Lecture 26 Chapter 41 Recombinant DNA Cloning and Creation of Chimeric Genes Outline Restriction Enzymes and their uses Cloning definition of plasmid construction of a chimeric plasmid transformation DNA sequencing Polymerase Chain Reaction Microarrays Nucleases Nucleases are enzymes that cut nucleic acids by hydrolyzing phosphodiester bonds DNase DNA RNase RNA Some do both Exonuclease starts at an end Endonuclease cuts internally Products may be oligonucleotides or nucleoside phosphates Most are relatively nonspecific Restriction Endonucleases Restriction Enzymes Recognize specific sequences in DNA Many cut specifically at the recognition sequence Products are relatively large polynucleotides Some restriction enzymes produce products with sticky ends Uses of Restriction Enzymes Cloning Any two pieces of DNA with compatible ends can be easily ligated together Analysis The relative position of restriction sites provides information about the sequence of the DNA Restriction analysis allows easy comparison of two different DNAs or confirmation of cloning events Restriction Endonucleases EcoRI Man Sticky ends can be ligated A A B B Horse B A A B Other common Restriction enzymes Southern blotting EcoR1 sites 5 Gene A 3 DNA probe P32 5 3 Southern blotting Pink Chromosome EcoR1 sites 5 Gene A Blue Chromosome 3 EcoR1 sites 5 Gene A Cut out from pink Chromosome Gene A Cut out from Blue Chromosome 3 DNA Fingerprinting All human DNA is 99 9 identical but 0 1 is variable This usually occurs in non protein coding regions Variable Number Tandem Repeat VNTR suspects How to Clone Cloning Picture of clone wars Cloned Gene inserted into Plasmid In vivo synthesis of Recombinant proteins Function Structure Pharmaceutical bio chemical use Inserted into cells organis m Function Distribution Effect of mutation Gene therapy Complementary DNA Human Proinsulin cDNA Cloning Transformation Infect E coli AMP Ampicllin resistance gene Generate recombinant proteins or more plasmids Eg Human insulin antibiotic Bacterial Division DNA Sequencing Sanger dideoxy method Controlled termination of replication Strand Elongation Reaction dNTP 3 5 A A G C C T T G A G G T template 3 NNNNNNCTAAGCTCGACT 5 dGTP 5 NNNNNNGATTCG A Primer template 3 NNNNNNCTAAGCTCGACT 5 5 NNNNNNGA Primer dCTP dTTP dTTP ddATP dCTP dATP dATP dGTP GA G GAT Polymerase Chain Reaction PCR 3 5 5 3 dsDNA Template 2 short synthetic DNA primers 15 20 bp one to anneal to top strand and one for bottom strand DNA polymerase dNTPs Thermocycler Polymerase Chain Reaction PCR 3 5 5 3 3 5 5 3 5 3 5 primers 5 5 3 3 5 5 3 5 3 5 3 5 5 3 5 5 3 3 5 3 5 3 5 5 3 3 3 5 5 5 5 5 5 3 5 3 3 3 5 5 5 5 3 5 3 5 3 3 3 5 3 5 3 3 5 3 3 5 5 5 3 3 3 5 3 5 3 5 3 3 5 5 3 3 5 5 3 5 3 3 5 5 3 3 5 3 5 5 3 5 3 3 5 3 5 5 3 5 3 3 5 3 5 5 3 5 3 Reference mRNA mRNA mRNA mRNA cDNA Reverse cDNA transcriptase cDNA cDNA qPCR Amplification by qPCR qPCR Cycle number of mRNA Microarrays are used to investigate the transcriptome Control cells Treated or cancer cells Isolate mRNA Reverse transcriptase Fluorescent labeled cDNA Combine equal amounts Adapted from http www cs wustl edu j buhler research array Hybridization step 4 Microarray chip slide http en wikipedia org wiki Fil e NA hybrid svg Scan Results induction repression Learning Goals Understand how restriction enzymes work Knowing what to clone means Understanding the principles of Southern blotting the polymerase chain reaction PCR microarrays and DNA sequencing
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