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Dr T-J’s Minilecture

Dr T-J’s Minilecture. Chapter 12. Restriction nuclease cutting followed by ligation of sticky ends creates closed circles from linear DNA fragments. Restriction nuclease cutting may generate sticky (with overhangs)- or blunt-ends.

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Dr T-J’s Minilecture

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  1. Dr T-J’s Minilecture Chapter 12

  2. Restriction nuclease cutting followed by ligation of sticky ends creates closed circles from linear DNA fragments

  3. Restriction nuclease cutting may generate sticky (with overhangs)- or blunt-ends

  4. DNA fragments may be amplified (cloned) by joining with plasmid DNA and replication of the recombinant DNA in bacteria

  5. Foreign DNA and vector DNA both must have matching sticky ends

  6. Size limits of foreign DNA that can be inserted into different cloning vectors

  7. Other Vectors: BACs and YACs

  8. Different DNA fragments created by a restriction nuclease may be joined in many different arrangements since they all have the same sticky ends

  9. RNA templates may be copied into double stranded DNA and then cloned [complementary DNA (cDNA) cloning] After being copied into DNA, the RNA template is usually destroyed (rather than displaced) before the synthesis of the second DNA strand.

  10. Useful features of a plasmid cloning vector

  11. Use of lacZ a-peptide coding sequence for color-dependent selection of recombinant clones

  12. Use of a radioactive probe and hybridization to immobilized DNA on a filter for selection of desired clones

  13. Contigs - Assembling full sequences from smaller parts

  14. Use of DNA microarrays (chips) Fluorescently tagged cDNA probes are hybridized to DNA spots in the microarray for studying differential expression of thousands of genes at a time in two mRNA samples

  15. Steps in the creation of a transgenic mouse

  16. Methodology for gene knockout or gene replacement using a “targeting” vector

  17. Site-specific mutagenesis of a cloned DNA sequence using a synthetic mutagenic primer

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