Recombinant DNA prepare foreign (target) DNA prepare vector (host) recombine target and vector DNA introduce rDNA to host screen for DNA of interest gDNA (fragments) cDNA (copy of RNA) Preparing gDNA restriction enzymes ‘random nuclease’  size fractionate

gDNA vs cDNA level of expression introns ease of preparation 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

Useful Plasmid Features Relaxed Replication Selectable Markers Streamlined Polylinker or MCS Identification of Recombinants most derived from pUC or pBR322 |SacI| |ScII| |XbaI||SpeI||BamH||SmaI||PstI||EcRI||EcRV||HIII||ClaI| |SalI||XhoI| |KpnI| GAGCTCCACCGCGGTGGCGGCCGCTCTAGAACTAGTGGATCCCCCGGGCTGCAGGAATTCGATATCAAGCTTATCGATACCGTCGACCTCGAGGGGGGGCCCGGTACC CTCGAGGTGGCGCCACCGCCGGCGAGATCTTGATCACCTAGGGGGCCCGACGTCCTTAAGCTATAGTTCGAATAGCTATGGCAGCTGGAGCTCCCCCCCGGGCCATGG Multiple Cloning Site:

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

Intramolecular vs. Intermolecular

Removal of 5’-PO4 Prevents Vector Self Ligation

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