Chapter 8 Notes D

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DesLee26
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Chapter 8 Notes D
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2015-03-31 15:44:31
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  1. How can you screen for cells that have been mutagenized?
    plate them at a permissive temperature and let them divide and form colonies. 

    Then, transfer some to another plate and grow both--one at permissive temp and one at different temp. 

    Cells with the temp sensitive mutation will die

    essentially, you are testing for conditional mutations
  2. epistasis analysis
    the order in which the genes act; can only provide information about gene order in a pathway when both mutations are null alleles

    By comparing the phenotypes of the diff. combinations of mutations, we can discover the order in which genes act

    ex: protein accumulation in ER and Golgi
  3. Differential display
    compares wild type to mutant; which genes are responsible for phenotype

    used to identify and compare changes in gene expression at the mRNA level
  4. Differential display
    - Techniques used?
    • PCR and how it works
    • acrylamide gels
    • cloning
    • sequencing (after this, searching the database and seeing if any info is on it)
  5. How can you adjust PCR?
    • annealing temperature: 
    • - too high: it doesn't stick
    • - too low: bind too tightly or binds to other places (primers start binding everywhere)

    salt concentrations
  6. Specific steps for differential display
    • Extract mRNA 
    • convert to cDNA 
    • run pcr under low stringency 
    • get bands on gel
    • Spread out
    • compare the samples to find differences btw the samples 
    • cut out differences 
    • go into database
  7. pros and cons of differential display
    Simplest, cheapest way 

    results are random. You don't know what you'll get (shot in the dark)
  8. What does an array provide info for?
    Only for the genes that are on the array; you don't know the sequence of the probe 

    purpose: used to measure the expression levels of large numbers of genes simultaneously or to genotype
  9. Microarray technology--what skills are needed?
    • MRNA extraction from two different tissues
    • cDNA lib
    • fluorescent tagging of cDNA
    • printer to shoot out DNA on chip
    • spectrophotometer
    • Database
  10. Steps for microarray technology
    • 1) Create microarray with thousands of different DNA strands (printer will make spots on array, each spot getting a different gene)
    • 2) collect mRNA from control (untreated) tissue and experimental (treated) tissue 
    • 3) convert mRNA into cDNA and add fluorescent labels--for our purposes, treated is red and untreated is green
    • 4) mix the probes and allow them to float over the top of the array for hybridization purposes
    • 5) use spec to measure the intensity of colors and ratio of reed to green
  11. What are the four hybridizations that can occur from the microarray technology?
    • 1) only in untreated cell (green)
    • 2) only in treated cell (red)
    • 3) expressed in both (will be color in between the two)
    • 4) expressed in none

    Put it in a spec to read the intensity: red means highly expressed in treated cell; green means it is highly expressed in untreated cell; black means no expression

    if green is present, that means there is increased activity in untreated cells; if red, there is increased activity in treated cells
  12. Method three: transcriptome

    what do we want info about?
    The entire transcriptome (set of all RNA molecules, including mRNA, rRNA, tRNA, and other non-coding RNA transcribed in one cell or a population of cells.)

    we do this by SAGE--serial analysis gene expression (allows digital analysis of overall gene expression pattern)
  13. What is the concept of SAGE?
    Go to cell/tissue type and extract mRNA expressed. From this, make a qualitative list of the types of mRNA and a quantitative in regards of the leveled of expression. 

    1) we do not need to know the entire sequence to get our mRNA
  14. Explain more of the sage method.
    There's a tag(short stretch of nucleotides unique to that mRNA )

    take mRNA--> go in and isolate tags--> link tags together (100-150 tags altogether)--> clone long string of tags--> sequence 

    this should give info about 50-100 items
  15. How do we get the tag?
    we use two restriction enzymes, one of which is within a linker (string of nucleotides (ds) wih complementary overhang to our sticky end with a recognition site for a tagging enzyme, which cuts downstream, cutting the tag from the poly-A tail)
  16. Adv and disadvantage of transcriptomes?
    Adv: you see every single mRNA created and the proportions

    dis: it's tricky
  17. Complete steps of transcriptome formation?
    1
    1) create dsDNA (oligodT primer with a little black dot representing a metal atom linked to oligodT; reverse transcription makes first strand. The second strand is then made to create cDNA; create dsDNA of every message on the basis of how many present)
  18. Complete steps of transcriptome formation?
    2-3)
    2) cut cDNA wih an anchoring enzyme that cuts in a bunch of places. Because we only want to cut closest to the poly-A tail, we put it ina eppendorf   tube and put in a magnet to allow the metal atom to bind the magnet, separating them from the rest of the cuts. 

    --> the tag is at the 5' end of the 3'- most fragment after the anchoring enzyme cute 

    3) put buffer back in and detach magnet
  19. Complete steps of transcriptome formation?
    4-5
    4) get linkers attached, which have a recognition sequence for a second enzyme, called the tagging enzyme, which binds to the sequence and cuts 15 nucleotides downstream, cutting off the tag

    5) now put the fragments back into tube with magnet, allowing metal atoms to bind. Pour off top, which contains our tags.
  20. Complete steps of transcriptome formation?
    6-7)
    6) ligate tags together, clone, and sequence 

    as we sequence, we get info about several genes on the basis of how many we put in

    7) make a list to measure quantitative (how many times the same tag is present) and qualitative (List of genes expressed) Go into a database
  21. Linker
    • Seq of 20-25 nucleotides that bind to overhang of sticky end on our existing strand
    • has recognition sequence for tagging enzymes, which bind to sequence and cut 15 nucleotides downstream, cutting off our tag
  22. Gene manipulation
    Create RNA molecule that is antisense to original molecule: alter genome of organism by cutting gene out, flipping it around, and putting it back in front of the promoter. This gives you your antisense strand

    this antisense strand will bind to the sense strand whenever it is present, inhibiting it's expression and causing it to be degraded
  23. How else can gene manipulation be performed?
    Take dsRNA of simple organism and inject or feed the organism it to stop it
  24. How do you create a specific mutation in a protein..
    • 1) Obtain the gene of interest and clone it in a plasmid as dsDNA 
    • 2) melt plasmid into single strands 
    • 3) hybridize a short nucleotide primer with a point mutation to the ss DNA (can be done under the right conditions, such as a hi temperature)  
    • 4) DNA polymerase will elongate the strands

    Result: plasmid with one nucleotide change is produced --> put in bacteria to allow one normal and one mutant daughter
  25. How is a knockout organism created?
    Go into cdna lib, find clone, and cut it out 

    make 2 modifications to gene: 1) cut in middle and insert into other gene (NEO in the case of myostatin, which is resistant to drug x) and 2) at end of gene, attach another gene, TK, which is sensitive to drug Y

    • inject into mouse ES cells--> that gene tries to incorporate itself into the genome of ES cell either:
    • 1) randomly for no specific reason via nonhomologous recombination 
    • 2) myostatin gene will relocate gene and, through homologous recombination, it will replace the myostatin gene in the genome
  26. How to test if the gene was replaced?
    Give drug x: if it did not take up the engineered gene, it will die since NEO isn't present to give resistance 

    if they survive, administer drug Y: only recombined cells (homologous) will survive in presence of drug Y because TK is not present to make it sensitive to drug Y. 

    This is infrequent homologous event is rare
  27. Myostatin
    Limit on muscle growth; prevents over proliferation
  28. after the recombined gene is obtained, what is the next step?
    Put in stem cell

    grow

    inject into embryo 

    modified stem cells becomes part of mouse 

    mice have knockout genes in parts of body depending on differentiation of stem cell 

    we want it in reproductive gametes

    if two mutant mice come together for reproduction, then we get knockout progeny

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