KelleyLect3_2009 - Manipulation and molecular...

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Unformatted text preview: Manipulation and molecular characterization of nucleic acids characterization • Reading nucleic acids sequences - Restriction analysis - Blotting - Role of synthetic DNA - DNA sequencing - PCR • Manipulating nucleic acids for - Recombinant DNA technology - Site directed mutagenesis - Transgenic animals - Transgenic plants • Other the study of gene function methodologies for studying nucleic acids - siRNA - microarrays Decoding nucleic acids DNA molecular structure Genomic structure • How can genomes be made into more manageable fragments for study? • How can base sequences be read? • How can chromosomal organization be studied? Genomic sequence: governs function Restriction nucleases provide a tool for DNA manipulation 1962: First restriction nuclease discovered Restriction nucleases recognize specific DNA sites Gel electrophoresis Single base resolution Chromosomal resolution Restriction nucleases provide a tool for DNA manipulation Restriction digest of λ-DNA λ=4 λ=4 Blotting techniques for nucleic acids analysis Probe design is critical for interpretable blots Good probe Bad probe Fluorescence in situ hybridization Modern technique for visualizing localization of specific probes within chromosomes Production of synthetic DNA Early days of molecular biology: probes made enzymatically 1980s: DNA synthesis developed and automated Production of synthetic DNA Chemist’s reagent Nature’s reagents Chemical DNA synthesis DNA sequencing G>A dimethyl sulfate under neutral conditions (reacts with N7) A>G dimethyl sulfate under acidic conditions C hydrazine in 1.5M NaCl (adds to C4-C6) C+T hydrazine DNA sequencing Sanger DNA sequencing Sanger Sanger DNA sequencing Sanger Automated DNA sequencing Automated Single molecule DNA sequencing Single www.helicosbio.com Genome sequencing Genome Sequencing of the human genome Sequencing Sequencing of the human genome Sequencing Amplification of nucleic acids using PCR Amplification PCR: polymerase chain reaction Amplification of nucleic acids using PCR Amplification PCR: polymerase chain reaction Application of PCR in forensic science Application Lengths of PCR products in highly variable regions used to “fingerprint” individuals: Chance of 2 individuals having the same pattern is 1 in 10 billion Application of PCR in forensic science Application Real-time PCR Real-time Diagnosis of chronic myeloid leukemia using real-time PCR using Recombinant DNA technology Recombinant Key advances enabling Key production of production artificial genes -Restriction digest allows Restriction generation of fragments with known ends with -PCR allows generation of PCR large quantities of known sequences sequences Recombinant DNA technology Recombinant Key advances enabling Key production of artificial genes artificial -Restriction digest allows Restriction generation of fragments with known ends with -PCR allows generation of PCR large quantities of known sequences sequences Recombinant DNA technology Recombinant Production of recombinant clones Production of insulin via gene cloning and bacterial synthesis cloning Production of human genome gene “library” gene Transgenic animals Transgenic Transgenic plants Transgenic Site-directed mutagenesis Site-directed Inventor: Michael Smith (UBC) – Nobel Prize 1983 Site-directed mutagenesis Site-directed of green fluorescent protein Microarray technology technology Applications of DNA microarrays: cancer diagnosis Applications AML: acute AML: myeloid leukemia myeloid ALL: acute ALL: lymphoblastic leukemia lymphoblastic AML and ALL have AML different treatments, different cure rates are diminished cure if wrong treatment is used used 1100 genes can be 1100 observed to have differential expression using microarray analysis analysis RNA interference (RNAi) RNA Knockdown of GFP using RNAi (GFP = green fluorescent protein) DAPI stain Fluorescence from GFP - RNAi + RNAi Summary: Manipulation and molecular Summary: characterization of nucleic acids characterization Tools for the manipulation of nucleic acids in vitro Restriction enzymes Recombinant DNA Site directed mutagenesis Chemical DNA synthesis Tools for the manipulation of nucleic acids in vivo Transgenic animals Transgenic plants RNA interference Tools for monitoring structure/sequence/function of nucleic acids DNA sequencing Blotting Microarrays Real-time PCR ...
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