Chapter 16 - 204-325 1 Summary of the Last Lecture...

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Unformatted text preview: 204-325 1 Summary of the Last Lecture Transposable elements are mobile genetic elements that can occasionally move from one position of the genome to another position in a process known as transposition. Transpositions are either DNA-based or RNA-based. IS, Tn in bacteria, Ac in corn and P element in Drosophila are DNA-based. The element is autonomous if transposase is supplied by the same element or nonautonomous if it is not. Retrotrnasposons, such as Ty of yeast, copia of Drosophila and human SINE and LINS, transpose through RNA intermediates and need reverse transcriptase. Transposable element can cause mutations of gene, changes in transcription regulation, or chromosome rearrangement. 2 Today's Topics 1. Molecular Cloning 2. Genomic and cDNA Libraries 3. Restriction Mapping 4. DNA Sequencing 5. Polymerase Chain Reaction (PCR) 3 Topic 1 Molecular Cloning 4 Restriction Enzyme Cuts at Specific Palindromic Restriction Sites Restriction Site =palindrome: Was it a car or a cat I saw? 5 Different Ways of Cleavage by Restriction Enzymes overhang=sticky end 6 List of Some Restriction Endonuclease 7 List of Some Restriction Endonuclease 8 Frequency of Restriction Sites Assumptions: 1. GC is 50% 2. Sequence is totally random 9 Engineer Recombinant DNA by Restriction and Ligation + Ligase 10 Ligation of Adjacent DNA Fragments 5' 11 Plasmid Cloning Vector PUC 19 Plasmid: Small, circular, non essential extrachromosomal DNA. Plasmids as Cloning Vectors: 1. ori sequence for replication (high copy number) 2. Selectable marker for maintenance 3. Multiple unique restriction sites for cloning (Polylinker) 4. Other convenient feature (color selection). 12 Recombinant DNA: Insertion of a Piece of DNA into Vector Vector recirculation without insert: lacZ functional 13 Molecular Cloning: Making Many Copies of the Same DNA Piece Transformation (Chapter 9) Clones 14 Animation 1: Molecular Cloning 15 Yeast Artificial Chromosome (YAC) Cloning Vector ARS ARS ARS 16 Bacterial Artificial Chromosome (BAC) Cloning Vector BAC is based on the 7-kb F plasmid of E. coli HindIII and BamHI are cloning sites at which foreign DNA is inserted CMR is chloramphenicol resistance gene oriS, repE, parA, and parB are F genes for replication and regulation of copy number . 17 Topic 2 Genomic and cDNA Libraries 18 Library Library: a collection of DNA sequence from an organism, each of which has been cloned into a vector and each DNA sequence from the source is represented at least once. Genomic libraries (made from genomic DNA) cDNA libraries (made from cDNA- copy of mRNA) 19 Constructing a Genomic Library 20 Synthesis of Complementary DNA (cDNA) from mRNA 21 Difference between Genomic and cDNA Libraries Individual, tissue and stage specific Individual specific 22 Screening a cDNA Library Using an Antibody Probe 23 Screen Genomic Library for Specific Sequence by DNA Probe 24 Topic 3 Restriction Mapping 25 Constructing a Restriction Map for EcoRI and BamHI 26 Restriction Mapping to Confirm a Plasmid 27 Gel Electrophoresis 28 Animation 2: Restriction Mapping 29 Topic 4 DNA Sequencing 30 A Dideoxynucleotide (ddNTP) 31 Features of DNA Polymerases (Chapter 11) 1. Can't make DNA strand de novo 2. Require DNA or RNA primer 3. New strand is extended in 5' to 3' direction 4. Use dNTP as substrates 5. Enforce base pairing (10-6 error rate) 6. Proofreading by 3' to 5' exonuclease activity, part of the polymerase (error rate reduced to 10-9) 32 Dideoxy DNA Sequencing of a DNA Fragment 33 A Sequencing Gel using Radioactivity - GTAC ddG ddA ddT ddG 5' + 34 Animation 3: DNA Sequencing 35 Automated DNA Sequencing Using Fluorescence 36 Output of Automated DNA Sequencer 37 Topic 5 Polymerase Chain Reaction (PCR) 38 PCR: Cell-free Molecular Cloning 39 PCR Cycles 40 PCR Amplification: 106 Folds in 10 Cycles; 109 Folds in 20 Cycles 41 Animation 4: PCR 42 Summary Using restriction enzymes and DNA ligase, a recombinant DNA molecule containing an insertion into a vector (plasmid, BAC or YAC) can be made in a test tube. By making genomic or cDNA library, a gene of interest can be isolated by screening the library with antibody or DNA probe. Restriction mapping is the determination of locations of restriction sites on a DNA fragment. Complete DNA sequence can be determined by using dideoxyribonucleotides and electrophoresis. Polymerase chain reaction (PCR) can amplify a specific DNA segment bracketed by two primers 43 ...
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