Chapter 8 Notes A
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. What would you like to do?
Explain the study with mice.
They used a regular B6 control mouse and an MRL (mutant mouse) to study the autoimmune disease, lupus.
They punched holes in the ears to distinguish between the mice. Permanent holes stood in the normal mice. the holes in the mutant mice were shut due to regeneration, which doesn't usually happen in mammals.
Led to the study of mammalian regeneration
What are the benefits of growing cells in culture and treating them with different chemicals, etc?
- apply to humans; looking at restricted group of cell types
- expand the cells
What are the drawbacks of growing cells in culture and treating them with different chemicals, etc?
More complex than just the cell is the body
- Cell growth may be disproportionate due to competition between the cells
- Not in the tissue specific environment that the cells came from
What had to be done to the ear of the mouse?
cut ear off and fix the ear, freezing the cells and molecules where they are and as they are
How do you fix something?
- 1) liquid nitrogen; good for RNA and DNA
- problem: hard to handle the sections ocne thawed
2) drop into formaldehyde; create chemical crosslinks that keep them where they are and prevent denaturation because proteins are crosslinked. So is RNA
What is an H and E stain?
Hematoxylin and eosin stain (H&E stain or HE stain)
H is basic (purple); E is acidic (red/pink)
- DNA and RNA are stained purple
- Proteins are pink
How can you find proteins, DNA, and RNA?
Immunohistochemistry: Use ATP to find proteins with intense section:
- 1) isolate protein
- 2) produce antibody by injecting pure state protein into organism other than the organism from which it came. It is recognized as an antigen and polyclonal antibodies are formed.
- Through a series of experiments, find the hybrid cell that produces the best antibody
variable region that recognizes different antigens
Antibody is used to __.
examine things in tissue sections. Antibody detected as enzyme stain
What is the difficulty with using antibodies to code?
false positives may be present; look for control
How else can they be tagged?
can fluorescently tag so they can be detected using fluorescent microscopy.
Explain fluorescent microscopy.
Advantage: use LM; use equal segments
Two filters: first barrier filter lets throguh only blue llight, while the second cuts out unwanted fluorescent signals, passing the specific green fluorescein emission. Then, it goes through a beam splitting mirror: reflects light
How else are organisms labeled?
bromodeoxyuridine: synthetic nucleoside that is an analog of thymidine
does nothing to your cells
Purpose of using bromideoxyuridine.
To detect proliferating cells in living tissues due to it being pulled in by DNA polymerase during replication. It can be stained with an antibody.
To study wound healing and regenerative process, we can poke a hole into tissue. Epithelial have to grow over and heal the section
Explain stringent versus nonstringent hybridization.
To use a DNA probe to find an identical match, stringent hybridization conditions are used; the reaction temperature is just a few degrees below that at which a perfect DNA helix denatures in the solvent used (its Tm), so that all imperfect helices formed are unstable.
When the DNA probe is being used to find DNAs with related, as well as identical sequences, less stringent conditions are used; hybridization is performed at a lower temperature, which allows even imperfectly paired double helices to form
Only the lower-temperature hybridization conditions can be used to search for genes that are nonidentical but related to gene A
Purpose of DNA-DNA hybridization?
Can be used to find complementary DNA sequences
find gene of interest in organism whose genome hasn't been sequenced
Purpose of RNA-DNA hybridization
Allows determination of whether a gene is being expressed or not
in situ hybridization
Uses a labeled complementary DNA or RNA to localize a specific DNA or RNA sequence in a portion or section of tissue
- used to:
- 1) reveal location of specific nucleotide sequence on chromosome or in tissue and thus patterns of gene expression
- 2) evenly time and place mRNA product is being expressed
- 3) determine structure of chromosomes
laser capture microdissection
Used to look at specific cell types and multiple genes And isolate specific cell types within a larger sample
Explain in depth laser capture microdissection.
- Microscope with arm that allows the cap of an eppendorf tube to sit on top of a tissue section.
- On the bottom surface and in contact with tissue is substance that melts when hit with laser; laser hovers over one cell
- laser shoots through cap, hits film, melts it, and causes it to go down into tissue section
Take the arm with the cap and move it over. There is tissue on teh cap due to the melted part
What do you do with the melted part of the cap?
You microdissect different cells.
Basal epidermal cells (for example) are attached to eppendorf cap. Put in eppendorf tube with TRI reagent to isolate proteins, RNA, and DNA
Essentially, what is the purpose of LCM?
You can microtarget different cells and answer very specific questions about individual cell types to detect which genes are on, what mutations occured, etc.
Why was DNA the hardest macromolecule to work with?
How was each problem resolved?
- 1) there's so many different types of genes (restriction enzymes)
- 2) there's only two genes (plasmid)
What were restriction enzymes originally used for?
Bacteria use restriction enzymes to cut up invading nucleic acids. Restriction enzymes that are most often used are six cutters.
They usually make sticky ends, but blunt ends are possible
How do restriction enzymes bind?
They bind as dimers to palindromes because there is a central axis
What is the result of a restriction nuclease cut?
partially single stranded overhang
ssDNA is usually a sign of __, leading to __.
What do we let happen to these strands?
we let them float and find each other--> hybridize
How do you clone?
- Take a big chromosome, chop it up into pieces wi restriction enzyme for cloning--> take plasmid and cut with the same restriction enzyme.
- The DNA is linearlized with sticky ends, allowing hybridization of the strands.
- Circular double-stranded plasmid DNA is a cloning vector that is cut with restriction nucleases. The DNA fragment to be cloned is placed into the plasmid (aka: cloning vector).
- DNA ligase covalently links the DNA together
A small piece of DNA taken from viruses, plasmids, etc. into which a foreign DNA fragment can be inserted for cloning
What is transfection? Explain it.
process of deliberately introducing nucleic acids into cells
- Can occur in two ways:
- 1) temperature induced: put in the plasmid; put the cells in an ice bath (get them cold)--> put them in heat (42 dC) for expansion--> plasmids bypass the membrane through gap
2) electroshock therapy: charge temperature introduces gaps, allowing entry into bacteria
What are the two libraries and how do you know which one to make?
cDNA and genomic
beginning with mRNA
convert mRNA into a clonal model; copying the entire DNA; In a plasmid, there are a lot of genes. They can be preserved
- start with complete genome of organism you're working on
- cut with restriction enzyme into little fragments and mix with the right amount of plasmids so as to have one fragment per plasmid
- The enzyme can cut out coding regions
Differences between genomic and cDNA libraries.
- Genomic: includes all fragments of DNA
- cDNA: only expressed gene sequences
- Genomic: larger
- cDNA: smaller
- Genomic: represents entire genome of organism having both coding and noncoding regions
- cDNA: only contains coding sequences; only the expressed part of the genome
- genomic: equal amounts made
- cDNA: different amounts made since up and down regulation can occur
How to make a genomic library
- 1) extract and purify DNA
- 2) digest DNA with restriction enzyme, creating fragments
- 3) insert fragments of DNA into vectors taht were cut with the same restriction enzyme
- 4) Seal with DNA ligase, creating a large pool of recombinant molecules
- 5) Transfect the bacteria with the plasmid, creating a library (all of your clones constitute your library)
- 6) Screen the library, which can be done by hybrodization. The whole library is plated over media and can be determined with a probe and autoradiography; can also be done with PCR
How does one determine the sequence of a genome?
You need to cut with several restriction enzymes and discover the overlap
Explain in detail the screening process of genomic libraries?
- After creating the plasmids, transfect the bacteria and plate them on a medium containing the antibiotic.
- Only bacteria carrying a plasmid are resistant to the antibiotic and grow.
- Test for the colony containing the desired DNA clone and inoculate into a large culture
- Purify the plasmid
How does one create cDNA?
- Lyce cells and purify mRNA.
- Hybridize it with a poly(T) primer.
- Make DNA by reverse transcription
- Degrade the RNA with RNase H, which removes the RNA template at the beginning
What are the differences between cDNA clones and genomic DNA clones derived from the same region of DNA?
- In the genomic DNA library, both introns and nontranscribed DNA are included in clones, and most clones, contain, at most, only part of the coding sequence of a gene
- In the cDNA clones, the intron sequences are remvoed during the formation of mRNA, and a continuous coding sequence is present in each clone
- In a cDNA library, there is a quantitative aspect to it as it reveals which genes are transcribed more frequently.
- In the genomic DNA library, the genes are represented in equal amounts
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