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DNA Technology

DNA Technology. DNA Extraction. Chemical treatments cause cells and nuclei to burst The DNA is inherently sticky , and can be pulled out of the mixture This is called “spooling” DNA. “Spooled” DNA. Cutting DNA. Restriction enzymes cut DNA at specific sequences

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DNA Technology

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  1. DNA Technology

  2. DNA Extraction • Chemical treatments cause cells and nuclei to burst • The DNA is inherently sticky, and can be pulled out of the mixture • This is called “spooling” DNA

  3. “Spooled” DNA

  4. Cutting DNA • Restriction enzymescut DNA at specific sequences • Useful to divide DNA into manageable fragments

  5. These cuts usually occur in the following forms: 1.The cut can be made straight across a base-pair sequence resulting in a "Blunt End“

  6. 2. The cut can be made in an offset manner leaving exposed nucleotide sequences. These exposed sequences are called "Sticky Ends"

  7. Gene Splicing • The presence of sticky ends allows segments of DNA to be joined together. • Recombinant DNA – joining of DNA from 2 or more species • DNA strands which have been cut by the same restriction enzyme can easily bond together according to base pairing rules.

  8. Gene splicing cont’

  9. Gene splicing use: • Allows genes to be "cut & pasted" between organisms. • E.g. production of human insulin. • The DNA sequence of insulin is identified, & cut out with restriction enzyme. • A plasmid from E. coli is removed & cut open using the same restriction enzyme • Both fragments have complimentary sticky ends, they bind, the gene for human insulin is integrated into plasmid • The plasmid is reinserted into bacterial cell which produces insulin. • Human insulin can now be extracted and provided to diabetics.

  10. Plasmid – a ring of DNA found in a bacterium in addition to its main strand of DNA

  11. Electrophoresis • DNA can be separated based on size & charge • The phosphate groups are negatively charged • DNA is placed in a gel and electricity is run through

  12. Electrophoresis machine

  13. Electrophoresis • Negative DNA moves toward the positive end • Smaller fragments move farther and faster through pores

  14. Electrophoresis

  15. Tutorial with animation • http://www.tvdsb.on.ca/westmin/science/sbioac/genetics/Electro.htm

  16. Animations • http://www.lewport.wnyric.org/jwanamaker/animations/Chrom%26Elpho.html

  17. Steps in DNA Sequencing • Many copies of a single strand of DNA are placed in a test tube • DNA polymerase is added • A mixture of nucleotides is added some of which have dye molecules attached • Each base (A,T,C,G) has a different color dye

  18. Steps in DNA Sequencing • By chance, some dyed nucleotides & some regular ones are added • Dye molecules are large and stop the chain from growing

  19. DNA Sequencing • The result is DNA fragments of multiple sizes with colors that can be identified

  20. DNA Sequencing • After the gel separates the resulting fragments by size, we 'read' the sequence from bottom to top.

  21. DNA Fingerprinting • DNA is now a powerful tool in identification. • Based on the fact that the amount of "junk DNA" differs uniquely between individuals. • Structural genes are often separated by large regions of repeating base pairs. • The number of these repeats is unique to an individual. • When DNA from a person is cut with a restriction enzyme, the length of the fragments will be unique to that individual.

  22. Fingerprinting cont’ • This will produce a unique banding pattern following a gel electrophoresis. • This test is highly accurate, and the probability of another individual possessing an identical banding pattern is estimated as around 1:14,000,000,000.

  23. Fingerprinting cont’

  24. Copying DNA • Polymerase Chain Reaction • Also called PCR • A method of making many copies of a piece of DNA

  25. Steps in Copying DNA • A DNA molecule is placed in a small test tube • DNA polymerase that can work at high temps is added

  26. Steps in Copying DNA • The DNA is heated to separate the two strands • Primers, short pieces of DNA complementary to the ends of the molecule to be copied, are added

  27. Copying DNA • The tube is cooled, and DNA polymerase adds new bases to the separated strands

  28. PCR Large amounts of DNA can be made from a small starting sample

  29. PCR Virtual Lab • http://learn.genetics.utah.edu/content/labs/pcr/

  30. Cloning • Clone- a member of a group of genetically identical cells • Produced by asexual reproduction (mitosis)

  31. Cloning organisms • A body cell from one organism and an egg cell from another are fused • The resulting cell divides like a normal embryo (many identical cells produced)

  32. Cloning “Dolly”

  33. GEL ELECTROPHORESIS VIRTUAL LAB • http://learn.genetics.utah.edu/content/labs/gel/

  34. REFERENCES - http://www.accessexcellence.org/RC/VL/GG/plasmid.php

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