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Finished Lecture 7 and Lecture 8

by: Bree Scalzo

Finished Lecture 7 and Lecture 8 BIOSC

Marketplace > Biology > BIOSC > Finished Lecture 7 and Lecture 8
Bree Scalzo
Foundations of Biology 2
Dr. Swiganova

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Foundations of Biology 2
Dr. Swiganova
Class Notes
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This 3 page Class Notes was uploaded by Bree Scalzo on Friday February 13, 2015. The Class Notes belongs to BIOSC at a university taught by Dr. Swiganova in Fall. Since its upload, it has received 194 views.


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Date Created: 02/13/15
Lecture 7 Methods of Genetic Engineering 1 Application in medicine Recombinant DNA technology a Human growth hormone i GH1 codes for a pituitary growth hormone signaling molecule 1 If no coding may end up with pituitary dwar sm slower growth shorter stature ii 2600 bp genomic sequence 1 Explain the size disproportion Introns iii Dot plot 1 Comparison of genomic sequence and mRNA a How many exons 5 b Early treatment i Cow and pigs GH were noneffective ii HGH from human cadavers associated with prion disease 1 Also drug rare and expensive iii 1984 usage of HGH from human cadavers banned c Current treatment i Geneticengineeringcloning HGH into E coli d Making cDNA from mRNA i Cloning into vectors was popular but didn t know where exactly GH was ii Look at mRNA in cell from pituitary gland 1 Harvest all mRNAs in tissue cells 2 RNA cannot be cloned into a vector so has to be rewritten into doublestranded DNA 3 Use reverse transcriptase and make DNA in double stranded plasmid a Although this will replicate many more mRNAs than needed iii Inserting cDNA into plasmids cloning vectors 1 Identify a palindromic recognition site 46 nucleotides attach same recognition site to the cDNA gene 2 Add restriction endonuclease a Restriction endonucleases cut DNA sequences at palindrome sequence forming sticky end 3 Sticky end results 4 Insert gene into plasmid a Add DNA ligase which makes a phosphodiester bond to put the plasmid together iv Construction of a cDNA library 1 cDNA library is a collection of cDNAs isolated from a particular cell type of tissue a Isolate mRNA b Synthesize cDNA c Make cDNA double stranded d Make recombinant plasmid e Transformation v ssDNA probe is used to locate the gene of interest in a pool of signle stranded sequences 1 Make labeled probe 2 Expose probe to singlestranded DNA 3 lsolate labeled DNA a Hybridization between the DNA probe and ssDNA sequence based on nucleotide is complementarity vi Screening cDNA library for GH mRNA Grow transformed cell Sample colonies through ler Make DNA single stranded Probe cDNA Find probe Identify labeled colony a Must express now vii What is the next step 1 To produce large quantities of the HGH protein the gene has to be cloned into expression vector and transformed into an E coli cell 2 Expression vector contains a Bacterial promoter b Antibiotic resistance gene c Coding region of the HGH d Fusion protein 3 Bacteria expression HGH grown in large quantities 4 HGH harvested and puri ed a Provide to all patients 2 Polymerase Chain Reactions a Ampli cation of target DNA sequencesmust know primers i Size of ampli ed fragments 10001500 bp b PCR primers must bind to sequence on either side of target sequence on opposite strands i When target DNA is made single stranded primers bind and allow DNA polymerase to work 1 Hydrogen bonds are broken in high temperatures 3 PCR reactions a Is an in vitro DNA replication i Needs 1 dNTPs 2 primers P P FP N 3 Taq polymerase because it can survivie in the high temperatures b PCR reaction i To each tube add 1 DNA template dH20 2 Buffer 3 dNTPS 4 primer F and P 5 taq polymerase ii To separate DNA go through 3 steps 1 Denaturationseparate the strands 2 Annealingprimers bind to the now separated strands 3 ExtensionTaq polymerase extends the DNA strand 4 Draw a graph depicting accumulation of ampli ed during PCR reactions a The reaction stops because it runs out of nucleotides the H enzyme conditions i also become not good I enough a 5 Sanger Sequencing a Dideoxy DNA sequencing b Get very small reads 5 c Get an early termination oof of DNA sequencing since the the ddNTPs do not have OH OH group Hm


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