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10 Cards in this Set
- Front
- Back
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Define cDNA? Characteristics?
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DNA complementary to mRNA. No promoters
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How we create massive amounts of cDNA? Outline the process. (Hint: R_ - P_ _ )
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Reverse Transcriptase - PCR (RT-PCR)
- mRNA poly A tail bound to singled stranded poly T oligonucleotide (this forms the primer) - RT uses primer to initiate single-strand DNA synthesis. -RNA digested/removed and poly dG adapter annealed at 3' end. -poly dC primer formed, used to initiate second DNA strand syn. - DNA pol. 1 (E.Coli) extends 2nd strand |
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How can we express massive amounts of a protein of interest?
(Hint: a special promoter that can do this two ways) |
The lac promoter.
-remove lacZ gene, KEEP lac promoter -insert desired gene (cDNA) - lactose (or lactose analog like IPTG) activates promoter, stimulating transcription Lac promoter can drive T7 RNA polymerase, which expresses gene under control of T7 promoter |
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Define Heterologous
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From a different organism. (Roy's words)
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Name the differences between Transient and Stable transfection.
(Hint: Stable transfection is a.k.a. Transformation) |
Transient:
-viral ORI -plasmid doesn't insert into genome -ineffective distribution among daughter cells of cell division - SOME CELLS express transgene Stable (Transformation): -plasmid vector introduced into genome. Perhaps done by enzymes of DNA repair & recombination -vector has neo^r gene for resistance against G-418 -ALL resistant cells EXPRESS transgene |
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Green Fluorescent Protein is an example of a _____ protein. What would you use it for? Example?
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It's a fusion protein
-it can be inserted in between a promoter and gene of interest Used to indicate where & when a gene's expressed. Ex: GFP-tagged histones & tubulin highlight mitosis in REAL TIME |
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Molecular Probes are used to screen ____ libraries.
Outline basic process |
Screen cDNA libraries (Genomic ones too)
-DNA reproduced on nitrocellulose membrane - DNA denatured, radiolabelled probes anneal -non-labelled and/or non-hybridized molecules washed away. |
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What enzyme labels olignucleotides?
How & Where on the 'tide? (Hint: enzyme is from a bacteriophage) |
Polynucleotide kinase
- adds a gamma phosphate to hydroxyl group at 5' end |
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PCR can make radiolabelled probes.
Where is the radiolabel incorporated? |
on the alpha phosphate.
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What gives a stronger, more specific signal: PCR probes or chemically synthesized probes? Why?
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PCR because many radioactive P^32 atoms are incorporated into the probes, which are longer in PCR
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