Term
| Cloning in plasmid vectors |
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Definition
| Digest and ligate a plasmid (w/Ampf, ori, and EcoRI binding spot) and a human cDNA fragment together to make one recombinant plasmids w/one human fragment each. Transform E.coli w/ the recombinant plasmids. Plate E.coli on amp. plate, only resistant, recombinant cells will grow. Isolate colonies and get recombinant E.coli. |
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Term
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Definition
| Reverse transcriptase generates cDNA copy of specific, not random, mRNA molecule. Oligonucleotide linkers w/restriction endonuclease cleavage sites are added to ends of cDNA to give it the right shape to bind into a specific vector. cDNA is ligated to appropriate vector. |
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Term
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Definition
| Allow DNA synth to go on in presence of fluorescent chain-terminating dideoxynucleotides, one ddNucleotide at a time (ddA, ddT, ddC, ddG). Where the ddGs stopped growth = G. Where the ddAs stopped growth = A. Where the ddCs stopped growth = C. Where the ddTs stopped growth = T. Use electrophoresis to separate the unfinished, terminated DNAs by length, shortest = first nucleotide, longest = last nucleotide. Scan w/lase for fluorescence, use computer to analyze. |
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Term
| Next-Generation sequencing |
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Definition
| Anchor single DNA molecules to solid surface and copy each in situ by PCR to amplify template. Add polyerase, universal primer, and 4 color labeled reversible terminators (like ddNucleotides but reversible) to get growth. Once a colored base binds it'll stop growth, detect that base w/laser to see which one it is, remove base, let polymerase put the normal base there, add colored base to following base. Repeat many times till every colored base is detected. |
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Term
| Expression of cloned genes in bacteria |
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Definition
| cDNA inserted at cloning site (next to binding sequences of bacterial promoter and ribosome) of plasmid (w/Ampf, ori). Transform E.coli, cDNA will be transcribed into mRNA and human protein will be expressed in E.coli. Similar process lets us express genes in yeast/mammalian cells and study their function. |
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Term
| Detection of DNA by nucleic acid hybridization |
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Definition
| Denature mixture of total cell's dsDNA. Add labeled DNA probe complementary to certain DNA sequence. Renature. Labeled probe hybridizes to complementary DNA sequence. |
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Term
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Definition
| Denature starting DNA. Gene-specific primers bind to ssDNA. Taq pol extends DNA from primers. Gives 2 new molecules. Can repeat and double DNA every time. Used to assay amounts of mRNA in cell if it is first converted to cDNA by reverse transcriptase. |
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Term
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Definition
| Digest DNA w/restriction endonuclease (different ones digest different lengths). Separate differently sized restriction fragments w/gel electrophoresis. DNA denatured and transferred to filter paper from gel. Hybridize filter w/labeled probe which will bind to complementary sequences. See where the probe binded = where the sequence is. Doable w/mRNAs - Northern blotting. |
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Term
| Screening a recombinant library by hybridization |
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Definition
| Insert 1 DNA fragment into each lambda vector, package into phage particles. Infect E.coli w/recombinant phages carrying diff. DNA fragments. Each phage forms a plaque. Transfer plaques to filter. Hybridize w/specific labeled probe, see where probe is = desired sequence, desired phage. |
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Term
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Definition
| Gel electrophoresis protein mixture to separate by size. Transfer to filter from gel. See protein bands. Incubate filter w/specific antibody against protein of interest. Detect antibody w/chemiluminescence = protein of interest location. |
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Term
| Subcellular fractionation |
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Definition
| Break up cell mixture gently using a blender, get lysosomes, peroxisomes, and membrane fragments. Centrifuge = pellet of nuclei. Centrifuge faster & longer = pellet of mitochondria, lysosomes, and peroxisomes. Centrifuge faster & longer = pellet of plasma membrane and ER. Centrifuge fast & longer = pellet of ribosomes. |
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Term
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Definition
| Cells lysed and protein stained w/fluorescent antiobdy. Immuno = antibody. |
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Term
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Definition
| Incubate radiolabeled protein mixture w/antibody for protein of interest. Collect antigen/antibody complexes by incubation w/antibody-binding beads. Boil to isolate beads. Use gel electrophoresis to separate antibody and antigens by size. Proteins visible w/radio film. Can do same thing w/interacting proteins by binding complex to BAIT molecule instead of beads = coimmunoprecipitation. |
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Term
| Mutagenesis w/synthetic oligonucleotides |
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Definition
| Plasmid w/gene of interest. Denature and hybridized w/mutagenic oligonucleotide. DNA synth and ligase to incorporate mutant. Plasmid w/mismatched base is now at site of mutation. Transform E.coli, some cells will be normal, some will be mutant. |
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