Molecular Biology Lecture Notes - BIOL10221

BIOL10221 “Molecular Biology” Dr David Boam Module 10 - Lecture 1: Genetic engineering  Aims of this lecture To introduce basic principles and themes in recombinant DNA technology To describe the process of Molecular cloning and restriction endonucleases To de...

BIOL10221 “Molecular Biology” Dr David Boam Module 10 - Lecture 1: Genetic engineering  Aims of this lecture To introduce basic principles and themes in recombinant DNA technology To describe the process of Molecular cloning and restriction endonucleases To describe gene library construction; differences between genomic and cDNA libraries To describe how to produce a recombinant protein with therapeutic function BL122 lecture 21 2  Genetic engineering Molecular Cloning - Cutting joining and propagating recombinant DNA Isolating genes - Gene libraries – cDNA – Genomic Therapeutic protein production BL122 lecture 21 3  What molecular cloning does for us Cloning DNA fragments: – Reduces the complexity of DNA Allows large scale production and analysis of purified single sequences BL122 lecture 21 4  Molecular cloning - overview Isolate DNA Cut DNA Join to a “vector” – recombinant Introduce recombinant vector into bacteria (transformation) Amplify recombinant DNA by bacterial growth BL122 lecture 21 5  Cutting and splicing DNA 5’-nnGAATTCnn-3’ PO4 HOGnn-5’ 3’-nnCTTAA Restriction endonuclease Cuts palindromic sequence ‘sticky ends’ can be rejoined There are many restriction endonucleases, each with different sequence specificities BL122 lecture 21 6  Molecular cloning Cut plasmid Cut DNA DNA ligase Contains a fragment selectable marker (antibiotic) and Phosphatase origin of Introduce into E. replication coli Select for antibiotic resistance  Unmodified plasmid  Recombinant plasmid  Circularised DNA fragment Phosphatase Will not replicate prevents recircularisation of BL122 lecture 21 plasmid 7  DNA libraries Isolation and separation of individual sequences within a cell Nuclear DNA - (genomic library) Transcription start Transcribed region All genes ATG TGA Enhancer Promoter Intron Exons mRNA – (cDNA library) only expressed genes (mRNA) Cell-type (tissue) specific Must be converted to DNA to be cloned (complementary or cDNA) mature mRNA AUG UGA 5'-UTR Translate 3'-UTR d region  DNA cDNA libraries Genomic Isolate mRNA Isolate DNA cut DNA Convert to cDNA – reverse transcriptase Insert into a vector and transform Colonies (clones) – 1 sequence per colony BL122 lecture 21 9  DNA libraries cDNA Genomic Comprises expressed Comprises sequences genes (transcriptome) representing the genome – ‘libraries’ from different same in all tissues tissues contain different Includes introns and sequences regulatory sequences as No untranscribed well as exons sequences “Raw material” for gene mapping and genome projects BL122 lecture 21 10  Genetic engineering in action – therapeutic proteins Why? – Many proteins have therapeutic value but hard to obtain – General principle Fuse coding region of gene to a strong promoter Insert recombinant gene into host Host multiplies and overproduces protein Purify protein BL122 lecture 21 11  Host systems Bacteria Animal cells – Advantages – Advantages Cheap Post-translational Fast growing modification Easy to maintain Soluble/properly stable folded – Disadvantages – Disadvantages Proteins may be Expensive insoluble/denatured Unstable No post- translational modification BL122 lecture 21 12  Case study - insulin Why? – Only animal sources – immunological problems, potential of cross-species infection Answer – produce human insulin in bacteria But – insulin is processed Answer – synthesise 2 chains apart then join BL122 lecture 21 13  Summary Molecular cloning – Why – How – Vectors – Enzymes – Selection Libraries – cDNA and genomic – Differences Recombinant protein production BL122 lecture 21 14 Intended learning objectives for this lecture You should have a basic understanding and knowledge of Basic principles of molecular cloning and molecular tools required to construct recombinant DNA molecules Use of recombinant DNA techniques to create and use gene libraries Differences in composition of and uses of genomic and cDNA libraries How recombinant DNA techniques have been employed to produce safe and pure therapeutic proteins Lecture 4/2 RNA polymerases