recombinant techniques and detecting expression

techniques to detect if a gene is being expressed

Reporter constructs, Northern blot, In situ hybridization, cDNA libraries, RT-PCR, qRT-PCR

cDNA

complementary DNA

reporter construct

regulatory sequences upstream of gene of interest used to drive expression of substrate such as luciferase or green fluorescent protein

how is cDNA produced

reverse transcriptase synthesizes a DNA strand using mRNA as a template

in-situ hybridization (qualitative or semi-quantitative)

radio-labeled or fluorescently-labeled oligonucleotide (probe) is applied to a fixed or frozen sample to visualize where in the cell mRNA is expressed

northern blot process

isolate RNA from tissues of interest, separate using gel electrophoresis, “blot” onto a membrane (nitrocellulose), apply labeled probe complementary to mRNA of interest, wash off unbound probe and visualize binding

cDNA library

represents only genes being expressed in the cells at the time the library was made

making cDNA libraries

RNA is isolated and used to synthesize cDNA, cDNA is fragmented using REs or sonification, fragments inserted into vector and transformed

real-time PCR (RT-PCR)

semi-quantitative method to measure the abundance of specific mRNA transcripts

northern blot (semi-quantitative)

shows band indicating presence and size of RNA of interest

quantitative real-time PCR (qRT-PCR)

perform PCR using transcript-specific primers in the presence of a fluorescence-deleting camera

enzyme-linked immunosorbent assay (ELISA)

antibody that binds to target protein is added, 2ndary antibody and it’s substrate added, if binding present, reaction produces measurable signal

western blot

run on SDS-PAGE, blot onto nitrocellular matrix, apply probe that binds proteins of interest, rinse away unbound probe, apply secondary antibody, labeled w/ an enzyme to cleave substrate, leading to color change or fluorescence

elisa

enzyme-linked immunosorbent assay

sds-page

polyacrylamide gel electrophoresis

what kind of cloning did Sahli use

blunt-end

bacteria transformation

makes many copies of your plasmid by replicating them in E coli

what method did Sahli use to deliver DNA into cell cultures

transfection- surround dna w/ vesicle that can fuse w/ target cells or enter them via endocytosis

luciferase assay is a type of what

reporter construct