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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 shortpalindrome 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 nwhere 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 withultraviolet 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 DNAcontaining 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 individualdigested wit

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