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Download Dr T-J’s Minilecture - Susquehanna University
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Transcript
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 DNA fragments may be amplified (cloned) by joining with plasmid DNA and replication of the recombinant DNA in bacteria Foreign DNA and vector DNA both must have matching sticky ends Size limits of foreign DNA that can be inserted into different cloning vectors Other Vectors: BACs and YACs Different DNA fragments created by a restriction nuclease may be joined in many different arrangements since they all have the same sticky ends 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. Useful features of a plasmid cloning vector Use of lacZ a-peptide coding sequence for color-dependent selection of recombinant clones Use of a radioactive probe and hybridization to immobilized DNA on a filter for selection of desired clones Contigs - Assembling full sequences from smaller parts 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 Steps in the creation of a transgenic mouse Methodology for gene knockout or gene replacement using a “targeting” vector Site-specific mutagenesis of a cloned DNA sequence using a synthetic mutagenic primer
