AQA A Level Biology复习笔记8.4.11 Gel Electrophoresis

• Gel electrophoresis is a technique used widely in the analysis of DNA, RNA and proteins
• During electrophoresis the molecules are separated according to their size / mass and their net (overall) charge
• Of the electrical charge molecules carry – positively charged molecules will move towards the cathode (negative pole) whereas negatively charged molecules will move towards the anode (positive pole) eg. DNA is negatively charged due to the phosphate groups and so when placed in an electric field the molecules move towards the anode
• Different sized molecules move through the gel (agarose for DNA and polyacrylamide – PAG for proteins) at different rates. The tiny pores in the gel result in smaller molecules moving quickly, whereas larger molecules move slowly
• Different restriction enzymes cut the DNA at different base sequences. Therefore scientists use enzymes that will cut close to the variable number tandem repeat (VNTR) regions
• Variable number tandem repeats (VNTRs) are regions found in the non-coding part of DNA. They contain variable numbers of repeated DNA sequences and are known to vary between different people (except for identical twins). These VNTR may be referred to as ‘satellite’ or ‘microsatellite’ DNA

Apparatus

• Agarose gel
• Electrophoresis tank
• Electrolyte solution
• Micropipette
• Electrodes
• DNA sample
• DNA standard
• Probes
• Nitrocellulose
• A dye

Method

• Create an agarose gel plate in a tank. Cut wells (a series of groves) into the gel at one end
  • Different gels have different sized pores which affect the speed that the molecules can move through them

   

• Submerge the gel in an electrolyte solution (a salt solution that conducts electricity) in the tank
• Pipette the DNA samples into the wells using a micropipette ensuring the DNA standard is loaded into the first well
  • DNA can be collected from almost anywhere on the body, e.g. the root of a hair or saliva from a cup. After collection DNA must be prepared for gel electrophoresis so that the DNA can be sequenced or analysed for genetic profiling (fingerprinting)
  • To prepare the fragments scientists must purify the DNA, increase (amplify) the number of DNA molecules by the polymerase chain reaction (PCR). Then restriction endonucleases (enzymes) are used to cut the DNA into fragments

   

• Connect the negative electrode to the end of the plate with the wells and connect the positive anode at the far end
  • The DNA fragments will then move towards the anode (positive pole) due to the attraction between the negatively charged phosphates of DNA and the anode

   

• The smaller mass / shorter pieces of DNA fragments will move faster and further from the wells than the larger fragments
• The fragments are not visible so must be transferred onto absorbent paper or nitrocellulose which is then heated to separate the two DNA strands
• Probes are then added, after which an X-ray image is taken or UV-light is shone onto the paper producing a pattern of bands which is generally compared to a control fragment of DNA
• Probes are single-stranded DNA sequences that are complementary to the VNTR regions sought by scientists. The probes also contain a means by which to be identified. This can either be:
  • A radioactive label (eg. a phosphorus isotope) which causes the probes to emit radiation that makes the X-ray film go dark, creating a pattern of dark bands
  • A fluorescent dye (eg. ethidium bromide) which fluoresces (shines) when exposed to ultraviolet (UV) light, creating a pattern of coloured bands

   

The separation of DNA fragments using gel electrophoresis. Gel electrophoresis can be used in DNA profiling where scientists separate the VNTRs (as these are unique to every person except identical twins).

Limitations

• The measurements are not precise and must be compared to a standard in order to gather data
• Electrophoresis requires quite a lot of sample and therefore depends on PCR to work correctly to amplify DNA fragments