Probing, Recombining, Cloning Flashcards
Steps for nucleic acid probing
- lyse organism to release nucleic acid
- plane dna in heat/alkaline conditions to break hydrogens break
- decrease temp/ph add prob
- wash away probe
- dete any bound probe through radiactive presence/light emission
Methods which utlize probing
southern blot, northern blot, direct probing, pcr
Souther blot
combines transfer of electrophoresis-separated DNA fragments to a filter membrane
Northern blot
by detection of RNA (or isolated mRNA) in a sample.
Polymerase chain reaction PCR
using dntps, enzymes nd primers millions of dsdna are made
How does PCR work
- lyse to release dna
- heat at 100degres c
- cool to <60
- add primers, dnTps and dna polymerase
- Head again and cycle through step 3 and 4 (thermocycling)
Why are these techniques powerful
- Specificity
- Speed
- Sensitivity (make more)
Recombinant Dna
take dna from 2 sources and splice together
Splice-join together
Gene Cloning
allow recombinant dna within cell to create clones of dna, removes genes from organism or make your own gene then insert gens into host (bacterial, human)
How to clone
- obtain dna by cell lysis
- cut off gene of interest and recipient dna to creat smae sticky ends
- ligate
- transform/transduce
How to lyse a cell
chemical treament/hypotonic environment
how to cut dna
using restriction enzymes,
how to seal surgar phosphate
dna ligas creates phosphodiester bond, glues gap
- DNA hybridization
bind a labeled piece (probe) of dna/rna to specific nucleic acid having complementary sequence
probe
looking for aaa search with ttt/uuu
