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Genotyping technique, types de marquers, genetic variation may serve,…

- - - - non denaturant et contient SDS verticale
      - [] =6 a 12%
      - contient
        
        APS (ammonium persulfate) polymerizing agent
        
        Temed catalyseur de polymerisation
      - haute resolution(meilleur separation des bandes)
    - - separe les frag selon la masse et la charge
      - act as a sieve (0.8 a 2%)
      - chauffe pr dissolver l'agarose in buffer
        
        then cooling down at room temp to make it polymerize
- - - - if some desease is often passed with specific marker gene it can be concluded that this genes are located on which xme
- - - - used when mutation didnt affect restriction site
        
        we modify 1 base at the 3' end of the primer
        
        1 primer matches the normal gene and 1 matches the mutant
        
        3 primers are used
        
        conserved reverse primer
        
        forward normal primer
        
        forward mutant primer
        
        seul celui parfait sera amplifie par PCR
        
        eza fi mutant b sir amplify by PCR w eza fi normal same thing
    - - also called Sanger's method or dideoxy sequencing
      - based on the use of ddNTP and dNTP found in DNA
      - ddNTP have a Hydrogen grp on 3'C instead of hydroxyl HO (prevent from adding more nucleotides
    - - restriction fragment length polymorphysm
      - restriction enzymes recognise and cut DNA and specific sites
      - diff size of framents are cut wether the restriction site exist or not
      - etapes
        
        1-extraction of DNA and digestion (with restrictio enzymes)
        
        2-electrophorese on agarose gel
        
        3-blotting transfer gel to the nitrocellulose membrane by capillary action
        
        4-incubation of the membrane with a radioactivity labeled probe
        
        5-autoradiography
- - - - SSCP
        
        pr detecter du polymorphisme ou des mutations in pol chain R amplified DNA
        
        under non denaturing conditions ssDNA fragment will have unique conformation determined by their primary seq & stabilized by intramol interactions
        
        a single base interaction can result in a conformational change, which can be detected by the altered mobility of the ssDNA
        
        steps
        
        product amplification and denaturation on a 94C followed by cooling with ice
        
        forms a stable secondary structure
        
        mix sample with loading buffer + foramide (prevents renaturation)
        
        loading on a non denaturing polyacrilamide gel
        
        migration of ssDNA depends on size and on secondary conformation
        
        depends on: temp, glycerol in gel, loading buffer ...
        
        mutation detectable with migration at 4C and not 20C, add 10% glycerol in the polyacrilamid gel
        
        staining: radioactivity, ethidium bromide, silver nitrate
        
        limits et inconvenients
        
        the electrophoretic behavior of ss fragments is impredectible (hsb l temp..)
        
        for larger strands (>300pb) mutations are not detectable
      - DGGE
        
        a partially denaturated DNA migrates slower than a dsDNA or a completely denaturated one
        
        progressively denaturing DNA
        
        DNA is electrophoresed in a gel gradient where it can change from native to denaturing: DNA will be ss (7M urea 40% formamide)
        
        temp cte
        
        DN denaturation occurs domain by domain (their melting speed depends on the bases)
        
        avantage: 2 mol de m taille qui different slt d'une pdb ne vt pas migrer en m temps
        
        steps
        
        1-DNA extraction
        
        2-PCR
        
        3-linear gradient electrophoresis
        
        4-migration in ds form (hsb l melting behavior)
        
        5-when it reaches Tm, it will partially denature and migrate slower
      - dHPLC
        
        is used
        
        to detect mutation and polymorphism: separation in denaturing conditions (based on detect heteroduplex fragments)
        
        analysis of DNA fragments : separation according to size (in non denaturing condition)
        
        steps
        
        PCR products are passed on a chromatographic column
        
        an organic solvent elutes the DNA
        
        heteroduplex are less stable than homo, therefore will be disconnected on a high enough temp
        
        which will lead to DNA mismatch on the melting temp
        
        which will decrease the interaction with the column and therefore reduced retention time compared to homoduplex in the chromatographic separation process
        
        advantages
        
        easy and fast
        
        not expensive
        
        no need to label the amplified products
        
        sensitive (98%)
- - - - to observe the loading and check the migration