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gDNA (fragments). cDNA (copy of RNA). Preparing gDNA restriction enzymes ‘random nuclease’ size fractionate. Recombinant DNA. prepare foreign (target) DNA prepare vector (host) recombine target and vector DNA introduce rDNA to host screen for DNA of interest . gDNA vs cDNA.
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gDNA (fragments) cDNA (copy of RNA) • Preparing gDNA • restriction enzymes • ‘random nuclease’ • size fractionate Recombinant DNA • prepare foreign (target) DNA • prepare vector (host) • recombine target and vector DNA • introduce rDNA to host • screen for DNA of interest
gDNA vs cDNA • level of expression • differential gene expression • accessibility • introns • complicates gDNA analysis • can preclude expression • ease of preparation • cDNA more work
Preparation of cDNA • 1) Isolate mRNA • 2) Synthesize DNA-RNA hybrid • reverse transcriptase • oligo-dT primer • random priming • 3) Synthesize 2nd DNA strand • 4) Add termini RNA dependent DNA polymerase
1) Isolate mRNA • 2) Synthesize DNA-RNA hybrid • 3) Synthesize 2nd DNA strand • self-priming • replacement synthesis • primed synthesis • 4) Add termini
1) Isolate mRNA • 2) Synthesize DNA-RNA hybrid • 3) Synthesize 2nd DNA strand • 4) Add termini • i.e., linkers CGGAATTCCG GCCTTAAGGC Eco RI
Recombinant DNA Vectors • autonomously-replicating DNA used to ‘carry’ and amplify foreign DNA within host cell • eg: plasmids, phage/viruses, or combinations • Plasmids • extra-chromosomal elements • 1-200 kb size range • transmitted during conjugation • antibiotic resistance • low copy number vs high copy number
Multiple Cloning Site: |SacI||ScII||XbaI||SpeI||BamH||SmaI||PstI||EcRI||EcRV||HIII||ClaI||SalI||XhoI||KpnI| GAGCTCCACCGCGGTGGCGGCCGCTCTAGAACTAGTGGATCCCCCGGGCTGCAGGAATTCGATATCAAGCTTATCGATACCGTCGACCTCGAGGGGGGGCCCGGTACC CTCGAGGTGGCGCCACCGCCGGCGAGATCTTGATCACCTAGGGGGCCCGACGTCCTTAAGCTATAGTTCGAATAGCTATGGCAGCTGGAGCTCCCCCCCGGGCCATGG Useful Plasmid Features • Relaxed Replication • Selectable Markers • Streamlined • Polylinker or MCS • Identification of Recombinants • most derived from pUC or pBR322
Generic rDNA Protocol • prepare foreign DNA • prepare vector • ligate foreign DNA and vector • introduce vector into host • screen for rDNA of interest
Ligation Reaction • mix foreign and vector DNA in presence of DNA ligase • optimal ratios of vector to insert generally 1.5-2:1 • intermolecular base-pairing can occur between compatible overhangs
DNA Ligase • catalyzes the formation of phosphodiester bond between 5’-PO4 and 3’-OH • i.e., joins DNA fragments • typically carried out at lower temperatures (8-16o) for extended periods
prepare foreign DNA • prepare vector • ligate target and vector • introduce rDNA to host • heat shock + Ca2+ • electroporation • select for transformants with antibiotic • screen for rDNA of interest
Colony Lift • Sources of Probes • cloned genes • synthetic oligonucleotides • PCR products
Identifying Recombinants • based on interruption of a gene • eg., lacZ gene = b-galactosidase • intact b-galactosidase produces blue color in presence of X-gal • -complementation or blue-white screening