Objectives for Exam 4 Genetics, Spring 2012 Chapter on Recombinant DNA – Chapter 17 •Be able to define the term recombinant DNA. •recombinant DNA- joining together DNA sequences from different organisms (sources) in a test tube•not found in nature- usually produced by artificial means•How it works:•1. DNA to be cloned is purified from cells or tissues•2. Proteins called restriction enzymes are used to generate specific DNA fragments. These molecules recognize and cut DNA molecules at specific nucleotides•3. Fragments produced by the restriction enzymes are joined to other DNA molecules that serve as vectors (plasmids), or carrier molecules. A vector joined to a DNA fragment is a recombinant DNA molecule.•4. The recombinant DNA molecule is transferred to a host cell. Within the host cell, the recombinant molecule replicates, producing identical copies (clones) of the recombinant molecule.•5. As the host cell replicates, the recombinant molecules within them are passed on to all their progeny, creating a population of host cells, each of which carries copies of the cloned DNA sequence•6. The cloned DNA can be recovered from host cells, purified, and analyzed•7. The cloned DNA can also be transcribed, its mRNA translated, and the encoded gene product isolated and used for research or commercial purposes. •Know what restriction enzymes do, what organisms have them, and what a palindromic DNA sequence is. (Lect. 30, Slides 5-6)•restriction enzymes (endonucleases)- cut the DNA in a sequence specific manner; recognize and bind to specific nucleotide sequences•the ʻrecognition sitesʼ are Palindromic sequences•can cleave the specific sequence with the restriction enzyme on the organism DNA and the vector DNA. •now you have fragments with complementary single stranded sequences.•These single stranded sequences can now anneal (or stick together via complementary base pairing) to form a recombinant DNA•DNA ligase seals the gaps•restriction enzymes are produced by bacteria as a defense mechanism against viral infection by degrading the DNA of invading viruses•Be able to do restriction enzyme mapping problems like the ones in the homework assignment. •I found this link, which helped me better understand how to do the restriction mapping problems, it has some practice problems as well:•http://people.rit.edu/rhrsbi/GEPages/LabManualPDF5ed/16%20mapping.pdf•Be able to describe how restriction enzymes are used in cloning DNA. •ex: •1. The DNA to be cloned is isolated and treated with a restriction enzyme to create fragments ending in specific single-stranded tails.•2. The fragments are then linked to plasmid molecules that have been cut with the same restriction enzyme, creating a collection of recombinant vectors. •3. The recombinant vectors are transferred into E. coli host cells. Inside of the host cell, a vector replicates to form many clones, or clones.•4. The bacteria are plated on nutrient medium, where they form colonies.•5. The colonies are screened to identify those that have taken up recombinant plasmids. •Know what a cloning "vector" is, and why different vectors (plasmid, lambda, and BAC vectors) are used in different situations. •Vectors (carrier DNA molecule) transfer and help replicated inserted DNA fragments.•the fragments produced by restriction enzymes must be joined to a vector before they can be inserted in a host cell•The first vectors were modified plasmids (dbl. stranded, extrachromosomal DNA found in certain bacterial strains)•Many different vectors are used for cloning--they differ in which host cells they are able to enter, the size of the inserts they can carry, and other properties•Vectors must:•replicated independently once inside the host cell (along with the DNA fragment it carriers)•contain several restriction-enzyme cleavage sites that allow insertion of the DNA fragments that are to be cloned•carry a selectable marker gene to identify host cells that contain recombinant vectors (usually an antibiotic resistance gene)•Types:•standard plasmids- inserts less than 10 kbp•Bacteriophage vectors- inserts 10-50 kbp•BACs- inserts 100 kbp•YACs- inserts 1000 kbp•Know the difference between a genomic library and a cDNA library, what each of these libraries are used for. •library- collection of clones•genomic libraries- cloned fragments of DNA isolated from chromosomes•used for cloning regulatory sequences (promoters), studying genome organization, sequencing of whole genomes•cDNA libraries- cloned DNA copies of mRNA isolated from cells or tissues•used for isolating expressed genes•cDNA = ʻcomplementary DNAʼ made from mRNA•synthesis of cDNA uses reverse transcriptase to make a DNA copy of mRNA•Be able to explain how mRNA is separated from bulk RNA in the cell, and how double stranded cDNA is made from mRNA.•Because many eukaryotic mRNAs have a polyA tail of variable length at one end, a short oligo-dT molecule annealed to the tail serves as a primer for the enzyme reverse transcriptase•Reverse transcriptase uses the mRNA as a template to synthesize a complementary DNA strand (cDNA) and forms an mRNA/cDNA double stranded duplex.•The mRNA is digested with an enzyme, producing gaps in the RNA strand•The 3ʼ ends of the remaining RNA serve as primers for DNA polymerase, which synthesizes a second DNA strand•The result is a double stranded cDNA molecule that can be cloned into a suitable vector.•Understand how you can screen a library to identify clones carrying a specific gene by use of DNA hybridization. (For a picture to go along with the steps--Lect. 30 Slide 14)•You can screen a gene library by DNA hybridization using a cloned gene as the ʻprobeʼ•How it works:•The library (present in bacteria on Petri plates), is overlaid with DNA-binding filter, and colonies are transferred to the filter.•Colonies on the filter are lysed, and the DNA is denatured to single strands.•The filter is placed in a hybridization bag (with solution) and the labeled single-stranded DNA probe•During incubation, the probe forms a double-stranded hybrid with any complementary sequences on the filter•The filter is removed from the bag and washed to remove excess•Hybrids are detected using a piece of X-ray film over the filter and exposing it for a short time•The film is developed and hybridization events are visualized as spots on the film•Colonies containing
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