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Hybridization of Nucleic Acids
DNA1 DNA2 RNA Probe 核酸 變性後可再黏合復性，回復原來的雙股核酸。若取兩種不同來源的核酸，經變性後混合在一起，假如兩種核酸的序列同質性高，則可能會雜合為混成的雙股核酸。 也可使用已知序列的核酸小片段，作為探針 (probe) 與 DNA 雜合；一般可先把樣本 DNA 經電泳分離後，轉印至尼龍膜上，再以放射性探針進行雜合，稱之為 Southern hybridization。 RNA 也可以在電泳後，與其互補的 DNA 序列雜合，則稱為 Northern hybridization。 Northern hybridization Southern hybridization Juang RH (2004) BCbasics
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Preparation of Traditional Nucleic Acid Probe
Amino acid sequence GLY-ASP-GLU-SER-SER-VAL-LEU----- GGG-GAC-GAG-TCC-TCC-GTT-CTC--- Nucleic acid sequence * * * * * * * * Codon degeneracy The nucleic acid sequence is Deduced from amino acid sequence Synthesizing oligonucleotide Chemical synthesis 探針 的來源有很多，上圖舉出一例，說明可以由已知蛋白質的一小段胺基酸序列，回推其核苷酸序列，並依此序列進行人工合成探針。注意對應同一種胺基酸的核酸密碼，可能有數種，例如 Ser 就有 UCU, UCC, UCA, UCG , AGU, AGC 等六種可能，稱為密碼的不確定性 (codon degeneracy)。 這樣合成人工探針就會比較麻煩，有幾個對應之道： (1) 把所有可能的密碼全部合成，也就是說變成是一種混合物，各種可能的序列都有，因此精確度降低；(2) 調查該種生物的密碼使用習慣，並選擇較常使用的密碼，(3) 參考不同生物來源的同一個基因序列，抄襲所用的密碼。 PROBE: GGGGACGAGTCCTCCGTTCT Juang RH (2004) BCbasics
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DNA Target gene denaturation Single colony Lysed
Probe is labeled with radioactive 32P Hybridization DNA denaturation Target gene Single colony 這段 探針經放射性磷酸標定後，可用在 Southern blotting 上，與未知樣本中的核酸進行雜合反應。目標基因若在細菌中，則必須先把細菌溶解，然後以化學反應使核酸變性露出單股 DNA，則可與探針進行雜合反應。 Lysed Juang RH (2004) BCbasics
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Colony Is Screened by Hybridization with Probe
Colony hybridization Cover with filter paper Transferring … Autoradiography Collect filter paper Dissolve cell DNA denatured Add probe 探針 雜合反應的做法實例。 菌落在培養皿長出來之後，先以濾紙複印得各菌落，利用氯仿把細菌溶並解釋出核酸，然後變性解開核酸的雙股後，再加入標有放射線的探針，進行雜合反應。 若某菌落中含有目標基因，便會與探針雜合成功，該菌落的位置便會出現放射線影像；然後再回去挑起原來培養皿上面的菌落，大量培養後可純化目標基因。 本圖可接續 N1-27 頁的分子群殖，在培養基生長的各個菌落，可以用探針經雜合反應挑出所要的目標菌落。 Juang RH (2004) BCbasics
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Biochip Based on Hybridization
Sample DNA Complementary DNA hybridize Biochip 核酸 晶片的基本原理，就是利用類似 Southern blotting 的核酸雜合反應。但是比起一般的毛細管轉印雜合，有幾點不同：(1) 以極細微的色點替代電泳色帶；(2) 一次可以處理上千萬個核酸色點，因此對同一樣本可有上千萬個測試；(3) 樣本所需的量極小；(4) 偵測方式簡便快速，也配合電腦比對分析。 因此晶片看起來很炫，也有其實質的效用。但晶片最重要的靈魂，還是在那一公分見方的小玻片，上面所點的到底是何種 DNA。也就是說，每一個點上的 DNA 是什麼，才是最重要的決定因素；若這些 DNA 能夠明確指示某種關鍵性疾病的有無，則這塊晶片便是無價之寶。這些重要的 DNA 要從哪裡來？ 當然還是要經由無數的基礎研究，獲得對生物的深刻瞭解後，所得到的基本知識點滴。 因此，晶片製作過程是一種技術 (technology)，而晶片上必須點上何種 DNA，便要靠基礎科學 (science) 來提供，兩者是不一樣的，但絕對相輔相成。 其他很多方面也有類似的情形，例如大學裡的教學與研究必須並進，不能偏廢，大家都知道。 然而，目前台灣有個很嚴重的問題，可能會影響國本。那就是在研究上，大家只重視可以馬上應用的技術，不太支持長期的基礎科學研究；在大學裡，教授又只重視如何發表大量論文 (不管有沒有用)，不太花時間在教學的努力與改進上。 [Friend SH, Stoughton RB (2002 四月) 神奇的 DNA 晶片。科學人 2: 40~48] Each spot contains known DNA Signal appears Schena (2000) Microarray Biochip Technology, p. A31 Juang RH (2004) BCbasics
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The Genetic Code Initiation and termination Codons
Initiation codon: AUG Termination codons: UAA, UAG, UGA Degeneracy: partial and complete Ordered Nearly Universal (exceptions: mitochondria and some protozoa)
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Key Points Each of the 20 amino acids in proteins is specified by one or more nucleotide triplets in mRNA. (20 amino acids refers to what is attached to the tRNAs!) Of the 64 possible triplets, given the four bases in mRNA, 61 specify amino acids and 3 signal chain termination. (have no tRNAs!)
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Key Points The code is nonoverlapping, with each nucleotide part of a single codon, degenerate, with most amino acids specified by two to four codons, and ordered, with similar amino acids specified by related codons. The genetic code is nearly universal; with minor exceptions, the 64 triplets have the same meaning in all organisms. (this is funny)
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Do all cells/animals make the same
Repertoire of tRNAs?
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The Genetic Code
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The Wobble Hypothesis: Base-Pairing Involving the Third Base of the Codon is Less Stringent.
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Base-Pairing with Inosine at the Wobble Position
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In molecular biology, a wobble base pair is a non-Watson-Crick base pairing
between two nucleotides in RNA molecules. The four main wobble base pairs are guanine-uracil, inosine-uracil, inosine-adenine, and inosine-cytosine (G-U, I-U, I-A and I-C). The thermodynamic stability of a wobble base pair is comparable to that of a Watson-Crick base pair. Wobble base pairs are fundamental in RNA secondary structure and are critical for the proper translation of the genetic code.
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Suppressor Mutations Some mutations in tRNA genes alter the anticodons and therefore the codons recognized by the mutant tRNAs. These mutations were initially detected as suppressor mutations that suppressed the effects of other mutations. Example: tRNA mutations that suppress amber mutations (UAG chain-termination mutations) in the coding sequence of genes.
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Making a (UAG) Mutation
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Translation of an amber (UAG) Mutation in the Absence of a Suppressor tRNA
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Translation of an amber Mutation in the Presence of a Suppressor tRNA
Note it is amber su3…why?????????
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Translation of an amber Mutation in the Presence of a Suppressor tRNA
If there was a single tRNATyr gene, then could one have a amber supressor of it?
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Fig1
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New Base
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Are the proteins produced a pure reflection of the mRNA sequence????
tRNA environment, protein modifications post-translationally
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Good things to Know RNApol II TATAA CCATGG (Nco I site and Kozak Rule) ATG AGGT….splice GT……………A………polypyrimidine AG PolyA recog sequence AATAAA The Reasons why ATG is a single codon and TGG is a single codon.
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SELEX yields a functional binding site
SELEX yields a functional binding site. A, COS7 cells were transfected in triplicate with either pEBG or increasing concentrations of BENwt (250, 500, and 1000 ng) and either p81TKluc (TK) luciferase reporter or p81TKluc-WT3X (WT3X). The luciferase values are reported as relative luciferase activity normalized to the amount of total protein. -Fold decrease in activity is measured relative to the basal transcriptional activity observed with pEBG empty expression vector alone. Western blot with anti-GST antibody shows dose-dependent expression of GST-BEN.
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B, COS7 cells were transfected in triplicate with either pEBG or
BENwt (1000 ng of each) and with either TK, or WT3X or Mut3X (600 ng of each) and the Renilla construct (pRL-TK).
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