9 10 2015 Early DNA Technology experiments selective breeding Modern DNA Technology In restriction nuclease digestion restriction enzyme nucleases cleave DNA at specific sequences Electrophoresis separates DNA molecules on the basis of size DNA is visualized with fluorescent dyes such as ethidium bromide It is a defense mechanisms for the bacterial cell to chop up any foreign DNA with the given sequence It cuts the palindromic sequence has four to six bases A molecule of DNA can undergo denaturation and renaturation hybridization Reading pp 327 328 DNA ligase can join together any two DNA fragments in vitro to produce recombinant DNA molecules 1 9 10 2015 DNA Cloning in Bacteria DNA Cloning refers to the production of many identical copies of a DNA sequence Is the most important feats of recombinant DNA technology makes it possible to separate a gene of interest from the rest of a cell s genome Recombinant DNA refers to DNA that has been artificially manipulated to combine genes from two different sources Cloning Vector Plasmid extrachromosomal circular double stranded DNA self replicable often carry antibiotic resistance gene Human genomic libraries containing DNA fragments representing the whole human genome A cloning vector for use in bacteria must contain the following 1 a replication origin to allow the plasmid to be replicated in a bacterial cell independently 2 at least one unique restriction site to allow easy insertion of foreign DNA 3 an antibiotic resistance gene or some other selectable marker gene to allow selection for bacteria that have taken up the recombinant plasmids Complementary DNA cDNA is prepared from mRNA 2 9 10 2015 To clone a gene from a eukaryote 1 has to be able to isolate the mature mRNA with the introns removed 2 Then use reverse transcriptase to synthesize a complmentary strand of DNA RNA hybrid DNA 3 Then alkali treatment destroys the RNA strand leaving a strand of DNA 4 This DNA is replicated by E coli DNA polymerase DS DNA and called complementary DNA 5 The cDNA can then be treated as bacterial DNA in E coli Polymerase chain reaction PCR generates millions of copies of a target piece of DNA Polymerase chain reaction PCR generates millions of copies of a target piece of DNA 72 C 94 96 C 50 60 C primers short DNA molecules that define the segment of DNA to be amplified DNA polymerase Taq synthesizes DNA by Reading adding dNTPs to the end of the primer pp 335 339 PCR is often the first step in cloning PCR template can be genomic DNA complementary DNA cDNA DNA copies of RNA are generated by reverse transcriptase antibiotic resistance gene Ligation DNA cloning Transformation Reading pp 330 333 http tainano com chin Molecular 20Biology 20Glossary htm Ligate the DNA molecules together with ligase Dideoxy DNA sequencing generates a population of sequences that terminate at each base in the DNA fragment being sequenced Transform E coli with the recombinant plasmid E coli that do not take up the plasmid die when plated on media containing antibiotics purify the plasmid DNA Reading pp 341 3 9 10 2015 Originally sequencing reactions were radioactively labeled and the DNA fragments were separated by electrophoresis on a gel Modern sequencing reactions are fluorescently labeled and the DNA fragments are separated via electrophoresis on a capillary gel 4
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