℃下30分钟后,通过在65℃下孵育5分钟使酶失活,随后进行琼脂糖凝胶电泳。
    为了在哺乳动物细胞中瞬时表达基因,质粒pcDNA3.1(Life Technologies,Invitrogen )(http://tools.invitrogen.com/content/sfs/vectors/pcdna3.1+.pdf )可以用作受体质粒。由于立即早期巨细胞病毒(CMV)增强子/启动子和牛生长激素(BGH)多聚腺苷酸化序列,其允许高表达水平。来自RT-PCR的cDNA必须插入多克隆位点。此外,应当在开放阅读框的起始密码子周围添加有效的Kozak序列(A/GCCATGG),其可以在RT-PCR期间实施到有义引物中。
  10. 用消化的DNA重复步骤3至8。切出仅含有线性化受体质粒的小凝胶切片。与环状质粒DNA的污染将导致原始质粒的有效重新转化,并且强烈降低携带含有插入片段的质粒的细菌菌落的量。
  11. 混合2微升消化和纯化插入,3微升消化和纯化的受体质粒DNA和5微升的Takara连接试剂盒的溶液I.在16℃孵育过夜。或者,可以测量DNA浓度,并且可以使用载体:Insert DNA比率为0.03pmol:0.03-0.3pmol用于连接(来自TaKaRa-DNA-Ligation-Kit版本2.1的制造商的说明书)。
    用于转化DNA和随后在细菌中扩增质粒的详细方案可以在生物方案"标准DNA克隆"(He,2011b)和"Plasmid DNA Extraction from E.coli Using Alkaline裂解法"(He,2011a)(请生成超链接)。
    简而言之,必须执行以下步骤:
  12. 通过热休克转化超感官XL-2细菌与1.5μl连接反应
  13. 使转化的细菌在37℃下在没有抗生素的SOC培养基中生长1小时
  14. 在含有适当抗生素的LB琼脂平板上在30至37℃下培养细菌过夜
  15. 挑选单个菌落,并使其在30至37℃下在含有适当抗生素的3至6ml LB培养基中生长过夜。
  16. 从1.5至3ml的细菌培养物中提取质粒DNA。保持其余的文化在4℃
  17. 用适当的限制酶消化质粒DNA以鉴定含有成功产生的质粒的细菌菌落。此外,对那些显示出适合的限制酶消化模式的质粒进行测序。该测序反应允许鉴定实际产生检测和克隆的RNA的剪接事件
  18. 从含有成功产生的质粒的鉴定的细菌菌落制备Midi,Maxi或Giga质粒制备物。不含内毒素的质粒制备试剂盒避免在用于转染或治疗动物的质粒DNA溶液中存在脂多糖。


    图1.用于检测和克隆剪接的RNA的RT-PCR方法的原理 A.正义和反义引物(水平箭头)在由剪接供体融合产生的外显子 - 外显子边界附近杂交1(SD1)和剪接受体1(SA1)序列。限制酶识别位点(RE1和RE2,垂直箭头)存在于质粒DNA中和从部分剪接的RNA产生的RT-PCR扩增的DNA中,其可用于将PCR片段插入质粒中。 B.有义和反义引物(水平箭头)在其5'端含有限制性酶识别位点(RE1和RE2,垂直箭头)。它们在5'和5'杂交 3'非翻译区(UTR)。通过RT-PCR扩增完全剪接的RNA允许使用限制酶1和2将DNA插入到质粒(例如pcDNA3.1)中。C.描述的是用于转染HEK293T细胞的质粒和其成绩单。通过SD1和SA5之间的剪接或在SD4和SA7之间的另外的剪接事件之后产生的RNA被重新插入V H基因组中,产生质粒V H H env或V sup H nef(Grewe等人,2012)。显示了使用含有正向引物和反义引物1或引物1的引物组1,从转染有质粒V H基因组的细胞分离的RNA中RT-PCR扩增剪接的RNA后获得的琼脂糖凝胶电泳结果包含正向引物和反义引物2的集合2(参见C中的水平箭头)(Grewe等人,2012)。 SD1-SA5或SD1-SA5,SD4-SA7表示cDNA测序后检测到的剪接事件。
    UTR,非翻译区; RE,限制性酶识别位点;白色条,内含子;深灰色条,开放阅读框; polyA,聚腺苷酸化信号; SD,剪接供体; SA,剪接受体;水平箭头,引物;垂直箭头,限制性酶识别位点的位置;黑点,RNA 5'CAP;黑色椭圆形; 3'聚腺苷酸尾; LTR,慢病毒长末端重复序列; CMV,巨细胞病毒立即早期增强子/启动子; GFP,绿色荧光蛋白。

食谱

  1. 1x TAE缓冲区
    40mM Tris-HCl(pH7.8)
    5mM乙酸钠 1mM EDTA
  2. 琼脂糖
    1.5%(w/v)琼脂糖的1x TAE缓冲液中 在微波炉中煮1分钟,仔细摇匀
    加入0.7μg/ml溴化乙锭[ 在溶液冷却后仍然是液体并小心摇动,然后加入7μl的1%溴化乙锭溶液(10mg/ml)/100ml]。
  3. 6x装载染料
    30mM Tris-HCl(pH7.6) 1mM EDTA
    35%(v/v)甘油
    0.35%(w/v)溴酚蓝
  4. SOC介质
    2%(w/v)胰蛋白胨
    0.5%(w/v)酵母提取物
    20mM葡萄糖 10mM NaCl 2.5mM KCl
    10mM MgCl 2/
    10mM MgSO 4 通过高压灭菌消毒。
  5. LB琼脂
    1.5%(w/v)Bacto-Agar 在LB-中
    通过高压灭菌消毒。
  6. LB培养基
    1%(w/v)胰蛋白胨
    0.5%(w/v)酵母提取物
    171 mM NaCl pH 7.4
    通过高压灭菌消毒。

致谢

该方案改编自我们的论文Grewe等人。(2012)。这项工作是由德国研究基金会(DFG)授予KlausÜberla(Ue45/11-1)资助的。 Bianca Hoffmann和Bastian Grewe得到了DFG研究生院(GRK 1045)的支持。在Bianca Hoffmann和Bastian Grewe,Katrin Ehrhardt,Maik Blissenbach,Sabine Brandt,KlausÜberla,Alexander Stang,Thomas Grunwald,Klaus Sure和Bettina Tippler之外,他们都是团队的成员。

参考文献

  1. Grewe,B.,Ehrhardt,K.,Hoffmann,B.,Blissenbach,M.,Brandt,S.和Uberla,K。(2012)。 HIV-1 Rev蛋白增强未剪接和剪接的含有RRE的慢病毒载体RNA的衣壳化, a>。 PloS One 7(11),e48688。
  2. 他(2011a)。 从 E的质粒DNA提取。大肠杆菌使用碱裂解法。 生物协议 1(3):e30。
  3. 他(2011b)。 标准DNA克隆。 生物协议 1(7):e52
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引用:Hoffmann, B. and Grewe, B. (2013). Detection and Cloning of Spliced Transcripts by RT-PCR. Bio-protocol 3(8): e486. DOI: 10.21769/BioProtoc.486.
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