1、PCR Amplification of Highly GC-rich Regions 高GC 含量DNA的PCR 擴增 ?1高GC 含量DNA序列擴增困難生物中廣泛存在高GC 含量DNA序列,擴增的DNA有的含70%-80%高GC,常規PCR條件下,難擴增,原因:易形成復雜的二級結構, 防止DNA模板變性,DNA聚合酶難以結合.2高GC 含量DNA序列擴增常遇到的問題1.常規PCR. 影響PCR產物合成.多重 PCR. 偏好低GC區擴增導致PCR產物比例失衡.定量 PCR.富GC區易與內標形成異源雙鏈核酸分子,導致PCR結果解釋錯誤.DNA測序. DNA聚合酶趨向于在富GC區停留使測序難以進

2、行.cDNA合成. 合成的是缺GC區的短的cDNA片段.3高GC 含量DNA序列擴增方法1.模板DNA變性處理 1) NaOH: 2)核苷酸類似物: dITP脫氧黃嘌呤, dGTP(7-脫氮- 2-脫氧鳥苷-5-三磷酸) 3)有機添加劑:二甲基亞砜（DMSO）、甲酰胺、 甜菜堿(betaine）、甘油、四甲基亞砜、酰胺、 聚已二醇（PEG) 2.使用熱啟動聚合酶和高Tm引物 它們的特異性比普通 Taq polymerases高,如. Tag 加 Pfu, Deep Vent, 4NaOH模板DNA在堿性溶液里不被降解,但一定濃度的堿性溶液可以破壞DNA高級結構,使DNA變性,變性的DNA在PC

3、R反應中容易與引物結合(退火),也容易延伸.模板DNA處理:1ml里含有0.4mol/LNaOH和0.4mmol/LEDTA(乙二胺四乙酸 ) 室溫10分鐘3MNaAc調pH 乙醇沉淀 PCR反應5DNA segments with very high GC content have proved difficult to handle in a wide range of molecular analyses. GC-rich regions may form rigid, constrained secondary structures that are difficult or impo

4、ssible for the DNA polymerases to enter under standard PCR conditions. Why is it difficult for DNA segments with GC-rich to be amplified ?6Highly GC-rich regions can obstruct PCR-dependent analyses in the following situations:1.Standard PCR amplification. Highly GC-rich regions prevent template dena

5、turation, and hence product synthesis.2.Multiplex PCR amplification. The preferential amplification of low-GC-content sequences results in misinterpretation of the balance between the two sequences.7多重 PCR (MPCR) 或復合PCR，它是在同一PCR反應體系里加上兩對以上引物，同時擴增出多個核酸片段的PCR反應，其反應原理，反應試劑和操作過程與一般PCR相同。所有引物對在統一指定的反應條件下

6、進行擴增。多重PCR Multiplex PCR8electrophoresisprimers12341234多重PCR（復合PCR）用于檢測特定基因序列的存在或缺失。9Highly GC-rich regions can obstruct PCR-dependent analyses in the following situations:3.Quantitative PCR amplification. GC -rich targets may form heteroduplexes (異源雙鏈核酸分子) with internal standards, which are designe

7、d to be very similar to target sequences, leading to misinterpretation of results.10Highly GC-rich regions can obstruct PCR-dependent analyses in the following situations:4.DNA sequencing. DNA polymerase tends to stall in GC-rich regions, which results in compression and difficulty in interpreting t

8、hese parts of the sequence.5.cDNA synthesis. cDNA synthesis of GC-rich templates may create shorter fragments that lack the GC-rich portions of the sequence. The result is a skewed(歪斜的) representation of the original mRNA molecules.11How to overcome these problemsOver the years, different approaches

9、 have been used to overcome the problems caused by secondary structure.Hot-start Taq polymerases have been especially designed to function only after an extended initial denaturing step at high temperature. The specificity(特異性) of these polymerases is higher than that of standard Taq polymerases. 12

10、To overcome the problem of DNA sequence compressions, the template can be denatured using either sodium hydroxide(NaOH) or the nucleotide analogs deoxyinositol triphosphate (dITP脫氧黃嘌呤), and 7-deaza GTP(7-脫氮-2-脫氧鳥苷-5-三磷酸)could partly substitute the dGTP. How to overcome these problems13Organic additi

11、ves such as dimethylsulfoxide (DMSO), formamide(甲酰胺), betaine(甜菜堿, 三甲銨乙內酯), glycine(甘氨酸), lowmolecular-weight sulfones that are chemically related to DMSO (e.g., tetramethylene sulfoxide四甲基亞砜), and, more recently, low-molecular-weight amides(酰胺) have all proved successful, to some degree, in solving

12、 the problems associated with highly constricted (收縮的)DNA and RNA structures.14BETAINE甜菜堿擴增增強劑甜菜堿Betaine (N,N,N, -trimethylglycine) 是氨基酸類似物和膽堿的主要代謝物，存在于肝和腎，調節植物的滲透壓。體內可保護蛋白質，體外可促進蛋白質的折疊. betaine 加到 PCR 混合液里可保護Taq polymerase 免被高溫變性傷害, 還能促進DNA-protein復合物的穩定，保證擴增的效率.高濃度的betaine能消除AT區和 GC 區熔解溫度的差異，但不改變雙

13、鏈B-型 DNA 的構型.15BETAINEBetaine (N,N,N-trimethylglycine) is an amino acid analog and a major metabolite代謝物 of choline (膽堿) metabolism, which is present in liver and kidney cells. Betaine is the major regulator of osmotic(滲透性的) pressure in plants, a role that serves to protect proteins in vivo and faci

14、litates refolding of proteins in vitro. 16BETAINEAddition of betaine to a PCR mix protects the Taq polymerase from denaturation during the high-temperature steps of the cycles, thereby maintaining the efficiency of the enzyme during amplification. 17BETAINEBetaine is a zwitterion(tsvitrai n兩性離子) at

15、neutral pH and, even at high concentrations (5 M), it has property facilitates the maintenance of stable DNAprotein complexes.18BETAINE High concentrations of betaine eliminate the differences in melting temperature between AT and GC domains, without changing the double-stranded DNA conformation of

16、the B form. 19LOW-MOLECULAR-WEIGHT SULFOXIDES, AMIDES, AND GLYCINES低分子量的亞砜、酰胺和甘氨酸In the search for more potent amplification enhancers for highly GC-rich templates, several classes of low-molecular-weight compounds, sulfoxides(亞砜) and amides(酰胺), have been analyzed. 20LOW-MOLECULAR-WEIGHT SULFOXIDES

17、, AMIDES, AND GLYCINES低分子量的砜、酰胺和甘氨酸Among the sulfoxides, tetramethylene sulfoxide(四甲基亞砜) and tetramethylene四亞甲基 sulfolane（環丁砜 ） were found to efficiently enhance the amplification of templates with GC contents from 52% to 73%. 21LOW-MOLECULAR-WEIGHT SULFOXIDES, AMIDES, AND GLYCINES低分子量的砜、酰胺和甘氨酸Aceta

18、mide(乙酰胺) , N,N-dimethylglycine, N-monomethylglycine (sarcosine肌氨酸), and trimethylamine N-oxide (三甲胺-N-氧化物TMANO), which are structurally similar to betaine, have been analyzed for their effect on T7 polymerase sequencing of supercoiled DNA. 22Enhencers 擴增增強劑Betaine proved to be the most efficient fa

19、cilitator, followed by TMANO, and then by N,N-dimethylglycine, which was intermediate in activity. Sarcosine(肌氨酸) showed only a minor ability to overcome the secondary structures of the DNA template. 23低分子量的砜、酰胺和甘氨酸擴增增強劑Betaine已經證明是最強的增強劑，其次是 TMANO(三甲胺-N-氧化物，與Betaine結構相似）、乙酰胺， N,N-二甲基甘氨酸。肌氨酸對克服DNA二級

20、結構僅有較小的能力. 24BETAINE APPLICATIONS甜菜堿的應用 DNA Sequencing DNA 測序 Multiplex PCR Amplification 多重PCR擴增 Reverse Transcription PCR 反轉錄PCR Quantitative PCR 定量PCR Microarray Studies 芯片研究25DNA Sequencing DNA 測序DNA regions with high melting temperatures, or with the consensus sequence of 嘧啶Py-G-C, may cause DN

21、A polymerases to stall, resulting in compression of the DNA sequencing ladder and difficulty in the correct interpretation of the sequence . 高Tm的或有Py-G-C(Pyrimidine 嘧啶-鳥嘌呤-胞嘧啶)共有序列的DNA能夠停止 DNA 聚合酶,導致難以正確測序 .26DNA Sequencing DNA 測序The addition of 2 M betaine 甜菜堿 to these difficult sequencing reaction

22、s can eliminate the problem more efficiently. The efficiency of TMANO, used at 0.25-2.0 M, was similar to that of betaine in these sequencing reactions. 測序反應里加2 M betaine 或0.25-2.0 M TMANO(三甲胺-N-氧化物)能有效解決這個問題。27Multiplex PCR Amplification多重PCR擴增GC-Rich fragments may be strongly underrepresented afte

23、r standard multiplex PCR amplifications. The addition of 1 M betaine and 5% (vlv) DMSO may equalize the amplification efficiency of different DNA fragments. 正常情況下,富GC區的多重 PCR擴增受抑制. 加 1 M betaine 和 5% (vlv) DMSO 能夠平衡不同 DNA 片段的擴增效率. 28Reverse Transcription PCR反轉錄PCRAddition of 2 M betaine alone, or ac

24、companied by 0.6 M trehalose(海藻糖), can dramatically enhance reverse transcriptase reactions. For example, in the presence of these additives, the yield of 12.5-kb cDNA fragments was increased 9-fold, as compared with reverse transcriptase (RT)-PCR amplifications without additives. 單加 2 M betaine alo

25、ne, 或同時加 0.6 M 海藻糖能大大促進反轉錄反應. 例如加增強劑的比不加的(RT)-PCR會使12.5-kb cDNA 量增加 9倍.29Quantitative PCR 定量PCRInternal standards（內標） for use in quantitative PCR amplifications are designed to be very similar to the target molecule. Target and the internal standard formed heteroduplexes, even after the first amplif

26、ication cycle. Addition of 2 M betaine, 5% formamide(甲酰胺), and 10% DMSO to the PCR mix reduced the amount of recombinants to below a detectable level.內標與目標分子非常相似,兩者容易形成異源二聚體,這甚至在第一輪擴增后就可以發生. 加入 2 M betaine, 5% 甲酰胺, 和 10% DMSO 到 PCR反應液里可以將異源二聚體的量減少到檢測線以下. 30Microarray Studies 芯片研究 In addition to its

27、use in enhancing amplification of GC-rich sequences, betaine proved useful in microarray studies. The addition of 1.5 M betaine to the DNA solution results in a significant reduction of the nonspecific background signal. betaine 除了能促進富GC序列的擴增, 在芯片研究方面也是有用的. 加 1.5 M betaine 到 DNA 溶液里會大大削減非特異的背景信號.31M

28、icroarray Studies 芯片研究 Betaine reduces the evaporation(蒸發作用) from the DNA samples in the microtiter plates during the manufacturing of the slides. After the DNA has been applied to the glass slide, the diminished evaporation may provide enough time for the DNA to be distributed uniformly within each

29、 spot, thereby inhibiting the “doughnut effect（空巢）” that disturbs the results.Betaine 能夠減少芯片制作中微量滴定板里DNA樣本的蒸發作用,保證有充足的時間讓DNA均勻地分布到每一個點, 從而抑制干擾結果的“空殼效應”.32The following procedure details the establishment of an amplification procedure for GC -rich sequences. In this example, three amplicons from two

30、different genes containing GC-rich sequences were used. PROTOCOL 1:ESTABLISHING AN AMPLIFICATION PROCEDURE FORHIGHLY CC-RICH REGIONS草案1：高GC 含量DNA序列擴增步驟33PROTOCOL 1:ESTABLISHING AN AMPLIFICATION PROCEDURE FORHIGHLY CC-RICH REGIONS 高GC 含量DNA序列擴增草案34Fragment 1 comprised the initial part of exon 1 from

31、the human release factor 3 (GSPT1/hRF3), a 387-bp sequence that contains two trinucleotide repeats: a (GGC) followed by an (AGC), and a total GC content of 75%. The second and third fragments come from the Klotho gene (LocusLink number for the genes Klotho 1: 9365 and GSPTL/hRF3: 2935). Fragment 2 c

32、omprised the 5 end of Klotho exon 1 (375 bp, 81% GC) and fragment 3, the 3 part of Klotho exon 1 (350 bp, 70% GC). 三個擴增子,來自兩個不同的富GC 含量基因片段 1: 來自人釋放因子 3 (GSPT1/hRF3),含 exon 1 起始部分,長 387-bp,其中有兩個三核苷酸重復”GGC后AGC”,總GC 含量 75%. 片段 2,3:來自Klotho基因 (LocusLink 號 Klotho 1: 9365 and GSPTL/hRF3: 2935).片段 2含 Kloth

33、o exon 1 的 5 端 (375 bp, 81% GC);片段 3含 Klotho exon 1 的 3 端 (350 bp, 70% GC). 35MATERIALS 材料BUFFERS, SOLUTIONS溶液, AND REAGENTS試劑BetainedNTP solution all four dNTPs, (each containing at 25 mM)Dimethyl sulfoxide (DMSO)Tetramethylene sulfone (Sulfolane環丁砜 )36ENZYMES AND ENZYME BUFFERSTaq polymerasePolyme

34、rase buffer (as supplied by enzyme manufacturer)NUCLEIC ACIDS AND OLIGONUCLEOTIDESHuman genomic DNA, 20 ng per amplification reaction模板與引物人基因組 DNA37hRF3 (hGSPTL) primers:hRF3f: CCGCCTCTGTCGTCGTCGChRF3r: CCGCGCTGAGGTTCTCCCKb 1 af: CTCGCAGGTAATrATrGCCAGKb lar: GATGGACGCACCCTGKb 1 cf: GTGCAGCCCGTGGTCAC

35、Kb icr: GACTCAGITCCCACACTTC3839Depending on the chosen conditions, include either DMSO (5%); betaine (1 M); betaine (2 M); betaine and DMSO (1 M and 5%, respectively); betaine and DMSO (2 M and 5%, respectively); tetramethylene sulfone (0.4 M); or no enhancers (final concentrations) in the PCR mix.條

36、件設定: PCR反應液里加 DMSO (5%); betaine (1 M); betaine (2 M); betaine 和 DMSO (1 M和5%); betaine 和 DMSO (1.5 M 和5%); PCR反應液里不加增強劑. 4041The results show that the optimal yield of the PCR product was seen with 5% DMSO and 1.5 M betaine as shown in Figure 4.結果加5% DMSO和1.5 M betaine 的PCR產物最好.42Some advice on solving the problems associated with GC-rich templates (50%GC).解決高于50%GC模板擴增問題的忠告Add 1.5 M betaine and 5% (v/v) DMSO to the PCR amplification.If no effect is seen, increase the betaine concentration to 2.5 M, add 10% (vlv) DMSO, and double the amount of Taq polymerase (DMSO inhibits Taq