6-3-6-electrophoresis

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

• This separation occurs because:

  • 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 thus when placed in an electric field the molecules move towards the anode

  • The different sizes of the molecules:

    • 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

  • The type of gel:

    • Different gels have different sized pores which affect the speed at which the molecules can move through them

DNA separation

• 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 first 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

  • 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

Method

• To separate the DNA fragments in gel electrophoresis the scientists :

  1. Create an agarose gel plate in a tank. Wells (a series of groves) are cut into the gel at one end

  2. Submerge the gel in an electrolyte solution (a salt solution that conducts electricity) in the tank

  3. Load (insert) the fragments into the wells using a micropipette

  4. Apply an electrical current to the tank. The negative electrode must be connected to the end of the plate with the wells as 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

  5. The smaller mass / shorter pieces of DNA fragments will move faster and further from the wells than the larger fragments

  6. 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 the 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 stain / dye (eg. ethidium bromide) which fluoresces (shines) when exposed to ultraviolet (UV) light, creating a pattern of coloured bands

The process of electrophoresis

Protein separation

• The different amino acids (because of the different R groups) determine the charge of proteins. The charge of the R groups depends on the pH and therefore buffer solutions are used during the separation of proteins to keep the pH constant

  • Proteins are prepared for electrophoresis by:

    • Denaturing (to break the disulfide bonds)

    • Then manipulating the proteins into rod shapes (which are negatively charged) to allow separation by size

• Gel electrophoresis can be used to show genotypes of individuals by separating polypeptide chains produced by different alleles

  • eg. The haemoglobin variants, α-globin, β-globin and the sickle cell anaemia variant of β-globin, have different net charges and therefore will separate out during electrophoresis to show the presence of the sickle cell allele