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Developmental Biology Student Presentation Notes for Exam 2

by: Madeline Abuelafiya

Developmental Biology Student Presentation Notes for Exam 2 BIOL 4331

Marketplace > Southern Methodist University > Biology > BIOL 4331 > Developmental Biology Student Presentation Notes for Exam 2
Madeline Abuelafiya

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Developmental Biology Student Presentation Notes for Exam 2 Includes Notes for: Quantitative PCR, ChIp, P-element transformation, RNA sequencing, and Western Blot
Developmental Biology
Dr. Strecker
Strecker, Developmental. Biology, Student Presentations, Quantitative PCR, ChIp, P-element transformation, RNA sequencing, and Western Blot
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This 5 page Bundle was uploaded by Madeline Abuelafiya on Tuesday March 29, 2016. The Bundle belongs to BIOL 4331 at Southern Methodist University taught by Dr. Strecker in Winter 2016. Since its upload, it has received 18 views. For similar materials see Developmental Biology in Biology at Southern Methodist University.


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Date Created: 03/29/16
Western Blot Method  used to identify specific proteins. They are carried out by gel electrophoresis and then tagging with antibodies.  important to keep samples on ice to prevent degradation  SDS-PAGE separates proteins based on size  Higher and lower voltage necessary  Sponge, filter paper, membrane, gel, etc. sandwich in buffer  Milk in the membrane  Wash the membrane  Secondary antibody and milk  Milk used to ensure specific binding only  Dark room to run gel and visualize results Objectives and Limitations  Objectives: detect a protein from a complex mixture, confirm identify of a protein, can test a protein’s response to a drug. Used with ELISA tests  Limitations: choosing the correct antibody, not quantitative (cannot tell how much of a protein is present), unexpected bands, time consuming, larger proteins are harder to detect. Application 2  Epigenetics and the study of Genomic Imprinting- an epigenetic silencing of an allele if it is imprinted  WB can study protein products in the imprinting  The DNA methyltransferase Application 3  Proteomics and Gametogenesis: WB used to study protein related to meiotic divisions  Proteomics: a large scale study of proteins produced by a cellular organism  Allows for the identification of proteins involved in gametogenesis Western Blot Analysis  GAPDH loading control sample  Allows to see early DNA reprograming as the proteins were expressed in Day 7 and by Day 25 were completely absent Chromatin Immunoprecipitation (ChIP) Used to determine if specific proteins are associated with specific regions of the genome. Method: 1) Cross-link bound proteins to DNA 2) Isolate chromatin and shear DNA Objectives: Suitable for various TF or weakly binding chromatin associated proteins Testable antibody specificity High specificity in antibodies Limitations: antibodies have to be generated TF Cant discriminate between different TF isoforms Genes expressed in low levels, in small number of cells makes it hard to detect. ChIP on chip Locate protein binding sites in order to identify functional elements in a genome using PCR and undergo a denaturation step – single strand Microarrays used to hybridized to complementary sequences Binding site ChIP Sequencing- used to analyze protein interactions with DNA Done through purification and denaturation sequence ChIP-exo Increased resolution Direct association with protein Prominent peak and limited noise Exonuclease can really help refine the fragment Exact bases it binds to Example of ChIP-Eco Glucocorticoid receptor- can influence DNA transcription with hormone binding Tells you what associates readily though the genome RNA Sequencing Measures gene expression- whole sequences or segments Whole transcriptome shotgun sequencing- can run million of transcripts all at the same time. 1) Choose your platform: Ie) Deep sequencing- transcriptome sequencing 2) Isolate and purify 3) Can run fragments directly, larger fragments must be fragmented with cDNA and reverse transcriptase 4) Examine/analyze Objectives: to see what sequences are being translated into mRNA Can be used for rare diagnostics Benefit: what genes are transcribed vs whole dna No need for prior knowledge of RNA sequencing allowing you to discover new sequences! Better than microarrays Limitations: Large amount of data therefore takes a lot of time Possible error with reverse transcripatase from RNA to cDNA Can be hard to put the reads together 1) Gene fusion in transcripts in breast cancer cell lines. - Can possibly make biomarkers for treatment or therapy - Might be able to determine specific cancer type related to that gene 2) West Nile Virus -no current cure, especially dangerous in immunocompromised patients. Identify resistant vs susceptible individuals -possible treatments or therapies 3) Alzheimers Brain Tissue -alternative splicing -compare transcripitome of healthy people brain vs alzheimers brain tissue -different levels of transcripts from two different tissue types alternative splicing/promoters sequence found in alzheimers- indication of disease progression 4) Use of mRNA profiling in embryonic stem cells - Sequences small mRNAs from human embryonic stem cells. - Pluripoteny in human embryonic stem cells change in gene expression contributes to differentiation - Identified ecpression differences in known and new sequences- 160 microRNA genes- some expressed and some not depending on whether or not the cell was differentiated or not Quantitative PCR: Real Time PCR Data is collected thoughtout the assay rather than only at the end of the assay Can keep track of amplication of DNA while its happening Temperature and location important Denaturation must come in contact with the cell Enzyme Reversetranscriptase: RNA  cDNA SYBR green qPCR Mastermix: fluorescent, binds, and amplifies target DNA One amplifies and one measures Obejctives/Limitnations -meant to amplify DNA starts -track gene of interest with fluorescent reporter -hybridize target gene increases sensitivity Limitations: -non specific markers can bind to unintended regions -nucleic acid extraction is difficult -Amplicons must be short and have high GC bp to have good results telomeres: cap of chromosomes and protects from degradation t:s ratio: a measurement of length: southern blots and PCR QPCR of foodborne pathogens: measure gene expression of the pathogens Allows faster recognition of food pathogens so people don’t get as sick Quantify Cytokine Gene Expression Delta RN: expression/time Quantifiable, reliable, and very specific -- Important aspect: involves using a special probe but the forward and reverse region will aplify and the amplicon Taq polymerase will displace the quencher As it is deepaced, the quencher is released and separated from the fluorophore and then is able to emit light. The third porbe is your detection probe Tac pol will replace each nucleotide with new ones, no longer inproximity by fluorophore/ quencher,, fluorophore can emit light. Signal from pcr amplification of template, release quencher, fuorophore emits You begin to see fluorophore as amplicon is amplified exponentially Signal will build in time as amplicon builds in concentration Follow amplification per cycle. You can watch amplicon build in concentration per time Probe that sits between flanking PCR probes are how you follow state of amplification P-element Transformation The P element is a transposon that is present specifically in the fruit fly drosophila melongaster and is used widely for mutagenesis and the creation of genetically modified flies used or genetic research Used to create transgenic flies via transposons (p-element) Method Plasmid with specific gene of interest with helper plasmid with transposase gene into a cell. Both must be there to work. Inject to posterior end of the egg because that is where germ cells are located. Objective Gene targeted expression: overexpress or introduce new genes in a cell Introduce genes with a marker coding sequence  may operate as a mutagens Limitations Efficiency: random integration, survivability, and size of P element Because genes can land anywhere it is hard to compare between constructs Success is varied. 10 lines with gene of interest is necessary… survival of embryos .. may have to inject wayyy more injections Larger the P element, the less frequent the insertion Use 1: P Element Mediated Transformation Rosy (ry) gene introduces into germ line though insertion and stable transposition by P elements Use 2: Transposon Mediated Mutagenesis Gene disruption project: p element insertion in every gene P element insertions occur within 400 of transcription start site Excise at a high frequency P elements have insertional preference: not found in hot spots genes that attract multiple p element insertions . Hybrid was most efficient: piggyback- allowed for random insertion into the genome P-element allowed for precise excision Use 3: Gal 4 UAD system Gal 4 used to regulate expression of specific genes in drosophila Using the P element- the GAL 4 gene is inserted at random sites of genome Reporter gene to see space-ial regulation by enhancer Data Example: Molecular Verification of Homologous Recombination


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