Biotech 2011-06-electrophoresis-blots

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  • 1. Electrophoresis and Blots
  • 2. Principles recap
    • Nucleic acids (DNA, RNA) move from the negative to the positive pole during gel electrophoresis
    • The resistance to movement is due to the sieving effect of the gel matrix
    • The rate of migration is related to the size of the nucleic acid
  • 3. Agarose versus acrylamide
    • Acrylamide is a crosslinked synthetic polymer useful in separating DNA molecules of a few hundred bases in length
      • The crosslinking reaction is carried out within the molding apparatus
      • The final gel is transparent and usually used in a vertical apparatus
    • Agarose is a polysaccharide similar to starch useful in separating DNA and RNA molecules of a few hundred to tens of thousands on bases long
      • A gel is created by boiling agarose into solution and allowing it to cool within a molding apparatus
      • The final gel is translucent and usually used in a horizontal apparatus
  • 4. Restriction enzymes I
    • Origin and native utility
      • Some restriction enzymes are used by bacteria to protect themselves from invading DNA
      • A “restriction-modification” system exists that marks bacterial DNA by methylating it on certain sequences
        • A restriction enzyme specific for those sequences cannot digest DNA if it is methylated
      • Invading viral DNA is unmarked and subject to cleavage at those sequences
  • 5. Enzymes
    • Restriction enzymes
      • Generally bacterial in origin
        • But may come from lower eukaryotes, viruses or transposable elements
      • Cleave DNA at short palindrome sites
        • The palindromes may be 2 or more nucleotides long
        • Unusual forms may recognize sequences up to 30 bases long
          • These are used in site specific recombination events
        • Cleavage usually results in a 5’ phosphate and a 3’ hydroxyl
        • The resulting ends may be single stranded over a short region, or double stranded (blunt ends)
        • The cleavage sites may be covalently linked back together by DNA ligase
  • 6. Restriction enzymes III
    • A restriction enzyme with a 6 base recognition site would produce on average a fragment size of 4096 bases in length when used to cut single copy DNA
      • The average fragment size can be determined for any recognition site by the formula 4 n where n is the number of bases in the recognition site
    Size of recognition sequence Average size of fragment 4 256 5 1024 6 4096 8 65536
  • 7. Agarose gel electrophoresis
    • Gels are loaded with samples of restricted DNA and electrophoresed for minutes to hours
    • The voltage is turned off and the gel is stained with the intercalator ethidium bromide
      • DNA may be visualized in the gel by illuminating it with ultraviolet light
      • Linear relationship between mobility and the logarithm of the DNA size
  • 8. Size determination
    • Size markers are commonly used in electrophoresis experiments
      • These are duplex DNA fragments of a known size
      • The migration distance of a fragment may be compared to the migration of the size markers directly on the gel
      • Alternatively the migration of a fragment may be determined by interpolation from a plot of the distance migrated by the size markers
    • The distance migrated is related to the inverse log of the molecular weight of the DNA fragment
  • 9. Digesting human chromosomal DNA
    • produces a range of fragment sizes due to cleavage of single copy DNA
      • Three billion base pairs cleaved by a restriction enzyme recognizing 6 bases results in 750,000 fragments
        • They appear as a background smear of DNA cleaved into all possible sizes by the restriction enzyme
        • The fragments are of random size with an average length of >4096 bases
          • Repetitive sequence can increase the average size, because usually a given restriction site will not be found within it
      • If a restriction site is in a repetitive sequence, digestion will produce a large number of fragments of identical length
        • This will appear as a band in the background smear of fragments
  • 10. Southern Blot
    • Once an agarose gel of digested chromosomal DNA has been stained, individual DNA sequences may be detected on the gel by hybridization
      • The DNA within the gel is denatured by exposing it to a solution of sodium hydroxide
      • The DNA is then neutralized and transferred out of the gel onto a membrane that binds DNA
        • This is a procedure known as blotting
        • It exposes the DNA to the surface so that it may hybridize to complementary sequences
      • The membrane bound DNA is then hybridized to a short specific sequence known as a probe
  • 11. Probe
    • A probe is an oligo or poly nucleotide that represents known DNA sequence
      • It is usually a sequence complementary to a gene
        • But it only need be complementary to DNA sequence within the chromosomal DNA itself
      • It is either single stranded to begin with or is denatured to make it single stranded
      • It is labeled with radioactive phosphorous or a fluorescent adduct
  • 12. Hybridization
    • The membrane is “prehybridized” with non-specific DNA to block non-specific binding sites on the membrane prior to hybridization
    • It is then hybridized to the probe
      • The temperature and salt conditions are controlled to insure optimal hybridization between the probe and the target sequences
      • The single stranded probe finds its complement in the membrane bound DNA and forms a double helix
    • Unbound probe is then washed off
  • 13. X-ray film exposure
    • The membrane is exposed to X-ray film
      • Radioactive probes expose the film whereever they hybridized on the blot due to emission of electrons (beta particles)
      • Fluorescent probes catalyze a light yielding reaction using energy supplied in a reaction cocktail
    • Development of the X-ray yields an autoradiogram
      • Bands on the film locate the chromosomal restriction fragments complementary to the probe DNA
  • 14. Overall
    • Southern Blots
      • DNA is digested with restriction enzymes and electrophoresed through an agarose gel
      • The contents of the gel are denatured in situ and then transferred onto a membrane that binds DNA
      • The DNA is hybridized to radioactive DNA complementary to the gene of interest
      • Unhybridized DNA is washed away and the membrane exposed to X-ray film
    • The technique can be adapted to identify any source of DNA containing a gene of interest or to RNA detection (northern blots)
  • 15. Restriction fragment length polymorphism (RFLP)
    • Chromosomal DNA from two people differs due to a random variations in sequence
      • There are millions of single nucleotide polymorphisms between two unrelated individuals
    • If one of these sequence variations results in the creation or destruction of a restriction site, then the fragment sizes of DNA will differ between these two people
    • This can be detected by southern blots
  • 16. Example I RFLP point mutation
    • A gene sequence contains two Eco R1 sites separated by 4000 nucleotides
    • A patient has suffered a mutation in one of his two homologous chromosomes such that a single nucleotide change has created a new EcoR1 site within the gene sequence
      • Now two fragments are created of 1500 and 2500 bases in length
      • The probe is complementary to sequence located within the 2500 base long fragment
      • Detection by southern blot of these two fragments will result in a 4000 base long fragment in normal DNA from one homologous chromosome and two fragments from the other homolog
  • 17. DNA from normal individual digested with Eco R1 and electrophoresed through agarose DNA from mutant individual digested with Eco R1 and electrophoresed through agarose
  • 18. Example II RFLP deletion mutation How could these two patterns arise by deletion?
  • 19. Example III Amplification
  • 20. Other blots Northern
    • Northern blots are mainly used to measure of the amount of a specific RNA in the cell
    • The blot is prepared and hydridized like a southern blot
    • There are a few minor technical alterations
      • The RNA is usually folded into a secondary structure so it must be denatured before and during electrophoresis
    • Bands on the autoradiogram are a measure of the presence of RNA from a particular gene
      • The more intense the band, the more RNA on the blot
  • 21. Protein electrophoresis
    • Proteins, unlike DNA, do not have a constant size to charge ratio
      • In an electric field, some will move to the positive and some to the negative pole, and some will not move because they are neutral
      • Native proteins may be put into gel systems and electrophoresed
        • However native proteins are sometimes difficult to prepare without degradation
      • An alternative to native protein gels forces all proteins to acquire the same size to charge ratio
  • 22. SDS-polyacrylamide gel electrophoresis
    • A sample of protein, often freshly isolated and unpurified, is boiled in the detergent sodium dodecyl sulfate and beta-mercaptoethanol
      • The mercaptoethanol reduces disulfide bonds
      • The detergent disrupts secondary and tertiary structure
      • On the molecular level, proteins are stretched out and coated with the detergent (which has a negative charge) by this treatment
      • They will then migrate through a gel towards the positive pole at a rate proportional to their linear size
        • Molecular weights with respect to size markers may then be determined
  • 23. Other blots II Western
    • Western blots measure the amount of a specific protein in a cell
    • This is a blot of an SDS- polyacrylamide gel onto which cellular protein is applied
      • The protein is denatured so it will enter the gel as a stretched out random coil
      • The proteins separate by size
      • They are blotted onto a membrane
    • Western blots are probed using antibodies
      • The antibodies are labeled. Those that remain bound to their specific targets identify specific proteins in the cell
    • The intensity of the signal reflects the level of expression
    • It can also reveal mutations by anomalies of migration
      • A deletion might cause a shorter protein for example