Objectives for Exam 4 Genetics Spring 2012 Chapter on Recombinant DNA Chapter 17 Be able to de ne 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 arti cial means How it works 1 DNA to be cloned is puri ed from cells or tissues 2 Proteins called restriction enzymes are used to generate speci c DNA fragments These molecules recognize and cut DNA molecules at speci c 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 puri ed 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 speci c manner recognize and bind to speci c nucleotide sequences the recognition sites are Palindromic sequences can cleave the speci c 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 speci c 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 rst vectors were modi ed 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 complementary DNA 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 3 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 speci c 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 probe How it works The library present in bacteria on Petri plates is overlaid with DNA binding lter and colonies are transferred to the lter Colonies on the lter are lysed and the DNA is denatured to single strands The lter 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 lter The lter is removed from the bag and washed to remove excess Hybrids are detected using a piece of X ray lm over the lter and exposing it for a short time The lm is developed and hybridization events are visualized as spots on the lm Colonies containing the insert that hybridized to the probe are identi ed from the orientation of the spots Cells are picked from this colony for growth and further analysis Know what a Southern blot is and what a Northern blot is be able to outline how they are done and understand what they are used for Southern Blotting detection of DNA sequences on a gel
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