Lecture 21

Lecture 21 - Lecture 21 Molecular Techniques DNA cloning:...

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Lecture 21 Molecular Techniques DNA cloning: General Strategy Study structure and functions of genes at molecular level requires a large quantity of individual genes in the pure form DNA cloning : Prepare large quantities of identical DNA molecules Taking one exact genome and reproducing the exact same one All cells in colony arise from a single transformed parental cell Initial fragment is referred to as cloned DNA Recombinant DNA : Any DNA molecule composed of sequences derived from different sources New DNA that did not originally exist in nature Key process : Link to a vector DNA Vector : Molecule used to transfer foreign genetic material into another cell Molecule that can replicate without a host cell Summary: 1 | P a g e
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Vectors can propagate in bacteria or in eukaryotic cells such as yeast DNA fragment can be of any origin of the same species o No way for transcriptional machinery to distinguish between eukaryotic base pair or a more primitive form Plasmids : Long pieces of circular DNA that exist natural in bacteria Modified to have recombinant DNA in there and replicate them fast DNA library : Collection of DNA fragments DNA cloning Vectors Bacteria Yeast - Plasmids up to 10 kb - Bacteriophages/comids up to 50 kb - Bacterial artificial chromosomes up to 2 Mb Plasmids Yeast artificial chromosomes Host Cells: Bacteria Culture Agar plate in solid medium for bacteria to grow in 2D Grow as fast as possible Bacteria contains plasmid DNA Method: 1.Pick a clone from Petri dish 2.Grow bacteria in a liquid culture 3.Purify plasmid DNA from bacteria Enzymes 2 | P a g e
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Objective : Obtain discrete, small regions of an organism’s DNA that constitute specific genes -DNA molecules must be cleaved into fragments in order to be inserted into a vector DNA Method: Cut vector at a particular site Paste the pieces in order to get new recombinant DNA Two types of enzymes: 1. Restriction Enzymes 2. DNA ligases Restriction Enzymes Restriction enzymes : Endonucleases that cut DNA molecules into small fragments Recognize 4bp-8bp sequences (restriction sites) Cleave DNA at this site Restriction sites: Palindromic sequences : Sequence is the same on each DNA strand when read 5’ to 3’ direction - Bacteria produce modification enzyme to protect themselves from cleavage (methylation) Sticky ends: Staggered cuts in 2 DNA strands at recognition site Single stranded tails at both ends Complimentary to fragments generated by same restriction enzyme Blunt ends: All nucleotides at the fragment ends are base paired to nucleotides in the complimentary strand 3 | P a g e
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- A given restriction enzyme will cut the DNA from a particular source into reproducible sets of fragments called restriction fragments Restriction Enzymes: Restriction Map Goal : Locate the sequence of interest Read as a map of potential places that can be cut DNA sequence or characteristic sequence that is artificially made
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Lecture 21 - Lecture 21 Molecular Techniques DNA cloning:...

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