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Published byLynne Daniel Modified over 7 years ago
DNA Technology n Now it gets real….
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Recombinant DNA n Definition: DNA in which genes from 2 different sources are linked n Genetic engineering: direct manipulation of genes for practical purposes n Biotechnology: manipulation of organisms or their components to perform practical tasks or provide useful products
Bacterial plasmids in gene cloning
DNA Cloning n Restriction enzymes (endonucleases): in nature, these enzymes protect bacteria from intruding DNA; they cut up the DNA (restriction); very specific link to animation link to animation link to animation n Restriction site: recognition sequence for a particular restriction enzyme n Restriction fragments: segments of DNA cut by restriction enzymes in a reproducible way n Sticky end: short extensions of restriction fragments resulting from the “staggered cut” n DNA ligase: enzyme that can join the sticky ends of DNA fragments n Cloning vector: DNA molecule that can carry foreign DNA into a cell and replicate there (usually bacterial plasmids)
Steps for eukaryotic gene cloning n Isolation of cloning vector (bacterial plasmid) & gene- source DNA (gene of interest) n Insertion of gene-source DNA into the cloning vector using the same restriction enzyme; bind the fragmented DNA with DNA ligase n Introduction of cloning vector into cells (transformation by bacterial cells) n Cloning of cells (and foreign genes) n Identification of cell clones carrying the gene of interest n Campbell Link Campbell Link Campbell Link
DNA Analysis & Genomics n PCR (polymerase chain reaction) link to PCR animation link to PCR animation link to PCR animation n Gel electrophoresis electrophoresis animation link electrophoresis animation link electrophoresis animation link n Restriction fragment analysis (RFLPs) n Southern blotting n DNA sequencing n Human genome project
Polymerase chain reaction (PCR) n Amplification of any piece of DNA without cells (in vitro) n Materials: heat, DNA polymerase, nucleotides, single- stranded DNA primers PCR animation link PCR animation link PCR animation link n Applications: fossils, forensics, prenatal diagnosis, etc.
DNA Analysis n Gel electrophoresis: separates nucleic acids or proteins on the basis of size or electrical charge creating DNA bands of the same length electrophoresis animation link electrophoresis animation link electrophoresis animation link
Restriction fragment analysis n Restriction fragment length polymorphisms (RFLPs) n Southern blotting: process that reveals sequences and the RFLPs in a DNA sequence n DNA Fingerprinting
DNA Sequencing n Determination of nucleotide sequences Sanger method, Sanger link 1 Sanger link 2 Sanger link 1 Sanger link Sanger link 1 Sanger link n Genomics: the study of genomes based on DNA sequences n Human Genome Project
Practical DNA Technology Uses n Diagnosis of disease n Human gene therapy n Pharmaceutical products (vaccines) n Forensics n Animal husbandry (transgenic organisms) n Genetic engineering in plants n Ethical concerns?
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