CHAPTER 10 Genomics Proteomics and Genetic Engineering Genomics deals with the DNA sequence organization function and evolution of genomes Proteomics identifies all proteins in a cell or organism Genomics was made possible by the invention of techniques originally devised for the manipulation of genes and the creation of genetically engineered organisms with novel genotypes and phenotypes recombinant DNA gene cloning genetic engineering DNA is cut into fragments by restriction enzymes transgenic organisms genetically engineered A transgenic organism is also called a genetically modified organism GMO 10 1 Cloning a DNA molecule takes place in several steps Restriction enzymes cleave DNA into fragments with defined ends Restriction enzymes are nucleases that cleave DNA wherever it contains a particular short sequence of nucleotides that matches the restriction site of the enzyme Restriction enzymes cleave DNA in palindromic sequences the breaks need not be directly opposite one another in the two DNAs and enzymes that cleave the DNA asymmetrically generate DNA fragments with complementary ends Most restriction enzymes make staggered cuts in the DNA producing single stranded ends called sticky ends that adhere to each other because they contain complementary nucleotides A number of restriction enzymes cleave both DNAs at the center of symmetry blunt ends Ligation of sticky ends re creates the original restriction site but any blunt end can join with any other blunt end and not necessarily create a restriction site Restriction fragments of DNA obtained from one organism have the same sticky ends as restriction fragments from another organism if they were produced by the same restriction enzyme Restriction fragments are joined end to end to produce recombinant DNA A vector is a relatively small DNA molecule that is able to replicate inside a cell and usually contains one or more sequences able to confer antibiotic resistance on the cell The simplest types of vectors are plasmids with double stranded circular DNA When a transformant containing the recombinant molecule has been isolated the DNA fragment linked to the vector is cloned A vector is a DNA molecule into which another DNA fragment can be cloned a carrier for recombinant DNA A vector is a carrier for recombinant DNA Vector DNA can be introduced into a host cell relatively easily it contains a replication origin and so can replicate inside the host cell and cells containing the vector can usually be selected by a straightforward assay by allowing growth of host cell on selective medium Recombinant DNA can be introduced into cells by a kind of electrophoretic procedure called electroporation Plasmid vectors typically include one or more genes for antibiotic resistance Plasmids are most convenient for cloning relatively small DNA fragments 5 to 10 kb Larger fragments can be cloned with bacteriophages larger can be cloned with a cosmid vector Specialized vectors can carry very large DNA fragments The vectors that can accept large DNA fragments are called artificial chromosomes Among the most widely used are bacterial artificial chromosomes BACs 300kb The BAC vector is based on the F plasmid replication system and the copy number control Cloning large molecules mixing large fragments of source DNA with vector ligation with DNA ligase introduction of recombinant molecules into host and selecting clones of interest Shotgun sequencing involves small single stranded fragments of 500 1000 bp randomly needs to be carried out at high redundancy Each genomic region covered by overlapping clones is a contig Typically a genomic sequence has many gaps prevents contigs from being assembled Vector and target DNA fragments are joined with DNA ligase Joining sticky ends does not always produce a DNA sequence that has functional genes Restriction fragments from the vector can join together in the wrong order this can be avoided by using a vector that has only one cleavage site for a particular restriction enzyme A recombinant cDNA contains the coding sequence of a eukaryotic gene Bacterial cells are not capable of RNA splicing Cloning from mRNA depends on reverse transcriptase which can use a single stranded RNA as a template and synthesize a complementary DNA or cDNA The stretch of A nucleotides at the 3 end of mRNA serves as a primer for reverse transcriptase The 3 end can hairpin serving as a primer for second strand synthesis which can be synthesized by DNA polymerase or by reverse transcriptase The joining of cDNA to a vector can be done by available procedures for joining blunt ends The efficiency of cloning rare cDNA molecules can be markedly increased by PCR amplification prior to ligation into the vector PCR amplification of the cDNA produced by reverse transcriptase is called reverse transcriptase PCR RT PCR Result is very little contaminating DNA Loss of beta galactosidase activity is often used to detect recombinant vectors To isolate a vector must ensure that the vector possesses an inserted DNA fragment and the fragment is the DNA segment of interest The cloning site is called a multiple cloning site MCS or polylinker because it contains unique cleavage sites for many different restriction enzymes and enables many types of restriction fragments to be inserted When a fragment of DNA inserted into the MCS interrupts the lacZ the recombinant plasmid yields Lac cells because the interruption renders the lacZ nonfunctional Lac colonies that contain nonrecombinant plasmids are blue and Lac colonies that contain recombinant plasmids are white Recombinant clones are often identified by hybridization with labeled probe In colony hybridization bacterial colonies are transferred to a filter treated with alkali to separate DNA strands denature flooded with mRNA or DNA hybridized washed dried and the labeled probe forms duplex with cDNA renatured 10 2 A genomic sequence is like a book without an index and identifying genes and their functions is a major challenge HUMAN GENOME PROJECT The protein coding potential of an organism is contained in its genome sequence A genome sequence without annotation is meaningless Genomic annotation is explanatory notes that accompany a sequence of DNA The annotation of genomic sequences is one aspect of computational genomics which is the use of computers in the interpretation and management of biological data Comparison among genomes is an aid to annotation Comparative genomics is a powerful strategy for