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Chapter 17 Vocabulary: Recombinant DNA Restriction endonucleases Gel electrophoresis Clone Vector DNA library cDNA DNA fingerprinting PCR Southern blot Northern blot Western blot Gene therapy Oultine 17.1 Recombinant DNA Recombinant DNA (DNA that contains information from 2 different sources) Uses restriction enzymes (restriction endonucleases) to cut DNA at specific sites Allows for creation of physical maps Allows for creation of recombinants Restriction enzymes usually cut at palindromes leaving “sticky ends” Can also make blunt cuts DNA ligase can seal recombinants Bacteria pick up recombinant DNA when the membrane is compromised by temperature of electrical impulses Gel electrophoresis uses an electric current to separate DNA based on size. 17.2 Introducing Foreign DNA into Cells Clone Vectors carry foreign DNA for cloning Plasmids Must have: origin of replication (so bacteria will copy the plasmid) Selectable marker (to check for successful recombination) Restriction sites (places DNA can be introduced) Artificial chromosomes (for larger DNA) Vectors for eukaryotic cells (no plasmids so harder to accomplish) Inject DNA into fertilized egg cell Inject stem cells into an embryo Gene gun Bacterial pathogens (tumor inducing plasmids) carrying gene of interest 17.3 Amplifying DNA Without a Vector - PCR PCR makes millions of copies of a sample of DNA Requires: DNA of interest (to be sequenced) Primers (to initiate replication and to bind to DNA of interest) Deoxyribo nucleotides (to add to growing strands of DNA) Taq polymerase (to catalyze reaction) Heating cycles cause dsDNA to separate Cooling cycles allows primer to bind and DNA polymerase to synthesize DNA This continues in an automated way for many cycles 17.4 Storing and Sorting DNA Fragments DNA libraries contain entire genome of an organism contained in vectors cDNA libraries contain the coding genome of an organism (can be different at different developmental stages or in different tissues) mature mRNA transcript reverse transcriptase mRNA / DNA hybrid RNase Single stranded DNA DNA polymerase Double stranded DNA 17.5 Analyzing and Creating DNA differences Southern blot procedure Cut DNA with restriction enzymes Separate DNA fragments with gel electrophoresis Denature dsDNA Blot gel onto filter paper Incubate filter paper with probe (complementary to specific gene) Expose bands that bound to probe Uses: To identify a single gene among thousands of fragments of DNA Detect sequences of DNA in an organism’s genome Used in gene discovery and gene mapping To analyze the genetic patterns in an organism’s DNA To identify gene mutations, deletions, duplications, and gene rearrangements involved in diseases To determine the number of copies of a particular DNA sequence presented in the genome of an organism To identify related DNA sequences in the genome and to determine if there is a gene family To detect certain cancers and genetic diseases Used in DNA fingerprinting, genetic engineering, & forensic science for tests such as: Paternity testing Personal identification Sex determination Species exclusion Northern blot is used for mRNA detection (does not require Denaturation) Western blot is used for protein detection (uses antibodies to bind to proteins) DNA fingerprinting uses restriction enzymes and gel electrophoresis to diagnose disease, establish biological relationships, identify individuals or in criminal cases DNA fingerprints are like consumer good barcodes; all a little different Takes advantage of short tandem repeats that vary in the human genome We can work backward to determine the gene of interest by using “knock out” experiments 17.6 Medical Applications Human genes inserted into bacteria for production Insulin, interferon, human growth hormone, erythropoietin, etc. Atrial peptides, tissue plasminogen activators Subunit vaccines DNA vaccines Plant based vaccines Gene therapy replaces a defective allele with a working copy to overcome disorders / diseases RNAi vectors Viral vectors Hurtles to gene therapy seem to be insertion points and regulation of the new gene 17.6 Agricultural Applications Plants get genes to alter their phenotype Disease resistance, insect resistance, herbicide resistance, drought resistance, changes in nutritional content, biopharming of useful compounds Concerns include cross pollination with wild type varieties, loss of biodiversity, long term effects of consumption of genetically modified food, possible allergic reactions Domesticated animals are also modified Improvements in desirable traits, pharmaceutical production in milk, environmental benefit in waste alteration Most important information from this chapter: How to make recombinants using restriction enzymes (plasmids, vectors) Gel electrophoresis. PCR How to make cDNA and why one would want to Southern, Western, Northern blotting techniques DNA fingerprinting (they should be able to read a gel to determine if a suspect is guilty. They should also be able to read a gel to determine paternity.) Gene therapy Mention applications in medicine and agriculture as examples not as information to be memorized.