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Biochemistry Week5, Wobble base pair 搖擺鹼基對, Other Forms of DNA, Complex…

- - - - Creation of identical copies of a piece of DNA(gene)
      - Isolate a specific gene from the source organism and amplify it in the target organism.
    - - Generate recombinant vector 載體: coining vector and DNA fragment of interest is cleaved with same restriction endonuclease限制酶.
      - Introduce DNA into organism: DNA is introduced into the host cell,Then,
        propagation(cloning) of transformed cell produces many copies of recombinant DNA.
      - Then,fragments are ligated by DNA ligase DNA連接酶 to the prepared cloning vector.
        Eventially become recombinant vector.
    - - Restriction Endonucleases 內切酶/限制酶
        
        Classifications
        
        Some make staggered cuts. -sticky ends 黏末端
        
        Some make straight cuts.-blunt ends 平末端
        
        Common in bacteria-eliminates infectious viral DNA
        
        Large number are known:
        commercially available/
        well documented-rebase
        
        Cleave DNA phosphodiester bonds at specific sequences.
      - DNA Ligase (DNA連接酶)
        
        Enzyme that covalently joins two DNA fragments
        
        Normally function in DNA repair
        
        Energy
        
        Human DNA ligase uses ATP.
        
        Bacterial DNA ligase uses NAD.
      - Cloning vector
        
        To clone whole chromosomes (up to 300,000 bp)
        
        BAC(bacterial artificial chromosome)-for use in bacteria
        
        Is very similar to using plasmids. The key point is the relationship between lacZ gene / β -galactosidase / X-gal.
        
        LacZ gene is required for the production of the enzyme substrate for β- galactosidase.
        
        藍白篩：
        If Colonies with active β-galactosidase—
        and hence no DNA insert in the BAC vector turn blue:
        If colonies without β-galactosidase activity—
        and thus with the desired DNA inserts—are white.
        (Because the lacZ gene is disrupted by restriction endonuclease,
        and then the β-galactosidase isn’t produced)
        
        As the reason of big chromosomes,it use eletroporation 電穿孔 to induce into cell.
        
        YAC(yeast artificial chromosome)-for use in yeast
        
        Plasmids質體
        
        Allows cloning of DNA up to 15000 bp
        
        Carry antibiotic resistance genes.
        
        Are replicate autonomously 自動複製(origins of replication for use in bacteria and or yeast.
        
        Circular DNA molecule that are separate from the bacterial genomic 基因組 DNA.
      - How to know whether the recombinant vector is introduced into the host cell or not ?/
        and the fragment of DNA is introduced
        into the plasmid or not?—
        Using antibiotic( antibiotic selection)
        
        Plasds can carry genes genes that give a host bacterium a resistance against antibiotics,then,allows growth selection of bacteria that have taken up the plasmid.
        
        Example: 做出含有ampR/ tetR / foreign DNA的incorporated plasmid後，將其個別放入agar containing tetracycline和 agar containing ampicillin+tetracycline。若cells that grow on agar containing tetracycline but not on tetracycline+ampicillin, means that cells are successfully introduced the foreign DNA. On the contrary,cells with plasmid without foreign DNA retain ampicillin resistance and grow on both plates.
  - - - Used to amplify DNA in the test tube
        
        can amplify regions of interest (genes) within linear DNA
        
        can amplify complete circular plasmids
    - - target DNA (目標DNA 的參與)
      - primers 有兩個(oligonucleotides 低聚核苷酸、寡核苷酸)complementary to target)
      - nucleotides: dATP, dCTP, dGTP, dTTP
      - thermostable 耐熱的,耐穩定的 DNA polymerase
    - - Cool separated strands to about 50–60°C.讓primer黏上
      - Primers anneal to the target. 使引子黏上
      - Melt DNA at about 95°C.解旋
      - Polymerase extends primers in the 5’→ 3’ direction.
      - After a round of elongation is done, repeat the steps.
      - Repeat step1-4 many times
    - - Cloning
        
        use flanking primers to replicate and amplify a specific segment of DNA.
      - Site-directed mutagenesis (oligonucleotide-directed)位點突變
        Altering the Genetic Code
        
        How
        
        use “sandwich” primers to make small changes to the segment of DNA and then amplify the altered DNA.
        
        To mutate an amino acid, change the nucleotide(s) in the coding DNA and express the mutated gene.
        
        Purpose
        
        Understanding the function of proteins often requires that a specific amino acid residue be mutated.故意把某位改變，以得知某位的protein function.
        
        Characteristics
        
        Site-directed mutagenesis usually relies on chemically synthesized mutated primers that are incorporated into newly synthesized DNA.
        
        Mutated plasmids are always sequenced to confirm that the desired (and only the desired) mutation is present.
      - DNA fingerprinting
        
        Purpose
        
        use primers for specific chromosomal locations to amplify and determine number of short tandem repeats for purposes of identification.Otherwise,it allows mathing “suspect” samples to know individual.
        
        Key points
        
        Humans have short sequences that repeat next to each other.— short tandem repeats (STR), Multiple STR locations exist in the human genome.
        
        Fragment sizes can be determined by using a capillary gel.毛細管電泳
        
        Thirteen well-studied locations are used in identifications.— based on number of alleles seen at each location, with misidentification less than 1 in 1018 (when good data are obtained)
        
        Differences in the number of repeats cause variations in the length of fragments that form when a sample is subjected to PCR using a primer specific for that region.
    - - Reverse transcriptase PCR (RT-PCR)
        
        used to convert RNA code to DNA and amplify specific segments(反轉錄得到cDNA)
      - Quantitative PCR (Q-PCR)
        即時定量聚合酶連鎖反應
        
        Purpose
        
        used to show quantitative differences in gene levels(顯示目前複製的情況)／
        一種在DNA擴增反應中，以螢光染劑偵測每次PCR循環後產物總量的方法。
        
        Why we need cDNA
        
        Reason
        
        with challenging RNA samples, we can get better results faster and easier by using DNA which is without introns.
        
        Barriers
        
        A eukaryotic gene from the eukaryotic genome will not express correctly in the bacterium.
        
        Eukaryotic genes have exons(coding regions) and introns(noncoding regions),introns in eukaryotic genes pose problems.
        
        Bacteria cannot splice introns out of coding DNA
        
        Solution
        
        mRNA is introns-free genetic material,and we can use mRNA as a template to synthesize DNA strand,which us means that the DNA strand is complementary to the mRNA.
        
        How to make
        
        All mRNA molecules have a poly A tail,which helps in purification of mRNA and serves as a universal template.when mRNA serves as a universal template,it is annealed to synthetic oligonucleotide primer,then,cDNA can be synthesized by reverse transcriptase反轉錄酶.the end result is a hybrid where the DNA strand is complemetaryt to the mRNA,and the hybrid can be converted to duplex DNA by DNA I and dNTPs,knows as cDNA.
        
        How
        
        利用專一的primer probe(引子探針)會在PCR過程中產生螢光，(fluorophore is separated from the quenching molecule,and the fluorescence spinal increase-probe被打開了才會發光))再利用螢光偵測系統來偵測每個循環所釋放出的螢光量，進而推算出每個循環所產生的產物含量，達到即時定量的目的。
  - - - Agarose gel hinders the mobility of DNA molecules.
      - Mobility depends on the size and the shape.
        (small molecules faster/compact molecules faster)
      - Negatively charged DNA migrates to the anode in the presence of an electric field.
    - - DNA analysis
      - DNA purification
      - DNA-proteininteractionstudies
  - - - When e want to study the protein product of the gene
    - - Use special plasmid—expression vecters,contain sequences that allow transcription of the inserted gene.
    - - Operator sequences
      - Code for ribosome-binding site
      - Promoter sequences
      - Transcription termination sequences
  - - - GST
        
        用glutathione來purify GST以辨識 gene for target protein
      - Fluorescence
        
        Green fluorescent protein (GFP)
        
        Is used to determine protein action in vivo.
        
        Use recombinant DNA technologies to attach GFP to protein of interest.
        
        Visualize with a fluorescent microscope.
        
        Visualization of Protein Location from a GFP–Tagged cDNA Library
        -cDNA+GFP=reporter construct,顯示標的物
        
        Immunofluorescence
        
        Tag protein ith primary antibody 一抗帶and detect with secondary antibody 二抗帶containing fluorescent tag.
        
        Protein can also be fused to a short epitope,and the primary antibody detecting the epitope can be fiuorescently labeled.
  - - - 可提供大量基因序列相關資訊，它是基因組和遺傳學研究的工具，研究人員應用基因晶片即可在同一時間定量的分析大量(成千上萬個)的基因表現，具有快速、精確、低成本之生物分析檢驗能力。
        allow simultaneous screening of many thousands of genes:
        high-throughput screening.
    - - 在數平方公分之面積上佈放數千或萬個核酸探針，檢體中的DNA、cDNA和RNA等與探針結合後，藉由螢光或電流等方式偵測。
    - - Tissue-specific gene expression: which genes are used to make proteins?
      - Mutational analysis: which genes have been mutated?
      - Genome-wide genotyping: which genes are expression
- - - - Syn conformation 同向: Angle near 0° corresponds
      - Anti conformation 反向: Angle near 180° corresponds
        (常見於一般的DNA裡)
- - - - Be responsible for bringing amino acids together during translation to form the growing peptide chain,which will become a protein.
      - RNA is generally single stranded,but tRNA folds into a “t” shape.
    - - Main component of ribosomes
      - rRNA combines with special proteins to form ribosomes whic then “read” mRNA to form proteins.
      - Essentially ribosomes contain rRNA and help build proteins.
    - - mRNA i the RNA type discussed most.
      - mRNA serves as genetic template since DNA can’t leave the nucleus.
      - Acts as the messenger between DNA and protein production.
      - Contains rebose instead of deoxyribose;
        contains uracil instead of thymine
      - One mRNA may code for more than one protein.(較有使用效能)
  - - - DNA melting
        
        Principle: can either heat solution by increasing un temperature or we can change the pH ,and one of these will basically break all these hydrogen bonds holding two strands of DNA together.Eventually, dissociate DNA helix and form these two individual strands of DNA.
      - DNA anealing
        
        Principle: when bring the temperature back down,these indivisual strands of DNA will begin to reassociate.->the hybridization process of DNA.
    - - Inside human body,e don’t change our pH nor temperature.Instead,inside ourselves,we use our enzymes knows as helicases,which use energy molecules (ATP) to break the bonds between bases,that means dissociate and melt our DNA structure.
    - - Tm depends on DNA length->longer DNA has higher Tm;it is important for short DNA.
      - Tm depends on pH and ionic strength->high salt increase Tm.
      - The midpoint of melting (Tm) depends on base composition.->high CG increase Tm.
      - AT-rich regions melt at a lower temperature than GC-rich regions.
        -> means that denaturation不一定會從序列首端發生。
  - - - Depurination 去瞟呤
        
        N-glycosidic bond is hydrolyzed
        
        Significant for purines: 10,000 purines lost/day in a mammalian cell
      - Deamination 去氨基
        
        Large number of residues
        
        Very slow reaction
        
        The net effect is significant: 100 C → U events/day in a mammalian cell.
    - - Chemical alkylation,methylation of guanine
      - Reversible: cells have mechanisms to coerrct most of these modification.
      - Oxidative damage.hydroxylation guanine,
        mitochondrial DNA is most susceptible易受影響.
    - - UV light induces dimerization oldf pyrimidines;
        this may be the main mechanism for skin cancers.
      - Ionizing radiation (x rays and γ rays)
        causes ring opening and strand breaking.
      - Irreversible: these are difficult to fix.Cells can repair some of these modification,but others cause mutation.Accumulate of mutations is linked to aging and carcinogenesis.癌變
- - - - May be attached to other positions for specialized function
      - Typically attached to 5’ position
        
        Nucleic acids are built using the 5’-triphosphates version of the nucleotide.ATP,GTP,TTP,CTP
        
        Two of the three phosphates used for building nucleic acids form a leaving group,
        and completed nucleic acids contain one phosphate moiety per nucleotide.
      - Negatively charged at neutral pH
    - - Pentose Forms Differ in Some Nucleic acids and Nucleotides
        
        β-2’-deoxy-d-ribofuranose in DNA
        
        β-d-ribofuranose in RNA
      - Pentode不一定是平面的，可能在c-2或c-3內凹或外凹
  - - - pyrimidines 瞟呤
        
        Thymine
        
        is found only in DNA.
        
        Uracil
        
        is found only in RNA.
        
        Cytosine
      - purines 嘧啶
        
        Adenine
        
        Guanine
    - - Absorb UV light around 250–270 nm
        
        is due to π → π* electronic transitions.
        
        Excited states of common nucleobases decay rapidly via radiationless transitions.
        
        effective photoprotection of genetic material
        
        no fluorescence from nucleic acids
      - Cytosine, adenine, and guanine are found in both DNA and RNA.
      - Planar or almost planar structures
      - All are good H-bond donors and acceptors.
      - Nitrogen-containingheteroaromaticmolecules
      - Neutral molecules at pH 7
    - - Prototropic tautomers are structural isomers that differ in the location of protons.
      - Classification
        
        Keto-enol tautomerism is common in ketones.
        
        Lactam-lactim 內醯胺-內醯亞胺 tautomerism occurs in some heterocycles.
      - Both tautomers exist in solution, but the lactam forms are predominant at neutral pH.
- - - - RNA lasted for a few years un water. mRNA is degraded in a few hours un cells.
      - Hydrolysis of RNA
        
        is also catalyzed by enzymes (RNase).
        
        RNase enzymes are abundant around us.
        
        S-RNase S-核糖核酸酶 in plants prevents inbreeding.
        
        RNase P 核糖核酸酶P is a ribozyme (enzyme made of RNA) that processes tRNA precursors.剪切tRNA分子中多餘的禍前體的多餘序列
        
        Dicer 切丁酶 is an enzyme that cleaves double-stranded RNA into oligonucleotides.寡肽
        
        protection from viral genomes
        
        RNA interference technology
    - - The 5’ end is different from the 3’ end.
      - We read the sequence from 5’ to 3’.
  - - - Purine pairs with pyrimidine
        
        A pairs with T->have two hydrogen-bonding
        
        C pairs with G ->have three hydrogen-binding
- - - - Is found in both bacteria and eukaryotes
        （胞嘧啶受到甲基化後的型態，是一種表觀遺傳休市，參與酵素為DNA甲基轉移酶)
    - - Is only found in bacteria
        （真核細胞mRNA中最常見的修飾，也見於tRNA rRNA snRNA
    - - Protect : mark own DNA so that cells can degrade foreign DNA(in prokaryotes)
      - Mark which genes should be active(in eukaryotes)
  - - - is sometimes found in the “wobbleposition”
        搖擺鹼基對of : the anticodon 反密碼子in tRNA.
      - Functions
        
        made by de-aminating adenosine 腺苷去氨酶
        
        provides richer genetic code
    - - Functions
        
        May stabilize the structure of tRNA
        
        May help in folding of rRNA
      - Introduction
        
        is found widely in tRNA and rRNA,is more.
        Common in eukaryotes but found also in eubacteria
        Made from uridine by enzymatic isomerization after RNA synthesis
        
        是尿苷的一種同份異構物，比尿苷多一個可形成氫鍵的位點。為RNA中最常見的修飾，有AUCG之外「第五種核苷」之稱