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