DNA cleavage (stage 1) - restriction endonuclease cleaves DNA into fragments
- produces large number of different fragments
- different endonucleases >> different fragments
- gel electrophoresis - procedure that separates fragments based on size
recombinant DNA production (stage 2) - DNA fragments inserted into vectors
- vectors cleaved w/ same restriction endonuclease as DNA
cloning (stage 3) - more recombinant DNA created
- vectors introduced into reproducing cells
- each reproduced cell contains recombinant DNA
screening (stage 4) - most challenging part of any genetics experiment
- 4-I - preliminary screening of clones
- gets rid of cells w/o any vectors, vectors w/o original DNA
- uses vector w/ gene for antibiotic resistance
- based on presence/absence of a certain phenotype
- 4-II - finding gene of interest
- hybridization - uses complementary nucleic acid (probe) to find particular fragment
- solution added to denature DNA, allowing radioactive probe to attach
polymerase chain reaction (PCR) - uses DNA polymerase to mass produce gene sequences
- denaturation (step 1) - excess of primer mixed w/ DNA fragment
- DNA strand heated to 98° C >> strands dissociate
- annealing of primers (step 2) - DNA solution allowed to cool to 60 C
- DNA strands reassociate w/ excess of primer instead of other complete complementary strand
- leaves large parts of DNA single-stranded
- primer extension (step 3) - uses Taq polymerase (heat-stable DNA polymerase) to copy rest of fragment
- supply of all 4 nucleotides added to solution
- creates double the amount of DNA as before (separate strands both replicated)
- cycle repeated to double amount of DNA each time
- can create large amount of DNA for study from just a tiny amount of DNA
southern blotting - identifies DNA w/ radioactivity
- DNA fragmented by endonucleases, spread apart by gel electrophoresis
- DNA strands denatured by basic gel solution, transferred to nitrocellulose sheet
- probe w/ single-stranded DNA complementary to gene of interest poured over sheet, will bind to particular sequence
restriction fragment length polymorphism (RFLP)
- identifies particular individual w/ specific gene marker
- mutations, sequence repetitions, transposons alters length of DNA fragments created by endonucleases
- pattern of bands produced by gel electrophoresis different for each person
- DNA fingerprinting - 2 individuals rarely produced identical RFLP analyses
- used in criminal investigations by forensic teams
- autoradiographs - parallel bars on X-ray film used for comparison