Slide 1Slide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Slide 23Slide 24Slide 25Slide 26Polymerase Chain ReactionSlide 28Slide 29Molecular analysis of genesForward genetics was the dominant paradigm for analyzing genes in the 20th century.•Genes were initially discovered by their mutant phenotypes, which yielded insight into the gene’s function.•But it was often years or decades before the gene responsible for a given phenotype could be located and subjected to molecular analysis.Mutant phenotype (white eyes)Find the geneSequence DNAWhiteIn 1953 Watson and Crick deduced the chemical structure of DNA, and proposed that genetic information is stored in the sequence of base pairs.With this realization, sequencing DNA (i.e. determining the order of base pairs along the length of the double helix) became the critical step in understanding the molecular biology of genes.As we discussed in Lecture 13, there are experimental methods for sequencing DNA molecules.The dideoxy chain termination method was developed in 1973, and is still used today.Watson, Figs. 7-15 & 7-16But the DNA sequencing of individual genes was complicated by the size and complexity of genomes:•Each gene is present in only one or two copies per cell, and represents only a tiny fraction of that cell’s DNA. •Traditional DNA sequencing methods require billions of copies of the same DNA sequence at a relatively high degree of purity.Lecture outlineToday we will focus on two experimental procedures that greatly facilitated the molecular analysis of individual genes in the 1970s:•Amplifying DNA sequences by cloning.•Constructing and screening DNA libraries as a way to find specific genes.We will finish by discussing the polymerase chain reaction (PCR), which revolutionized molecular biology in 1983 by making it possible to amplify specific DNAs without cloning.DNA cloning is a two-step procedure.Watson, Fig. 7-8First, the DNA of interest must be in a form that will be stably maintained inside of cells.This is usually accomplished by recombining that DNA into some form of vector.First, the DNA of interest must be in a form that will be stably maintained inside of cells.This is usually accomplished by recombining that DNA into some form of vector.Second, the recombinant DNA vector is transformed into a compatible host cell that is propagated indefinitely.Second, the recombinant DNA vector is transformed into a compatible host cell that is propagated indefinitely.The bacterium E. coli is widely used for the propagation of cloned DNAs:•E. coli can be raised in large volumes at minimal expense.•Cells double every 20 minutes with adequate nutrients.•Bacteria can serve as hosts for certain naturally occurring DNA vectors:-plasmids-bacteriophage viruses NOTE: for some experiments it is preferable to propagate cloned DNAs in non-bacterial cells, e.g. yeast or immortalized animal cell lines. For the sake of time we will not explore these alternatives in MCB 250.A 1 liter culture of E. coli can yield hundreds of micrograms of plasmid DNA, all carrying the same cloned DNA sequence.This is enough for hundreds or thousands of experiments.Bacterial plasmids•Plasmids are self-replicating extrachromosomal DNAs.•Once established in the bacterial cytoplasm, the plasmid is replicated and its genes expressed in parallel with the bacterial chromosome.•Plasmid DNA is easy to purify. Because the plasmid is much smaller than the bacterial chromosome, the two can be readily separated after lysing the cell. Watson, Fig. A-6The plasmids that are commonly used as cloning vectors (e.g. pBR322) were generated by modifying naturally occurring E. coli plasmids.A cloning vector must have several key features:1. An origin of replication that will be recognized by the host cell.2. One or more selectable marker gene(s). This intact plasmid has two genes that convey antibiotic resistance. 3. Cloning sites. pBR322 has 40 different unique restriction sites that can be used for DNA insertion. (Inserts are gener-ally < 10 kb in length).NucleaseDNA ligase+ ATPRestriction enzymes are endonucleases, i.e. they hydrolyze a phosphodies-ter bond to leave a 3’-OH and a 5’-phosphate.This phosphodiester bond can be reformed by adding DNA ligase plus ATP.Restriction enzymes recognize specific dsDNA sequences, and digest these sequences by making a single cut in each of the two DNA strands.+ H2OWatson, Fig. 7-4There are hundreds of restriction enzymes that recognize different DNA sequences and are used in cloning. Each one generates 1 of 3 different types of cut end:-blunt, e.g. HpaI-5’ overhang, e.g. EcoRI or HindIII-3’ overhang, e.g. PstIIn this example, chromosomal DNA is digested with two enzymes: EcoRI, which leaves a sticky 3' overhang, and PvuII, which leaves blunt ends. The resulting fragment is shown.The cloning vector is cut with these same 2 enzymes.DNA ligase is added to form phosphodiester bonds between the compatible ends of the cut plasmid and the chromosomal DNA fragment.3'-5'--5'-3'-5'-3'3'-5'-RECOMBINANT PLASMID WITH INSERT OF CHROMOSOMAL DNAThe ability to clone human DNA sequences into E. coli has had a major impact on medicine.Cloned human insulin can be synthesized in bacteria, and in 1982 was the first genetically engineered drug to become commercially available. Prior to that time diabetics had to purchase insulin purified from other animals such as swine. Cloned human insulin is signifi-cantly less costly, exceedingly pure, and virtually eliminates the risk of an allergic response.To express a protein like insulin in bacteria, the protein-coding DNA must be cloned into an expression vector which contains regulatory sequences for gene expression: •a bacterial promoter upstream of where the DNA is inserted. •a ribosome-binding site between the promoter and the insertion. •a transcriptional termi-nator downstream of the insertion.A DNA library is a collection of host cells which carry the