Gel electrophoresis
- separation of strands of DNA of different lengths
- analysis of proteins and DNA
- place mixture of molecules into wells cut in agarose gel and apply electric field
- factors affecting movement speed of molecules:
- net charge of molecules
- size of molecule
- composition of gel: size of ‘pores ‘ within gel
Electrophoresis of proteins
- separates polypeptides produced by different alleles of many genes
- charge on protein is dependent on the ionization of the R groups on the amino acid residues
- charge depends on pH hence use of buffer solution to keep constant pH
- polypeptides separate due to different net charges
- proteins are usually negatively charged
Electrophoresis of DNA
- a region of DNA is chosen
- DNA is extracted
- DNA is chopped into pieces using restriction endonucleases
- fragments are transferred to absorbent paper and placed onto gel; heated to separate DNA strands
- short sequences of single-stranded DNA are added. They contain radioactive P isotopes; X-ray results, darken film, the separated fragments become visible
Describe how electrophoresis is used in genetic fingerprinting
(Outline how genetic fingerprinting is carried out)
- ref. to VNTR (sequences) ;
- quantity of DNA increased by PCR ;
- DNA fragmented by, restriction enzyme(s) / endonuclease(s) ;
- loaded (into wells) in agarose gel ;
- (at) negative end/ cathode end ;
- ref. to buffer/ electrolyte ;
- direct current applied ;
- phosphate groups of DNA give negative charge ;
- (negatively charged) DNA attracted to, anode/ positive electrode ;
- short pieces/ smaller mass, move further/move faster ;
- (pieces) transferred to, membrane/ nylon/nitrocellulose/ absorbent paper or Southern blotting ;
- heated to separate strands ;
- probes/ fluorescent dye, added ;
- X-ray film/ UV light/ lasers ;
- pattern of stripes/ ref. banding pattern ;