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3/24生化整理 - Coggle Diagram

- - - - puckered
        
        2' / 3' endo
        
        2' / 3' exo
    - - pyrimidine
        
        Cytosine
        
        Thymine
        
        Uracil
      - purine
        
        Adenine
        
        Guanine
      - May contain heteroaromatic
      - Planar
      - Absorb Uv light
        
        250nm-270nm (π → π* electronic transitions)
        
        excited state decay rapidly —protect genetic material
      - Tautomerism互變異構
        
        Prototropic tautomers are structural isomers
        
        ketone : keto-enol
        
        heterocycles : lactam - lactim （lactam較多）
    - - negatively charged
      - attach 5' position
        
        β 𝒓 間 leavinng group
    - - Pentose attach nitrogenous base
        
        pyrimidines N1
        
        Purines N9
        
        free rotation
        
        朝內 = syn conformation
        
        朝外 = anti conformation
      - anomeric cabin — β configuration
      - stable (pyrimidines 👍)
      - Catalyzed by acid
  - - - Moadifucation after synthesis
      - 5-Methylcytosine (eukaryotes + bacteria)
      - N6-Methyladenosine (bacteria)
      - epigenetic marker
        
        prokaryotes : mark own DNA avoid degraded
        
        eukaryotes : mark genes to active
    - - Inosine found in wobble position
        
        provide richer genetic code
      - pseudouridine in tRNA & rRNA
        
        common in eukaryotes
        
        uridine enzymatic isomerization
        
        stabilize tRNA structure
        
        help fold rRNA
  - - - nitrogenous base
      - pentose
    - - nucleotides' polymers
        
        transmission
        
        processing
        
        storage genetic info
        
        synthesis
    - - pentose
      - phosphate
      - nitrogenous base
  - - - S-RNase prevents inbreeding
      - RNase P process tRNA precursors
      - Dicer cleave double-stranded
  - - - A form
      - B form (waston-crick model)
      - C form
    - - Two chains are antiparallel
  - - - use DNA template synthesize
      - ribose、uracil ( ❌deoxyribose 、thymine)
      - Work with tRNA
    - - Hairpins & cruciforms
  - - - Covalent bonds remain intact
      - Hydrogen bonds break
      - lose base stack
        
        Increase up absorbance
      - reversible : annealing
    - - Thermal
        
        DNA dissociate at elevated temperatures
        
        Rena Neal when temperature low
        
        monitored by UV spectrophotometry (分光光度法）
      - pH
    - - Tm base composition
        
        pH / ionic strength
      - DNA length
        
        longer has higher Tm
        
        shor DNA
  - - - slow
      - residue so many
    - - N-glycosidic bond is hydrolyzed
    - - oxidative damage
      - hydroxylation
      - alkylation / methylation
- - - - copy DNA
      - Isolate specific gene and amplify
    - - generate recombinant vector
        
        get interes DNA fragment
        
        ligated both
        
        DNA ligase
        
        covalently joins fragment (enzyme)
        
        human --> ATP
        
        bacterial --> NAD
        
        cloning vector + cleave it
        
        restriction endocleases
        
        Common in bacteria
        
        cuts
        
        staggered cuts
        
        sticky ends
        
        straight cuts
        
        blunt ends
        
        Cleave DNA phosphodiester bonds at specific sequences
      - introduce DNA into organism
        
        introduced into the host cell
        
        transformed cell producers copies
      - antibiotic selection
        
        introduction
        
        plasmid has different genet to against antibiotic
        
        antibody --> kill bacteria
        
        identification
        
        put every clonies into agar that contain tetracycline
        
        individual colonies transfer to matching position
        
        one plate contain tetracycline , the other tetracycline + ampicillin
        
        cells only grow ion tetracycline contain recombinant plasmids
    - - cloning vectors
        
        plasmid
        
        circular DNA molecules
        
        can replicate autonomously
        
        antibiotic resistance genes
        
        clone whole chromosomes
        
        bacterial
        
        bacterial artificial chromosome
        
        with active β-galactosidase and no insert ,BAC vector turn blue
        
        using X-gal check wether it have β-galactosidase
        
        比較長又一大團揪在一起那種
        
        yeast
        
        yeast artificial chromosome
        
        linear
        
        simplest eukaryote recombination
        
        removable
    - - step
        
        heat to separate strands
        
        add primers
        
        target DNA
        
        add DNA polymerase catalyze
        
        repeat
        
        catalyzed synthesis by thermocycler
      - separation
        
        Agarose gel hinders the mobility of DNA
        
        small faster
        
        compact faster
        
        Negatively charged DNA migrates to the anode
        
        analysis / purification / interaction
      - introduction
        
        amplify DNA
        
        mix
        
        thermocycler
      - application
        
        site-directed mutagenesis
        
        change the nucleotide in the coding DNA
        
        chemically synthesized mutated primers
        
        function of proteins require specific Amio acid residue mutation
        
        DNA fingerprinting
        
        Humans have short sequences repeat
        
        short tandem repeats
        
        variations in the length of fragments
        
        sizes determined by capillary gel
        
        13 location used in identifications
        
        cloning
      - adaptation
        
        Reverse transcriptase PCR
        
        Quantitative PCR
      - qPCR
        
        probe + fluorophore
        
        no light
        
        fluorophore / quenching molecule separate
        
        light
      - Construction of cDNA
        
        using mRNA as a template
        
        catalyzed by the reverse transcriptase
        
        hybridbe converted to duplex DNA
      - Expression
        
        operator
        
        ribosome-binding site
        
        promoter
        
        transcription termination sequences
      - purification
      - Fluorescence Determine Protein Location In Vivo
        
        Green fluorescent protein
        
        Immunofluorescence
      - DNA Microarrays