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Molecular Week 11 Notes

by: Kiara Lynch

Molecular Week 11 Notes Bio 413

Marketplace > La Salle University > Biology > Bio 413 > Molecular Week 11 Notes
Kiara Lynch
La Salle

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Notes from Week 11...Ch 8 cont. Manipulating Proteins, DNA, RNA
Dr. Stefan Samulewicz
Class Notes
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This 5 page Class Notes was uploaded by Kiara Lynch on Wednesday April 6, 2016. The Class Notes belongs to Bio 413 at La Salle University taught by Dr. Stefan Samulewicz in Spring 2016. Since its upload, it has received 8 views. For similar materials see Molecular in Biology at La Salle University.


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Date Created: 04/06/16
Week 11 Notes  Recombinant proteins o Amplify DNA o Take domain 1 and link to domain 2 o Embed palindrome  Cut with restriction enzyme- sticky ends o Mix  hybridize o How you add an epitope tag  Designing genes o Cells  isolate DNA  DNA to be cloned  separate strands and add primers  PCR for amplification  genomic clones st o Cells  isolate mRNA  mRNA sequence to be cloned  add 1 primer, reverse transcriptase and deoxyribonucleoside triphosphates  DNA/RNA double strand  separate strands and add second primer  PCR amplification  cDNA clones  PCR for forensic analysis o VNTRs- variable nucleotide tandem repeats  In introns or spaces between genes  Dinucleotide  Quantify? Real time PCR o Instead of Eppendorf tubes  thin wall capillary tube – holds 5 uL sample o Instead of metal heating block  air temperature in chamer – small sample changes temperature quickly o PCR cycles and spectrophotometer  Every PCR cycle, ring spins and each sample is exposed to spec  Certain wavelength of light shines through o PCR mix contains SYBR green  Fluorescent dye- unique dye  Only binds to double stranded DNA  Only fluoresces when bound to DNA whereas ethidium bromide always fluoresces  SYBR indicates how much double stranded DNA there is o Results  Same amount of cDNA – amount of product determines time  Housekeeping gene – 2 lines overlap, same level in pre- adipocytes and adipocytes o Promoter  Temperature sensitive, pH sensitive, etc.  Express gene  mRNA to protein  extract protein from bacterial cells o DNA helicase  Temperature sensitive promoter  Increase temperature, increase amount of DNA helicase  Good way to make abundance of proteins for other experiments but might not always work  Limit of effectiveness – won’t know to fold or add post- translational modifications, etc. o Protein  determine partial amino acid sequence  synthesize DNA probe  screen cDNA or genomic DNA library  gene or cDNA  insert into expression vector  introduce into E. coli or other host cell  extract protein  repeat  Epitope tagging o Gene for protein of interest (expression vector) o Insert DNA encoding epitope tag o Introduce into cell o Epitope tagged protein use antibodies to locate or purify  Immunolocalization using antibodies to protein tag  Rapid purification of tagged protein and any associated proteins  Pulldown experiment o What proteins interact with your protein? o Recombinant DNA techniques are used to make fusion between protein X and GST (glutathione S-transferase)  GST binds to glutathione which binds to protein o Fusion protein bound to glutathione coated beads o When cell extract is added, interacting proteins bind to protein X o Glutathione solution elutes fusion protein together with proteins that interact with protein X  FRET o Under what conditions do the 2 proteins interact? o Unique epitope tags on each protein  Blue fluorescent protein – shine violet light  comes out blue  Green fluorescent protein – shine in blue light  comes out green o Interacting proteins  Shine violet light  comes out green o Non-interacting proteins  Shine violet light  blue light detected  Yeast to hybrid system o Find as many binding partners for this protein as you can o Reporter gene that under certain condition o Epitope tag on DNA binding domain – BAIT o Create cDNA library with all proteins produced by cell line and binding factors  Attach epitope tag – TAD – find potential binding partner with TAD o BAIT and target protein + binding partner and transcriptional activation domain  Recombinant genes encoding BAIT and prey introduced into yeast cell  Transcription of reporter gene  blue o Reporter gene on only if binding partner binds to BAIT  TAD turns on reporter gene  transcription  blue cell o Used to find an alcoholism gene  Wild type- feed together  Mutant- eat alone  Identify genes o Mutagenized – wild type cells exposed to something (ex: radiation, heat) o Mutagenized cells plated out in petri dish grow into colonies  colonies replicated onto two identical plates and incubated at two different temperatures  mutant cell that divides at the permissive temperature but fails to divide at the restrictive temperature o At elevated temp, some don’t replicate o Pathway of cell replication  Find step by step processes  ER  Golgi apparatus  secretory vesicles  Normal cell – protein secreted  Secretory mutant A – protein accumulates in ER  Secretory mutant B – protein accumulates in Golgi apparatus  Double mutant AB – protein accumulates in ER  Differentiated Display o Need to know PCR and how to run a big gel o Annealing temperature is critical  Only want a specific sequence to be amplified  Too high- breaks H bonds and primer won’t bind  Too low- don’t need as much energy to bind primer – can bind in other places o Cheap, quick, easy o 4 samples in each experiment- compare o Take cDNA from each sample  PCR with low annealing temp  a lot of bands appear  Find conserved bands – look for differences o Don’t know what genes the bands represent  Sequence  find gene  Find cause of disease/phenotype  Antisense RNA o Eliminating expression of your gene o Antisense RNA- double stranded mRNA – won’t work o Ex: Cell division interrupted because protein responsible for division is inhibited  Antisense RNA bound to normal RNA  Create specific mutation o Clone enzyme o Alter 1 amino acid o Melt double stranded plasmid into single strands o Add primer that hybridizes almost perfectly except for 1 nucleotide o Point mutation o Add DNA polymerase o Plasmids- 1 strand of wild type and 1 strand with point mutation  Microarray o More expensive and challenging- need technology for creating chips and reading them (spec and quantify) o Look for hundreds and thousands of genes at 1 time o Know what’s on blot, don’t know what the probe is o Take single stranded DNA from many different clones and spot onto an array on a microchip o cDNA library  put each in different spot on chip o Liver cells  treatment and control groups  expression of genes  extract mRNA  convert to cDNA (reverse transcriptase- single stranded)  incorporate label – fluorescently tagged  take cDNA and mix and float over chip – hybridization, complementary base pairs, gene was expressed if binding occurs  Housekeeping genes- yellow – expressed equally in both types of cells  Colors indicate activity of genes in cells o Gene expression pattern for nontoxic and toxic substances  Liver reacting to drugs as if it was a toxin o Limiting factor- what if gene responsible for your disease is not on chip  won’t come up  SAGE- serial analysis of gene expression o Population of mRNA that might get extracted o Looking for gene expression o Tags  Within every mRNA is a short stretch of nucleotides totally unique to that mRNA  Search database  determine genome it came from o Don’t need to create cDNA and sequence the whole thing o Single stranded mRNA  double stranded cDNA  Find and cut out tags  Link tags in longer stretches of DNA  sequence multiple at a time  Analyze expressed genes- qualitative and quantitative o mRNA  cDNA (oligo dt primer tagged with iron) o Anchoring enzyme binds to specific palindromic sequence and chop cDNA into pieces o Want piece closest to poly A tail o Iron sticks to magnet o Tag is at 5’ end of 3’ most fragment of cDNA o Synthesize linker – binds to sticky ends  Contains another palindromic sequence for the tagging restriction enzyme  Binds to sequence within linker but cuts about 12 nucleotides downstream o Take tags, link together (50-100 tags) o Clone and sequence o List of genes – tally o Can discover new genes o Time consuming, have to manipulate DNA and RNA, technical steps, sequencing, expensive o If you don’t know what the gene is, it will still show up  Creating a knock out organism- eliminating gene completely from organism o Clone gene you want to knock out  Myostatin- limits size your muscles grow to o Cut and insert another gene  Neo- codes for protein that gives cell resistance to a certain drug X  Neo in  cells live o Attach another gene to end  TK- makes cells sensitive to a drug Y o Insert gene into nucleus of cell o Allow recombination to occur  Gene could be randomly inserted into host – nonhomologous recombination  Myostatin gene randomly inserted with neo and TK  Drug Y in  cells die  Gene could find myostatin gene and undergo homologous recombination  Gene inserted with Neo but without TK  Drug Y in  cells live b/c no TK o Knockout mouse  Altered genes into cells, grow into colony, test for the rare colony in which the DNA fragment has replaced one copy of the normal gene  Take embryonic stem cell (homologous recombination)  Inject into embryo of mouse  Embryo into mother  Reproduce  Gamete producing cells – ¼ of progeny will be knockouts  Myostatin knockout mouse- big muscles


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