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Chapter 3

Chapter 3. Recombinant DNA Technology and Genomics. Agarose Gel Electrophoresis. Electrophoresis is a molecular technique that separates nucleic acids and proteins based on: Size and +-+ Charge +-+. Shape. Agarose Gel Electrophoresis.

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Chapter 3

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  1. Chapter 3 Recombinant DNA Technology and Genomics

  2. Agarose Gel Electrophoresis • Electrophoresis is a molecular technique that separates nucleic acids and proteins based on: Size and +-+Charge+-+ Shape

  3. Agarose Gel Electrophoresis • DNA is a negatively charged molecule and therefore is attracted to positive charges.

  4. Agarose Gel Electrophoresis • Agarose provides a matrix through which DNA molecules migrate. • Larger molecules move through the matrix slower than small molecules • The higher the concentration of agarose, the better the separation of smaller molecules

  5. Agarose Gel Electrophoresis • How to make an agarose gel: • Weigh out a specified amount of agarose powder. • Add the correct amount of buffer. • Dissolve the agarose by boiling the solution. • Pour the gel in a casting tray. • Wait for the gel to polymerize.

  6. Agarose Gel Electrophoresis • How to make an agarose gel: • Place gel in chamber and cover with buffer • Add loading dye to the sample • Load sample on to the gel.

  7. Agarose Gel Electrophoresis • How to make an agarose gel: • Stain the gel • Take a picture of the gel • Analyze results

  8. Agarose Gel Electrophoresis Electrophoresis Animation

  9. Recombinant DNA • Recombinant DNA technology • Allows DNA to be combined from different sources • Also called genetic engineering or transgenics

  10. Recombinant DNA • Vector – DNA source which can replicate and is used to carry foreign genes or DNA fragments. • Recombinant DNA – A vector that has taken up a foreign piece of DNA.

  11. Restriction Enzymes • Restriction enzyme – an enzyme which binds to DNA at a specific base sequence and then cuts the DNA • Restriction enzymes are named after the bacteria from which they were isolated. • Bacteria use restriction enzymes to chop up foreign viral DNA

  12. Restriction Enzymes • Recognition site – specific base sequence on DNA where a restriction enzyme binds. • All recognition sites are palindromes, which means they read the same way forward and backward. example: RACECAR or GAATTC CTTAAG • Each restriction enzyme has its own unique recognition site.

  13. Restriction Enzymes

  14. Restriction Enzymes • After cutting DNA with restriction enzymes, the fragments can be separated on an agarose gel. • The smaller fragments will migrate further than the longer fragments in an electric field. • The bands are compared to standard DNA of known sizes. This is often called a DNA marker, or a DNA ladder.

  15. Restriction Enzymes Running a Restriction digest on an agarose gel

  16. Restriction Enzymes • After analyzing your results, you draw a restriction map of the cut sites. • A restriction map is a diagram of DNA showing the cut sites of a series of restriction enzymes.

  17. Restriction Enzymes

  18. Restriction Enzymes

  19. Restriction Enzymes • Most restriction enzymes cut within the recognition site. • When restriction enzymes cut in a zig zag pattern, sticky ends are generated.

  20. Restriction Enzymes • Overhanging sticky ends will complementarily base pair, creating a recombinant DNA molecule. • DNA ligase will seal the nick in the phosphodiester backbone.

  21. Restriction Enzymes Restriction Enzyme Animation

  22. Transformation • Transformation – the process by which organisms take up and express foreign DNA Griffith’s experiment

  23. Transformation • Bacterial Transformation • Bacteria, such as E.coli, can take up and express foreign DNA, usually in the form of a plasmid.

  24. Transformation • Gene cloning – using bacterial transformation to make lots of copies of a desired gene. Gene Cloning Animation

  25. Transformation • Steps of Bacterial Transformation • Choose a bacterial host • E. coli is a model organism • Well studied • No nuclear membranes • Has enzymes necessary for replication • Grows rapidly (20 min. generation time) • Inexpensive • Normally not pathogenic • Easy to work with and transform

  26. Transformation • Steps of Bacterial Transformation • Choose a plasmid to transform • Characteristics of a useful plasmid • Single recognition site • Plasmid only cuts in one place, so this ensures that the plasmid is reformed in the correct order. • Origin of replication • Allows plasmid to replicate and make copies for new cells. • Marker genes • Identifies cells that have been transformed.  gene for antibiotic resistance – bacteria is plated on media with an antibiotic, and only bacteria that have taken up a plasmid will grow  gene that expresses color – bacteria that have taken up a recombinant plasmid are a different color than bacteria that have taken up a NONrecombinant vector.

  27. Transformation • Steps of Bacterial Transformation • Prepare bacterial cells for transformation • Treat with calcium chloride – softens the phospholipid bilayer of the cell membrane, which allows the plasmid to pass through • Electroporation – brief electric pulse • Directly inject plasmid into bacterial host

  28. Transformation • Steps of Bacterial Transformation • Plate transformation on appropriate media • Contains nutrients for bacteria and antibiotic to distinguish transformed bacteria from NONtransformed bacteria • Incubate plates overnight • E.coli grows at body temp. (37 °C) • Analyze plates Gene Cloning Animation

  29. Gene Cloning • What makes a good vector?

  30. Gene Cloning • What makes a good vector?

  31. Gene Cloning • How do you identify and clone a gene of interest? • BUILD A LIBRARY!! • DNA library – a collection of cloned DNA fragments from a particular organism • Can be saved for a relatively long period of time and screened to pick out different genes of interest • Two types of libraries • Genomic library – contains DNA sequences from entire genome • cDNA library – contains DNA copies of mRNA molecules expressed Construction of a DNA library Animation

  32. Gene Cloning

  33. Gene Cloning • Steps to screen a library • Plate cells and transfer to nylon membrane • Lyse bacterial cells • Denature DNA • Add radioactively labeled probe that is complementary to gene of interest

  34. Gene Cloning • Steps to screen a library • Wash off non-specifically bound probe • Expose membrane to x-ray film • Align exposed film with original plate • Grow cells containing gene of interest in culture.

  35. Gene Cloning • Rarely is an entire gene cloned in one piece, even in a cDNA library, therefore must “walk” the chromosome until a start and stop codon are found.

  36. Sequencing • Sequencing – determining the order and arrangement of G’s, A’s, T’s and C’s in a segment of DNA.

  37. Sequencing • Let’s review replication…..

  38. Sequencing • The Sanger sequencing method uses dideoxy-nucleotides to generate all possible fragments of the DNA molecule to be sequenced. deoxynucleotide dideoxynucleotide

  39. Sequencing • Set up four different reactions:

  40. Sequencing • Load the four reactions in different wells of a polyacrylamide gel to separate the fragments

  41. Sequencing Sequencing Animation

  42. Human Genome Project • Initiated in 1990 with plan to identify all human genes • Analyze genetic variation among humans • Map and sequence genomes of model organisms • Develop new lab technology • Disseminate genome information • Consider ethical, legal, and social issues that accompany genetic research

  43. Human Genome Project Francis Collins Craig Venter

  44. Human Genome Project • Consider ethical, legal and social issues • Who owns your DNA?

  45. Human Genome Project • Develop new lab technology • Automated Sequencing

  46. Human Genome Project • Disseminate genome information • GenBank database

  47. Human Genome Project • Analyze genetic variation among humans • The genome is approximately 99.9% identical between individuals of all nationalities and backgrounds.

  48. Human Genome Project • Map and sequence genomes of model organisms • E.coli • Arabidopsis thaliana • Saccharomyces cerevisiae • Drosophila melangaster • Caenorhabditis elegans • mus musculus

  49. PCR • Polymerase chain reaction (PCR) • A lab technique used to amplify segments of DNA "PCR has transformed molecular biology through vastly extending the capacity to identify, manipulate and reproduce DNA. It makes abundant what was once scarce -- the genetic material required for experimentations."

  50. PCR • Reaction requirements • Template DNA – total genomic DNA isolated from an organism that contains a target region to be amplified • DNA primers - Short pieces of single stranded DNA that flank the target • Taq DNA polymerase - Attaches nucleotides on the growing strand of DNA • Nucleotides (GATC) – Polymerase adds complementary nucleotides to the template

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