Chapter 15 DNA technology and Genomics recombinant DNA molecule made by combining genes from different organisms restriction nucleases used to cut DNA molecules in places manageable sections from bacteria vector molecules carrier that transports DNA into a cell ex bacteriaphages and plasmids plasmids circular small DNA molecule may be present replicate inside bacteria cell ex e coli recombinant plasmid has foreign DNA spliced into it foreign DNA are copied cloned restriction nucleases molecular scissors restriction site in DNA sequence cleavage site that is cut by restriction enzyme usually named after bacteria originally isolated from restriction enzyme can fight off bacteriophage infections Palindromic Sequences base sequence of one strand reads same as complement when read in same direction blunt end staggered end complementary ends DNA Ligase enzyme that covalently links 2 DNA fragments to form stable new DNA molecule DNA CAN BE CLONED INSIDE CELL genome total DNA in a cell genomic DNA library collection of fragments that make up all DNA in a genome use to isolate and study specific genes chromosome library contains all DNA fragments in single chromosome make fragments using restriction enzymes have identical sticky ends plasmid DNA vector gets same enzyme linear molecule sticky ends are compliments make recombinant DNA DNA ligase covalently bonds human and plasmid plasmid anti biotic sensitive cell via transformation only cells with new DNA are an tibiotic resistant cell divides and makes new DNA copies colony clone of genetically identical cells from single cell 1 2 3 4 5 6 contain all same recombinant plasmid DNA at this time also cloned Genomic Probe segment of DNA that is homologous to sequence of interest usually radioactively labeled single strand of DNA hybridizes attached by base pairing to complementary base sequence transfer cells from bacterial colonies containing recomb plasmids to nitrocellulose nylon membrane replica of colonies lyse cells to release DNA and making single strand treat with radioactive probe any where complimentary to that particular probe detected by autoradiography cDNA wish to clone intact genes but avoid introns do not code for protein complimentary DNA cDNA copies of mature mRNA from which introns have been removed complementary to mRNA without intron reverse transcriptase enzyme to synthesize single cDNA separate from mRNA and double strand strand with DNA polymerase cDNA Library formed using mRNA from single cell type double strand inserted to plasmid virus and multiply inside random sample of all DNA sequences in genome contain repeats introns exons gene regulatory sequences and spacer DNAs non tran Clones Genomic DNA clones scribed and sit between random repeats determine complete genome cDNA clones expressed at that time only protein coding sequences of mRNAs in cell cDNA library represents mRNA molecules from distinct tissue types and therefore genes contain uninterrupted coding sequences of genes PCR Polymerase Chain Reaction can detect presence of viral genome in blood sample used in amplifying DNA in vitro outside living organisms without cloning amplified x millions in a few hours DNA polymerase uses nucleotides and primers synthesized mucleotides during DNA replication to give 3 end to which DNA polymerase adds nucleotides to replicate DNA denature heat separates the 2 strands and duplicates again only need 20 heat cool cycles to get 1 million copies use heat resistant DNA polymerase con too sensitive