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Southern Blotting

Southern Blotting. Invented by British biochemist Edwin Southern . Purpose. Used to determine whether a particular nucleotide sequence is present in different sources of DNA, and also to discover the size of the restriction fragment that contains the specific sequence . Example. Sickle Cell

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Southern Blotting

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  1. Southern Blotting Invented by British biochemist Edwin Southern

  2. Purpose • Used to determine whether a particular nucleotide sequence is present in different sources of DNA, and also to discover the size of the restriction fragment that contains the specific sequence

  3. Example • Sickle Cell • Can separate someone who is a heterozygous carrier of the sickle-cell allele, as well as someone who does and does not have the disease I II III • An individual with 2 normal alleles • An individual with 2 mutant alleles • Someone who has both (combination)

  4. Restriction • Restriction enzyme: An enzyme found in bacteria that cuts DNA at a certain sequence • Restriction fragment: a small segment of a DNA strand that was “cut” with a restriction enzyme CCGG GGCC Blunt end Sticky end

  5. Step 1: Preparing restriction fragments How to do it Why it works • Each sample of DNA is mixed with the same restriction enzyme, resulting in thousands of restriction fragments • The restriction enzyme detects specific sequences and will make cuts in the sugar backbone of the DNA strand accordingly

  6. Step 2: Gel electrophoresis How to do it Why it works • The fragments are dyed with ethidium bromide so they are visible • Restriction fragments are inserted into an agarose gel and then are separated by electrophoresis • The smaller fragments move faster through the agarose gel • They will move toward the anode if negatively charged and the cathode if positively charged

  7. Step 3: DNA transfer/“blotting” • Transferred to a nitrocellulose membrane

  8. Step 4: Radioactive probe How to do it Why it works • Radioactively labeled nucleic acid probe is added • The probe will only bind to complimentary DNA restriction segments

  9. Step 5: Probe detection How to do it Why it works • Cover membrane with an x-ray film and develop • After development, only the places where the probe has combined with the restriction fragments will be visible

  10. Northern Blotting • Used to study gene expression by detecting which RNA (or mRNA) is “active” http://www.biochemj.org/bj/328/0807/bj3280807a02.gif

  11. Process of N. Blotting • Transcription: synthesis of mRNA from DNA • Gel electrophoresis: agarose gel • Separates RNA by size – denatures during G.E. • Negatively charged molecules = anode • Positively charged molecules = cathode • Separated mRNA transferred to nylon membrane • RNA detected by probe • Audioradiogram: single gene transferred to X-Ray film http://www.youtube.com/watch?v=KfHZFyADnNg

  12. Uses • Gives information about RNA and RNA sequences • Observe how a particular gene changes during embryonic development • Ability to track RNA expression over the course of development or treatment (used in cancer research) • Identify closely related species

  13. Western Blotting • Also called immunoblotting • Analysis of proteins • Finds certain proteins in specific tissues or cells. • Determine the molecular weight of a protein. • Shows the abundance of protein in cells or tissues.

  14. Basic Process • Gain a cell and lyse it to release proteins • Use SDS-polyacrylamide gel electrophoresis to separate proteins based on size • Place proteins on a membrane using electricity • Primary antibody is used to find the particular protein • A secondary antibody binds to the primary antibody • This antibody has horseradish peroxidase enzyme which releases light • The light is detected on film which shows the size and abundance of the protein.

  15. Lyse, SDS-PAGE, and Membrane • Lyse destroying a cell by breaking the cellular membrane using detergents, usually either SDS or RIPA. • The detergents have a hydrophilic head and hydrophobic tail. • They are water-soluble and can disperse hydrophobic compounds into aqueous solutions.

  16. Antibodies • The membrane is incubated in primary antibody (antibodies raised against the antigenic, or protein). • Then washed to eliminate unbound primary antibodies. • Next incubated in secondary antibody and wash again. • The horseradish peroxidase enzyme attached to the secondary antibody produces a luminescent derivative when placed in an oxidation reaction.

  17. Scientific Uses • Forensics use Western Blotting to determine DNA data. • Testing for viruses or diseases uses Western Blotting, examples: • HIV tests • Mad Cow Disease • Hepatitis B • Some forms of Lyme disease

  18. Citations • Lebrasseur, Nicole D. "Blotting analysis." World of Genetics. Ed. K. Lee Lerner and Brenda Wilmoth Lerner. Online. Detroit: Thomson Gale, 2007. Science Resource Center. Gale. 28 September 2009 http://galenet.galegroup.com/servlet/SciRC?ste=1&docNum=CV2433500062 • "Western Blot." Molecular Station. N.p., 2008. Web. 29 Sept. 2009.      <http://www.molecularstation.com/protein/western-blot/>. • "Detergents for Cell Lysis." Thermo Scientific. Thermo Fisher Scientific Inc.,      2009. Web. 29 Sept. 2009. <http://www.piercenet.com/      browse.cfm?fldID=5558F7E4-5056-8A76-4E55-4F3977738B63>. • Sandwich for Nitrocellulose Membrane Transfer. Chart. Georgia Institute of      Technology School of Biomedical Engineering, 2001. Web. 29 Sept. 2009.      <http://www.bme.gatech.edu/vcl/WesternBlotting/Background/transfer.htm>.

  19. Bibliography • http://askabiologist.asu.edu/expstuff/mamajis/southern/southern.html • http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/R/RestrictionEnzymes.html • http://en.wikipedia.org/wiki/Restriction_fragment • http://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter14/animation_quiz_5.html • http://www.bio.davidson.edu/COURSES/GENOMICS/method/Southernblot.html • http://evolutionary-research.net/science/intro/the-engine-of-evolution/figures/DNA-DoubleHelix-ImgLibPDB-1bna_prepi_4.gif • http://www.biotechlearn.org.nz/var/biotechlearn/storage/images/themes/from_genes_to_genomes/images/restriction_enzyme/4713-1-eng-AU/restriction_enzyme_large.jpg • http://www.accessexcellence.org/RC/VL/GG/ecb/ecb_images/10_14_1_Southern_blotting.jpg • http://escience.ws/b572/L22/images/spact.gif • http://en.wikipedia.org/wiki/Northern_blot#Applications • http://www.protocol-online.org/prot/Molecular_Biology/RNA/Northern_Blotting/index.html • http://en.wikipedia.org/wiki/Messenger_RNA • http://www.peircenet.com/proteomics/browse.cfm?ldID=C2FABBDE-F46E-47IE-A907-3CB9802A393D

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