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 ;