Biotech 2011-06-electrophoresis-blots-120101022428-phpapp02


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Biotech 2011-06-electrophoresis-blots-120101022428-phpapp02

  1. 1. Electrophoresis and Blots
  2. 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. 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. 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. 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. 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 4n 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. 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. 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 Migration versus size 100 1000 10000 100000 0 2 4 6 8 Distance migrated in cm Nucleotidebasepairs
  9. 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. 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. 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. 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. 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. 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. 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. 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. 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. 18. Example II RFLP deletion mutation How could these two patterns arise by deletion?
  19. 19. Example III Amplification
  20. 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
  21. 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. 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. 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