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Amplified DNA Product Separation for Forensic Analysts

Gel Matrix

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Two types of gels are commonly used in for amplicon separation in forensic DNA analysis:

  • Agarose
  • Polyacrylamide gel (PAG)

Agarose is made from seaweed and consists of long chains of polysaccharides, which interlink to form a matrix with large pores. Agarose is non-toxic and has high gel strength. Agarose gels are generally used to separate larger DNA molecules.

Polyacrylamide gels have much smaller pore sizes, are generally stronger than agarose gels, and are used to separate smaller DNA molecules. PAGs use two major monomers: monoacrylamide and a cross-linking agent, N, N' -methylene- bis -acrylamide, often referred to as bis -acrylamide. The polymerization process involves the mixing of the monomer solutions in the presence of free radical catalysts and accelerators. The PAG is formed by covalent bonding of the acrylamide and bis -acrylamide. This covalent linking produces a molecular sieving gel.


Monoacrylamide is a known neurotoxin and must be handled with care.

Both types of gels have a texture very much like a colorless gelatin, ranging from a solid support medium to a viscous liquid. The amount of sieving can be controlled by adjusting the concentration of the gel. The main difference between polyacrylamide gels and agarose gels is that PAGs are created chemically versus the biological origin of agarose gels. For separation of amplified DNA products on slab-gels, polyacrylamide gels (PAGs) are the matrix of choice.

Characteristics of PAGs:

  • Transparent in the visible and portions of the UV spectrum
  • Reproducible, completely chemically synthetic compared to biological agarose gels
  • Strength of gel and easy to handle
  • Non-reactive with samples since there is no charge associated with it; therefore, the DNA fragments are not subjected to electroendosmotic effects
  • Stable
  • Thin gels can be cast which can promote better separations at higher field strengths due to efficient heat dissipation
  • Permanent record of separation
  • Pore size can be manipulated to increase molecular sieving
  • Different buffers can be used such that resolution and run times can be manipulated
  • Toxic
  • Need to formulate gel mixture and pour the gel
  • Need to degas the gel mixture under a vacuum to reduce bubbles
  • Possibility of bubble formation, even if degassed
  • Wait for polymerization – time-consuming

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