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Telomerase Repeated Amplification Protocol (TRAP)
端粒酶重复扩增实验(TRAP)   

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Abstract

Telomeres are found at the end of eukaryotic linear chromosomes, and proteins that bind to telomeres protect DNA from being recognized as double-strand breaks thus preventing end-to-end fusions (Griffith et al., 1999). However, due to the end replication problem and other factors such as oxidative damage, the limited life span of cultured cells (Hayflick limit) results in progressive shortening of these protective structures (Hayflick and Moorhead, 1961; Olovnikov, 1973). The ribonucleoprotein enzyme complex telomerase- consisting of a protein catalytic component hTERT and a functional RNA component hTR or hTERC- counteracts telomere shortening by adding telomeric repeats to the end of chromosomes in ~90% of primary human tumors and in some transiently proliferating stem-like cells (Shay and Wright, 1996; Shay and Wright, 2001). This results in continuous proliferation of cells which is a hallmark of cancer. Therefore, telomere biology has a central role in aging, cancer progression/metastasis as well as targeted cancer therapies. There are commonly used methods in telomere biology such as Telomere Restriction Fragment (TRF) (Mender and Shay, 2015b), Telomere Repeat Amplification Protocol (TRAP) and Telomere dysfunction Induced Foci (TIF) analysis (Mender and Shay, 2015a). In this detailed protocol we describe Telomere Repeat Amplification Protocol (TRAP).
The TRAP assay is a popular method to determine telomerase activity in mammalian cells and tissue samples (Kim et al., 1994). The TRAP assay includes three steps: extension, amplification, and detection of telomerase products. In the extension step, telomeric repeats are added to the telomerase substrate (which is actually a non-telomeric oligonucleotide, TS) by telomerase. In the amplification step, the extension products are amplified by the polymerase chain reaction (PCR) using specific primers (TS upstream primer and ACX downstream primer) and in the detection step, the presence or absence of telomerase is analyzed by electrophoresis. TSNT is, an internal standard control, amplified by TS primer. NT is its own reverse primer, which is not a substrate for telomerase. These primers are used to identify false-negative results by if the gel lacks internal control bands.

Keywords: Replicative senescence(复制性衰老), Telomeres(端粒), Cancer(癌症), Stem cells(干细胞), PCR(PCR)

Materials and Reagents

  1. Cancer cells (H1299 non-small cell lung, A549 non-small cell lung cancer cells)
  2. Tris-HCl (pH 8.3 and pH 8.0)
  3. Magnesium Chloride (MgCl2) (Thermo Fisher Scientific, catalog number: BP241 )
  4. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
  5. TweenTM 20 (Thermo Fisher Scientific, catalog number: BP337 )
  6. Ethylene glycol-bis(2-aminoethylether)-N, N, N′, N′-tetraacetic acid EGTA (Sigma-Aldrich, catalog number: E3889 )
  7. Ethylenediaminetetraacetic Acid (EDTA) (Thermo Fisher Scientific, catalog number: BP-120 )
  8. Nonidet-P40 (Fluka BioChemika, catalog number: 74385 )
    Note: Currently, it is “Sigma-Aldrich, catalog number: 74385 ”.
  9. Glycerol (Sigma-Aldrich, catalog number: G5516 )
  10. 2-mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
  11. 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) (Sigma-Aldrich, catalog number: A8456 )
  12. dNTP (Invitrogen)
  13. Cy5-TS primer (Sigma-Aldrich)
  14. ACX (reverse primer) (Sigma-Aldrich)
  15. TSNT (36-bp internal standard control) (Integrated DNA Technologies)
  16. NT (reverse primer for internal standard) (Sigma-Aldrich)
  17. Takara Taq DNA polymerase, hot start version (Takara Bio Company, ClonTech, catalog number: R007A )
  18. 40% acrylamide and bis-acrylamide solution (19:1) (Bio-Rad Laboratories, catalog number: 161-0144 )
  19. Ammonium persulfate (APS) (Bio-Rad Laboratories, catalog number: 161-0700 )
  20. N, N, N’, N’-Tetramethylethylenediamine (TEMED) (Sigma-Aldrich, catalog number: T9281 )
  21. Primer mix (see Recipes)
  22. Cy5-TS primer (see Recipes)
  23. 10x TRAP reaction buffer (see Recipes)
  24. 50x dNTP (see Recipes)
  25. NP-40 lysis buffer (see Recipes)

Equipment

  1. Polymerase Chain Reaction (PCR) Thermo Cycler (Bio-Rad Laboratories, model: PTC-1148 )
  2. Typhoon PhosphorImager® scanner system (Amersham Biosciences, GE Healthcare, model: Typhoon TRIO )

Software

  1. ImageQuant Software (Molecular Dynamics)

Procedure

  1. Prepare cell lysates
    1. Collect 100,000 cells into a DNase/RNase -free microfuge tube.
    2. Centrifuge cells at 3,000 x g for 5 min.
    3. Remove the supernatant.
      Note: The pellet doesn’t need to be washed. If samples are to be used at a later time point, put in -80 °C.
    4. Resuspend cell pellet on ice-cold NP-40 lysis buffer at a concentration of 2,500 cells per μl (40 μl lysis buffer for 100,000 cells).
    5. Leave on ice for 30 min or snap-freeze cell lysates in liquid nitrogen and then place at -80 °C.

  2. Preparation for PCR-Cy5 fluorescent gel-based TRAP
    1. Keep samples on ice.
    2. Choose a positive control (i.e. HCT116, H1299) and prepare three different serial dilutions (1:10) in NP-40.
    3. Prepare the TRAP master mix. Final volume of the PCR reaction is 50 μl.
      Master mix for one sample:
      H2O
      40.2 μl
      Trap buffer
      5 μl
      dNTP
      1 μl
      Cy5-TS primer
      1 μl
      Primer mix
      1 μl
      BSA
      0.4 μl
      Taq polymerase
      0.4 μl
    4. Add 1 μl of sample to 49 μl of the master mix.
      Note: For negative control, 1 μl NP-40 to 49 μl of the master mix.

  3. Amplification of the extension products by PCR
    1. 25 °C for 40 min for extension.
    2. 95 °C for 5 min to deactivate telomerase.
    3. 24 to 29 cycles at:
      95 °C for 30 sec
      52 °C for 30 sec
      72 °C for 45 sec
    4. 72 °C for 10 min
    5. 4 °C for up to five days
      Note: Samples can be stored at -20 °C for 1 month.

  4. Running acrylamide gel
    1. Add 5 μl of loading dye to each sample.
    2. Making the 10% nondenaturing acrylamide gel.
      32.5 ml H2O (MilliQ® water)
      12.5 ml 40% acrylamide (19:1 acrylamide:bisacrylamide)
      5 ml 5x TBE
      250 μl 10% APS
      50 μl TEMED
    3. 0.5x TBE running buffer
    4. Running time is about 2.5 h at 200/220 volts.
    5. Load 25 μl of the sample to each well.
    6. Visualize using Typhoon® that can read Cy5 fluorescein.


      Figure 1. TRAP gels show partial telomerase inhibition with 10 μM GRN163L in A549 non small cell lung cancer cells for 72 h. It is known that a lipid modified N3→P5 thio-phosphoramidate oligonucleotide, GRN163L (also referred to as Imetelstat), inhibits telomerase more potently than its parental non-conjugated thio-phospharamidate sequence, GRN163, in cancer cells (Herbert et al., 2005). This figure also shows that GRN163L (L= lipidated with palmitoyl C16) inhibits A549 cells more potently compared to GRN163. H1299 non-small lung cancer cells were used with three different dilutions as a positive control. 2500, 250 and 25 cells for H1299 were used in lane 1, lane 2 and lane 3, respectively. No cells were added in lysis buffer in lane 4, which is a negative control (A). ImageQuant Software was used to determine the intensity of the telomerase products (6 bp-ladder) and the ITAS (Internal Standard Control, 36 bp) band. The ratio of intensity levels of TRAP sample ladders and ITAS band (intensity of sample’s TRAP ladder/intensity of ITAS band) was calculated and each sample was normalized to the positive control as a percentage (positive control; H1299 2500 cells) after background subtraction (B).

Recipes

  1. Primer mix
    Primer mix (50X) includes the reverse primer (ACX), the substrate for 36-bp internal standard control (TSNT), and the reverse primer for the internal standard (NT) or ITAS (internal telomerase activity standard).
    Sequences:



    Stock
    Final concentration
    ACX
    1.0 μg/μl
    100 ng/μl
    NT
    1.0 μg/μl
    100 ng/μl
    TSNT
    1.0 attomol/μl
    0.01 attomol/μl
    Notes:
    1. It is highly recommended to purchase TSNT oligo from a company other than where the ACX and NT primers are bought. This helps prevention of the contamination of TSNT with ACX and NT.
    2. Preparation of stock TSNT should be done in a separate room from the TRAP bench and use a pipetman that is not used in the TRAP area.
    3. An attomol is 10-18 mol.

    Preparation of stock TSNT:
    Dilute the dry TSNT oligonucleotide to 100 μM concentration with DEPC treated water. Then prepare the serial dilutions (1:1,000, 1:1,000, 1:100); 100 nM, 100 pM and 1 pM. So, the final concentration of the TSNT stock will contain a 100x stock of TSNT (1.0 attomol/μl).
    Note: To get the correct ITAS signal, you can adjust the amount of 100x stock of TSNT. For example, try a variety of dilutions of 100x stock of TSNT such as 1,000x, 300x, 100x, and 30x and see how the ITAS looks on TRAP ladder.

    Preparation of primer mix:
    1. Mix ACX and NT primers together with water.
    2. Move to the area where the TSNT was prepared and add the TSNT to the mix.
    3. Clean the outside of the tubes and rack with diluted bleach (spray 10% bleach on the tubes, then dry them with paper towel or spray 10% bleach on paper towel and clean the outside of the tubes).
    4. Return to the TRAP area with the tube and prepare aliquots to store at -20 °C up to 1 year.

  2. Cy5-TS primer
    TS oligo is purchased modified with Cy5 on the 5’end (HPLC or PAGE purified). Final concentration of TS primer is 100 ng/μl (diluted in DEPC water).
    Sequence: 5’-AAT CCG TCG AGC AGA GTT-3’
  3. 10x TRAP reaction buffer

    Final concentration
    Tris-HCl (pH 8.3)
    200 mM
    MgCl2
    15 mM
    KCl
    630 mM
    Tween 20
    0.5% (v/v)
    EGTA
    10 mM

  4. 50x dNTP
    Use a mix containing all four DNA nucleotides (dTTP, dATP, dCTP, dGTP) at an equivalent and final concentration of 2.5 mM
  5. NP-40 lysis buffer

    Final concentration
    Tris-HCl (pH 8.0)
    10 mM
    MgCl2
    1 mM
    EDTA
    1 mM
    NP-40
    1% (v/v)
    Sodium deoxycholate
    0.25 mM
    Glycerol
    10% (v/v)
    NaCl
    150 mM
    2-mercaptoethanol
    5 mM
    AEBSF
    0.1 mM

Acknowledgments

Some of these protocols were adapted from previously published studies. Some of TRAP protocols are referenced here (Herbert et al., 2003; Norton et al., 1998; Wright et al., 1995). We thank Zeliha Gunnur Dikmen for her help in acquisition of TRAP gel and Abhijit Bugde from the Live Cell Imaging Facility at UT Southwestern for his assistance with the imaging and analysis part of Telomere dysfunction Induced Foci (TIF) analysis.

References

  1. Griffith, J. D., Comeau, L., Rosenfield, S., Stansel, R. M., Bianchi, A., Moss, H. and de Lange, T. (1999). Mammalian telomeres end in a large duplex loop. Cell 97(4): 503-514.
  2. Hayflick, L. and Moorhead, P. S. (1961). The serial cultivation of human diploid cell strains. Exp Cell Res 25: 585-621.
  3. Olovnikov, A. M. (1973). A theory of marginotomy. The incomplete copying of template margin in enzymic synthesis of polynucleotides and biological significance of the phenomenon. J Theor Biol 41(1): 181-190.
  4. Shay, J. W. and Wright, W. E. (1996). Telomerase activity in human cancer. Curr Opin Oncol 8(1): 66-71.
  5. Shay, J. W. and Wright, W. E. (2001). Telomeres and telomerase: implications for cancer and aging. Radiat Res 155(1 Pt 2): 188-193.
  6. Kim, N. W., Piatyszek, M. A., Prowse, K. R., Harley, C. B., West, M. D., Ho, P. L., Coviello, G. M., Wright, W. E., Weinrich, S. L. and Shay, J. W. (1994). Specific association of human telomerase activity with immortal cells and cancer. Science 266(5193): 2011-2015.
  7. Herbert, B. S., Gellert, G. C., Hochreiter, A., Pongracz, K., Wright, W. E., Zielinska, D., Chin, A. C., Harley, C. B., Shay, J. W. and Gryaznov, S. M. (2005). Lipid modification of GRN163, an N3'-->P5' thio-phosphoramidate oligonucleotide, enhances the potency of telomerase inhibition. Oncogene 24(33): 5262-5268.
  8. Herbert, B. S., Shay, J. W. and Wright, W. E. (2003). Analysis of telomeres and telomerase. Curr Protoc Cell Biol Chapter 18: Unit 18 16.
  9. Mender, I. and Shay, J. W. (2015a). Telomere dysfunction induced foci (TIF) analysis. Bio-protocol 5(22): e1656.
  10. Mender, I. and Shay, J. W. (2015b). Telomere restriction fragment (TRF) analysis. Bio-protocol 5(22): e1658.
  11. Norton, J. C., Holt, S. E., Wright, W. E. and Shay, J. W. (1998). Enhanced detection of human telomerase activity. DNA Cell Biol 17(3): 217-219.
  12. Wright, W. E., Shay, J. W. and Piatyszek, M. A. (1995). Modifications of a telomeric repeat amplification protocol (TRAP) result in increased reliability, linearity and sensitivity. Nucleic Acids Res 23(18): 3794-3795.

简介

端粒存在于真核线性染色体的末端,并且结合端粒的蛋白质保护DNA不被识别为双链断裂,从而防止端对端融合(Griffith等人,1999) 。然而,由于末端复制问题和其他因素如氧化损伤,培养细胞的有限寿命(Hayflick极限)导致这些保护结构的逐渐缩短(Hayflick和Moorhead,1961; Olovnikov,1973)。由蛋白质催化组分hTERT和功能性RNA组分hTR 或组成的核糖核蛋白酶复合物端粒酶端粒酶通过添加端粒重复来抵消端粒缩短到约90%的原发性人类肿瘤和一些短暂增殖的干样细胞中染色体的末端(Shay和Wright,1996; Shay和Wright,2001)。这导致细胞的连续增殖,这是癌症的标志。因此,端粒生物学在衰老,癌症进展/转移以及靶向癌症治疗中具有中心作用。端粒生物学中常用的方法如端粒限制性片段(TRF)(Mender和Shay, 2015b),端粒重复扩增方案(TRAP)和端粒功能障碍诱导Foci(TIF)分析(Mender和Shay,2015a)。在这个详细的协议中,我们描述端粒重复扩增协议(TRAP)。
TRAP测定法是确定哺乳动物细胞和组织样品中端粒酶活性的常用方法(Kim等人,1994)。 TRAP测定包括三个步骤:扩增,扩增和检测端粒酶产物。在延伸步骤中,通过端粒酶将端粒重复加入到端粒酶底物(其实际上是非端粒寡核苷酸,TS)中。在扩增步骤中,通过使用特异性引物(TS上游引物和ACX下游引物)的聚合酶链反应(PCR)扩增延伸产物,并且在检测步骤中,通过电泳分析端粒酶的存在或不存在。 TSNT是内标物对照,通过TS引物扩增。 NT是其自身的反向引物,其不是端粒酶的底物。这些引物用于鉴定凝胶是否缺少内部对照带的假阴性结果。

关键字:复制性衰老, 端粒, 癌症, 干细胞, PCR

材料和试剂

  1. 癌细胞(H1299非小细胞肺,A549非小细胞肺癌细胞)
  2. Tris-HCl(pH 8.3和pH 8.0)
  3. 氯化镁(MgCl 2)(Thermo Fisher Scientific,目录号:BP241)
  4. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9541)
  5. Tween TM(Thermo Fisher Scientific,目录号:BP337)
  6. 乙二醇 - 双(2-氨基乙醚)-N,N,N',N'-四乙酸EGTA(Sigma-Aldrich,目录号:E3889)
  7. 乙二胺四乙酸(EDTA)(Thermo Fisher Scientific,目录号:BP-120)
  8. Nonidet-P40(Fluka BioChemika,目录号:74385)
    注意:目前,它是"Sigma-Aldrich,目录号:74385"。
  9. 甘油(Sigma-Aldrich,目录号:G5516)
  10. 2-巯基乙醇(Sigma-Aldrich,目录号:M3148)
  11. 4-(2-氨基乙基)苯磺酰氟盐酸盐(AEBSF)(Sigma-Aldrich,目录号:A8456)
  12. dNTP(Invitrogen)
  13. Cy5-TS引物(Sigma-Aldrich)
  14. ACX(反向引物)(Sigma-Aldrich)
  15. TSNT(36-bp内标控制)(Integrated DNA Technologies)
  16. NT(内标用反向引物)(Sigma-Aldrich)
  17. Takara Taq DNA聚合酶,热启动型(Takara Bio Company,ClonTech,目录号:R007A)
  18. 40%丙烯酰胺和双丙烯酰胺溶液(19:1)(Bio-Rad Laboratories,目录号:161-0144)
  19. 过硫酸铵(APS)(Bio-Rad Laboratories,目录号:161-0700)
  20. N,N,N',N'-四甲基乙二胺(TEMED)(Sigma-Aldrich,目录号:T9281)
  21. 底漆混合物(见配方)
  22. Cy5-TS引物(参见配方)
  23. 10x TRAP反应缓冲液(见配方)
  24. 50x dNTP(请参阅配方)
  25. NP-40裂解缓冲液(见配方)

设备

  1. 聚合酶链反应(PCR)热循环仪(Bio-Rad Laboratories,型号:PTC-1148)
  2. Typhoon PhosphorImager 扫描仪系统(Amersham Biosciences,GE Healthcare,型号:Typhoon TRIO)

软件

  1. ImageQuant软件(分子动力学)

程序

  1. 准备细胞裂解液
    1. 将100,000个细胞收集到DNase/RNase-free微量离心管中
    2. 离心细胞在3,000×g离心5分钟。
    3. 取出上清液。
      注意:颗粒不需要洗涤。如果要在稍后的时间点使用样品,请放在-80°C。
    4. 重悬细胞沉淀在冰冷的NP-40裂解缓冲液中 浓度为2,500个细胞/μl(40μl裂解缓冲液为100,000 细胞)。
    5. 在冰上放置30分钟或在液氮中快速冷冻细胞裂解物,然后置于-80℃

  2. PCR-Cy5荧光凝胶型TRAP的制备
    1. 将样品保存在冰上。
    2. 选择阳性对照(即 HCT116,H1299),并在NP-40中制备三种不同的系列稀释液(1:10)。
    3. 准备TRAP主混合。 PCR反应的最终体积为50μl 一个样品的主混合:
      H sub 2 O
      40.2μl
      陷阱缓冲区
      5微升
      dNTP
      1微升
      Cy5-TS引物
      1微升
      底漆混合物
      1微升
      BSA
      0.4μl
      Taq聚合酶
      0.4μl
    4. 将1μl样品加入49μl主混合液中 注意:对于阴性对照,1μlNP-40至49μl的主混合物。

  3. 通过PCR扩增延伸产物
    1. 25℃延伸40分钟。
    2. 95℃5分钟以钝化端粒酶。
    3. 24到29个周期,在:
      95°C 30秒
      52℃30秒
      72℃45秒
    4. 72℃10分钟
    5. 4℃,最多5天
      注意:样品可以在-20°C保存1个月。

  4. 运行丙烯酰胺凝胶
    1. 每个样品加入5μl加载染料
    2. 制备10%非变性丙烯酰胺凝胶 32.5ml H 2 O(MilliQ水)
      12.5ml 40%丙烯酰胺(19:1丙烯酰胺:双丙烯酰胺) 5 ml 5XTBE
      250微升10%APS
      50μlTEMED
    3. 0.5x TBE运行缓冲区
    4. 在200/220伏下运行时间约为2.5小时。
    5. 每孔加入25μl样品。
    6. 使用可以读取Cy5荧光素的Typhoon ?进行可视化

      图1.TRAP凝胶显示10μM的部分端粒酶抑制 GRN163L在A549非小细胞肺癌细胞中72小时。已知 脂质修饰的N3→P5硫代 - 氨基磷酸酯寡核苷酸, GRN163L(也称为Imetelstat),更多地抑制端粒酶 比其亲本非缀合的硫代 - 磷酰胺酯序列有效, ?GRN163,在癌细胞中(Herbert等人,2005)。这个数字也显示 GRN163L(L =用棕榈酰C16脂化)抑制A549细胞更多 与GRN163相比。使用H1299非小细胞肺癌细胞 ?用三种不同稀释度作为阳性对照。 2500,250和25 H1299的细胞分别用于泳道1,泳道2和泳道3。没有 ?细胞加入到泳道4中的裂解缓冲液中,泳道4是阴性对照 ?(一个)。 ImageQuant软件用于确定 ?端粒酶产物(6bp梯)和ITAS的强度 (内标标准对照,36 bp)条带。强度水平的比率 的TRAP样品梯子和ITAS谱带(样品的TRAP强度 梯度/ITAS条带的强度),并且每个样品为 相对于阳性对照标准化为百分比(阳性对照; H1299 2500个细胞)(B)。

食谱

  1. 底漆混合物
    引物混合物(50X)包括反向引物(ACX),36-bp内标物对照底物(TSNT)和内标物(NT)或ITAS(内部端粒酶活性标准物)的反向引物。 顺序:



    库存
    最终浓度
    ACX
    1.0μg/μl
    100 ng /μl
    NT
    1.0μg/μl
    100 ng /μl
    TSNT
    1.0 attomol /μl
    0.01 attomol /μl
    注意:
    1. 强烈建议从其他公司购买TSNT oligo 比购买ACX和NT引物的位置。这有助于防止 用ACX和NT污染TSNT。
    2. 库存的准备 TSNT应在与TRAP台不同的房间中进行,并使用a 在TRAP区域中未使用的pipetman。
    3. < em> attomol是10-18mol。

    储备TSNT的准备:
    用DEPC处理的水稀释干燥的TSNT寡核苷酸至100μM浓度。然后准备系列稀释液(1:1,000,1:1,000,1:100); 100nM,100pM和1pM。因此,TSNT原液的最终浓度将包含100x的TSNT原液(1.0attomol /μl)。
    注意:要获得正确的ITAS信号,您可以调整TSNT的100x库存量。例如,尝试各种稀释度的100x TSNT样品,如1,000x,300x,100x和30x,并查看ITAS在TRAP梯形图上的外观。

    底漆混合物的制备:
    1. 将ACX和NT引物与水混合。
    2. 移动到准备TSNT的区域,并将TSNT添加到混合物中。
    3. 用稀释的漂白剂(喷雾剂)清洁管道和架子的外部 10%漂白剂在管上,然后用纸巾干燥或喷雾10% 漂白在纸巾上并清洁管的外侧)
    4. 返回到带有管的TRAP区,并准备等分试样在-20°C存储至1年

  2. Cy5-TS引物
    TS寡核苷酸购买在5'端用Cy5修饰(HPLC或PAGE纯化)。 TS引物的终浓度为100ng /μl(在DEPC水中稀释) 序列:5'-AAT CCG TCG AGC AGA GTT-3'
  3. 10x TRAP反应缓冲液

    最终浓度
    Tris-HCl(pH 8.3)
    200 mM
    MgCl 2
    15 mM
    KCl
    630 mM
    吐温20
    0.5%(v/v)
    EGTA
    10 mM

  4. 50x dNTP
    使用含有所有DNA核苷酸(dTTP,dATP,dCTP,dGTP)的混合物,其等效和终浓度为2.5mM
  5. NP-40裂解缓冲液

    最终浓度
    Tris-HCl(pH 8.0)
    10 mM
    MgCl 2
    1 mM
    EDTA
    1 mM
    NP-40
    1%(v/v)
    脱氧胆酸钠
    0.25 mM
    甘油
    10%(v/v)
    NaCl
    150 mM
    2-巯基乙醇 5mM
    AEBSF
    0.1 mM

确认

其中一些协议改编自以前发表的研究。在此引用了一些TRAP协议(Herbert等人,2003; Norton等人,1998; Wright等人,1995) 。我们感谢Zeliha Gunnur Dikmen帮助从UT西南的活细胞成像设备获得TRAP凝胶和Abhijit Bugde,他对端粒功能障碍诱导Foci(TIF)分析的成像和分析部分的帮助。

参考文献

  1. Griffith,J.D.,Comeau,L.,Rosenfield,S.,Stansel,R.M.,Bianchi,A.,Moss,H.and de Lange,T。(1999)。 哺乳动物端粒以大双链体环结束。 细胞 97(4):503-514。
  2. Hayflick,L。和Moorhead,P.S.(1961)。 人类二倍体细胞株的系列培养 Exp Cell Res < 25> 585-621。
  3. Olovnikov,A.M。(1973)。 边缘切除术的理论。在多核苷酸的酶合成中模板边缘的不完全复制和现象的生物学意义。 41 Theor Biol 41(1):181-190。
  4. Shay,J.W.and Wright,W.E。(1996)。 人类癌症中的端粒酶活性。Curr Opin Oncol 8 (1):66-71。
  5. Shay,J.W.and Wright,W.E。(2001)。 端粒和端粒酶:对癌症和衰老的影响 Radiat Res < em> 155(1 Pt 2):188-193
  6. Kim,N.W.,Piatyszek,M.A.,Prowse,K.R.,Harley,C.B.,West,M.D.,Ho,P.L.,Coviello,G.M.,Wright,W.E.,Weinrich,S.L.and Shay,J.W。(1994)。 人类端粒酶活性与永生细胞和癌症的特异性关系 /em> 266(5193):2011-2015。
  7. Herbert,B.S.,Gellert,G.C.,Hochreiter,A.,Pongracz,K.,Wright,W.E.,Zielinska,D.,Chin,A.C.,Harley,C.B.,Shay,J.W.and Gryaznov, GRN163(一种N3'→P5'硫代氨基磷酸酯寡核苷酸)的脂质修饰增强了端粒酶抑制的效力。癌基因24(33):5262-5268。
  8. Herbert,B.S.,Shay,J.W.and Wright,W.E。(2003)。 分析端粒和端粒酶。 Curr Protoc Cell Biol Chapter 18:Unit 18 16.
  9. Mender,I.和Shay,J.W。(2015a)。 端粒功能障碍诱导灶(TIF)分析 生物方案 5 22):e1656。
  10. Mender,I.和Shay,J. W.(2015b)。 端粒限制性片段(TRF)分析 B io- proto col 5(22):e1658。
  11. Norton,J.C.,Holt,S.E.,Wright,W.E.and Shay,J.W。(1998)。 增强人端粒酶活性检测 DNA Cell Biol 17(3):217-219。
  12. Wright,W.E.,Shay,J.W.and Piatyszek,M.A。(1995)。 端粒重复扩增方案(TRAP)的修饰导致可靠性,线性度和灵敏度的提高。 a> Nucleic Acids Res。23(18):3794-3795。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Mender, I. and Shay, J. W. (2015). Telomerase Repeated Amplification Protocol (TRAP). Bio-protocol 5(22): e1657. DOI: 10.21769/BioProtoc.1657.
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Dani Wicaksono
WTCCB - University of Edinburgh
Hi,

I am a little bit confused about the primer dilutions. It was shown in the protocol that ACX and NT primers stock concentration are both 1 ug/ul respectively. Do I need to dissolve the primers to 1 ug/ul for the stocks? The "final concentration" of ACX and NT primers is 100 ng/ul. Is this the final concentration of both primers in the 50X primer mix?If it is, does it mean that the final concentration of both primers in the TRAP reaction is 2 ng/ul? Lastly, It was stated that the concentration of TS primer is 100 ng/ul. Is the final concentration of TS primer in the TRAP reaction = 2 ng/ul? Thank you

Dani Wicaksono
11/22/2016 10:41:22 AM Reply
Jerry Shay
Department of Cell Biology, University of Texas Southwestern Medical Center, USA

the final primer concentration in the TRAP reaction is 2ng/uL.

The final primer concentrations are 100ng/ul in the primer mix. As listed in the table under 'recipes' the stock is 1ug/uL and the final in the primer mix is 100ng/uL. When 1ul of the primer mix is added to a 49 uL reaction (final volume of 50ul thus 100ng/50uL = 2ng/uL final primer concentration) the final concentration of primers is 2ng/uL in the trap extension/PCR.

The primers are used at lower concentration than typical PCR because of potential primer artifacts.

11/23/2016 7:28:31 AM