Help
Options
Didn't know it?
click below
click below
Knew it?
click below
click below
Don't Know
Remaining cards (0)
Know
retry
shuffle
restart
0:00
Embed Code - If you would like this activity on your web page, copy the script below and paste it into your web page.
Normal Size Small Size show me how
Normal Size Small Size show me how
MMBio Quiz 4
| Term | Definition |
|---|---|
| PCR | polymerase chain reaction making millions of copies of one specific piece of DNA |
| PCR requires (6 things) | thermostable DNA polymerase primers nucleotides appropriate pH (8.3-8.8) divalent cations (Mg2+) DNA template |
| denaturation | heat separates 2 DNA strands 95 C |
| primer annealing | add primers to bind strands 55-60 C |
| primer extension | Taq polymerase builds off primers 72 C |
| why the range 55-60? | because in order for primers to bind, it must be perfect too hot - primers can't form H-bhonds too cold - primers stick to incorrect places |
| Taq polymerase | from bacteria (thermus aquaticus) that can withstand high temps this is so the polymerase does not denature in high temps |
| primers | single stranded DNA that is complementary and anti-parallel to a target DNA sequence bonds to a specific sequence, highlights which DNA to copy |
| primers have a free 3' OH group which allows | nucleotides to be added on to the end of it |
| two primers (reverse primer and forward primer) both | build 5' to 3' towards each other |
| PCR generates a 'single band" in a gel because | the other fragment sizes aren't made exponentially, they don't show up in comparison |
| DNA sequencing is used to | identify genotypes, heritage (who the father is), and infecting pathogens |
| dideoxynuclotides | missing the O in the hydroxyl group, so it just has an H on the sugar "chain terminator" because you can add another nucleotide to it |
| (regular) dNTP | deoxynucleotide triphosphate |
| (modified) ddNTP | dideoxynucleotide triphosphate |
| only ___% of nucleotides in PCR are ddNTPs and are attached to ________ _____ | only 1% of nucleotides in PCR are ddNTPs and are attached to colored dyes |
| sanger DNA sequencing | determines nucleotide order using fluorescently labeled dideoxynucleotides (ddNTPs) to terminate DNA synthesis |
| ddNTPs are labeled with 4 different florescent dyes, one for each | base (A T C G) |
| sanger DNA sequencing (steps/how it works) | start with primer all on exact same base ddNTPs randomly stop at different bases PAGE separates fragments sizes smallest fragment stopped at base 2, smallest fragment stopped at the 3rd base and so on interpret sequence on a chromatogram |
| DNA sequencing on an unknown/new organism | digest genome with restriction enzyme ligate pieces into plasmid vector primers bind to known sequence, DNA polymerase moves to unknown insert, now you can read it |
| you don't have to know the DNA sequence, | just something right next to it |
| site-directed mutagenesis | using PCR to mutate a specific DNA sequence |
| site-directed mutagenesis (why) | figure out which amino acid in a protein is important by mutating it and see if protein stops working can later target that protein with drugs to stop from working if desired |
| site-directed mutagenesis (steps) | make primer with the desired base changes DNA polymerase makes the DNA with your mutation cut up old DNA, copy desired DNA |
| wild-type | original DNA |
| mutant | targeted, changed DNA |
| separate wild-type from mutant | use methylation sensitive restriction enzymes wild-type DNA is methylated in bacteria --> destoryed mutant DNA is not methylated or cleaved --> copy |
| quantitative PCR (q-PCR) | find out how much DNA/nucleic acids are present |
| Taqman probe | short DNA tag that glows only when your DNA target was copied |
| how a Taqman probe works | contains florescent dye and a quencher (that blocks the dye) when DNA polymerase copies DNA, it cuts the probe and this separates the dye from the quencher --> GLOWS |
| southern blots | DNA |
| northern blots | RNA |
| western blots | proteins |
| blotting | transferring DNA, RNA or proteins from a gel onto a paper to look at specifics |
| SNOW | DROP |
| why blot? | detect/quantify specific molecules, since cells have a huge diversity |
| probe | short, single stranded piece of DNA that is used to find a desired complementary DNA target designed to stick to one specific target |
| annealing | sticking together |
| probe hybridization | annealing two single-stranded nucleic acids to make 1 double stranded molecule must have good base pairing (as close to 100% as possible) must be anti-parallel to each other probe must be labeled or you cannot detect hybridization |
| radioactive probes | label probe with radioactivity to later detect radioactivity and see where/how much the target sequence is |
| fluorescent probes | FITC/fluorescein lights up your target safer than radioactivity |
| procedure for southern/northern blots | run gel electrophoresis, migrate to + end gel put under + filtration paper, move up to nitrocellulose paper filter peeled off, hybridize with probe probes attach to target DNA sequence use Xray film and autoradiogram to see |
| western blots | use antibodies to detect proteins |
| western blot steps | run SDS-PAGE separate proteins blot wash with first antibody binds only to target protein wash with second antibody binds to first antibody auoradiogram |
| GAPDH | enzyme that is a "loading control" lets you compare it with RNA that you got |
| ISH (situ hybridization) | detection of nucleic acids in tissue/cells requires lysis of cells/tissues probes labeled with florescent dyes for detection |
| lysis | breaking open a cell/cell membrane to release its contents |
Created by:
anyasalmon