Lecture 26 Chapter 41 Recombinant DNA Cloning and Crea on of Chimeric Genes Outline Restric on Enzymes and their uses Cloning de ni on of plasmid construc on of a chimeric plasmid transforma on DNA sequencing Polymerase Chain Reac on 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 Restric on Endonucleases EcoRI Man S cky ends can be ligated A A B B Horse B A A B Other common Restric on enzymes Southern bloPng EcoR1 sites 5 Gene A 3 DNA probe P32 5 3 Southern bloPng 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 Fingerprin ng Mr G DNA found at the crime scene suspects Mr T Mr Z Mr Y Mr X vic m Fragment size All human DNA is 99 9 iden cal but 0 1 is variable This usually occurs in non protein coding regions Variable Number Tandem Repeat VNTR How to Clone Cloning Picture of clone wars Cloned Gene inserted into Plasmid In vivo synthesis of Recombinant proteins Func on Structure Pharmaceu cal biochemical use Inserted into cells organism Func on Distribu on E ect of muta on Gene therapy Complementary DNA Human Proinsulin cDNA Cloning Transforma on Infect E coli AMP Ampicllin resistance gene Generate recombinant proteins or more plasmids Eg Human insulin an bio c Bacterial Division DNA Sequencing Sanger dideoxy method Controlled termina on of replica on Strand Elonga on Reac on dNTP 3 A 5 A A C C G G G G T T T template 3 NNNNNNCTAAGCTCGACT 5 dGTP 5 NNNNNNGATTCG A Primer template 3 NNNNNNCTAAGCTCGACT 5 5 NNNNNNG A Primer dTTP dCTP dTTP ddATP dCTP dATP dATP dGTP GA G GAT Polymerase Chain Reac on PCR 3 5 5 3 dsDNA Template 2 short synthe c DNA primers 15 20 bp one to anneal to top strand and one for bogom strand DNA polymerase dNTPs Thermocycler Polymerase Chain Reac on PCR 3 5 5 3 3 5 5 3 5 3 5 3 5 3 5 5 3 primers 5 5 3 3 5 5 3 5 5 5 3 3 5 3 5 3 5 5 3 3 5 5 5 5 5 5 3 5 3 3 3 5 3 3 5 3 5 3 5 5 5 3 5 3 5 3 3 3 5 3 3 3 5 5 5 3 3 5 3 5 5 3 5 5 3 3 5 5 3 5 3 3 5 3 3 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 Ampli ca on by qPCR qPCR Cycle number of mRNA Microarrays are used to inves gate the transcriptome Control cells Treated or cancer cells Isolate mRNA Reverse transcriptase Fluorescent labeled cDNA Combine equal amounts Adapted from hgp www cs wustl edu jbuhler research array Hybridiza on step 4 Microarray chip slide hgp en wikipedia org wiki File NA hybrid svg Scan Results induc on repression Learning Goals Understand how restric on enzymes work Knowing what to clone means Understanding the principles of Southern bloPng the polymerase chain reac on PCR microarrays and DNA sequencing
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