IB Biology SL

Revision Notes

3.4.1 Electrophoresis & PCR

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

Electrophoresis-1, downloadable AS & A Level Biology revision notes Electrophoresis-2, downloadable AS & A Level Biology revision notes
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

Exam Tip

Remember gel electrophoresis is the separation of molecules according to their size and charge (negatively charged DNA molecules move to the positive pole). Examiners like to ask questions about gel electrophoresis.

Polymerase Chain Reaction (PCR)

  • Polymerase chain reaction (PCR) is a common molecular biology technique used in most applications of gene technology
    • For example, DNA profiling (eg. identification of criminals and determining paternity) or genetic engineering
  • It can be described as the in vitro method of DNA amplification
  • It is used to produce large quantities of specific fragments of DNA or RNA from very small quantities (even just one molecule of DNA or RNA).
    • Using PCR, scientists can produce billions of identical copies of the DNA or RNA samples within a few hours, these can then be used for analysis

The requirements of PCR

  • Each PCR reaction requires:
    • Target DNA or RNA being amplified
    • Primers (forward and reverse) – these are short sequences of single-stranded DNA that have base sequences complementary to the 3’ end of the DNA or RNA being copied. They define the region that is to be amplified by identifying to the DNA polymerase where to begin building the new strands
    • DNA polymerase – is the enzyme used to build the new DNA or RNA strand. The most commonly used polymerase is Taq polymerase as it comes from a thermophilic bacterium Thermus aquaticus which means it does not denature at the high temperature involved during the first stage of the PCR reaction and secondly, its optimum temperature is high enough to prevent annealing of the DNA strands that have not been copied yet
    • Free nucleotides – used in the construction of the DNA or RNA strands
    • Buffer solution – to provide the optimum pH for the reactions to occur in

The key stages of PCR

  • The PCR process involves three key stages per cycle
  • In each cycle the DNA is doubled (so in a standard run of 20 cycles a million DNA molecules are produced)
  • The PCR process occurs in a piece of specialist equipment called a thermal cycler, which automatically provides the optimal temperature for each stage and controls the length of time spent at each stage
  • The three stages are:
    1. Denaturation – the double-stranded DNA is heated to 95°C which breaks the hydrogen bonds that bond the two DNA strands together
    2. Annealing – the temperature is decreased to between 50 – 60°C so that primers (forward and reverse ones) can anneal to the ends of the single strands of DNA
    3. Elongation / Extension – the temperature is increased to 72°C for at least a minute, as this is the optimum temperature for Taq polymerase to build the complementary strands of DNA to produce the new identical double-stranded DNA molecules

Polymerase chain reaction (1), downloadable AS & A Level Biology revision notesPolymerase chain reaction (2), downloadable AS & A Level Biology revision notes

The substances required for polymerase chain reaction to occur and the three key stages of the reaction

Exam Tip

It is important to know the three stages and the temperatures the reactions occur at during the different stages. You must also know why the Taq polymerase is used in PCR.

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