Pulse-field gel electrophoresis (PFGE):

Molecular typing method

Uses: Epidemiological analysis to identify the spread of a clone of Salmonella.

Advantages:

• Interpretation of the entire bacterial genome in a single gel, high discrimination, reproducibility, and typability.
• Guidelines for interpretation of PFGE results prepared by Tenover et al., offering a distinct advantage over other genotyping methods.

Principle:

• PFGE is originated from conventional gel electrophoresis.
• Conventional gel electrophoresis applied that the DNA molecules pass through an agarose gel matrix in an electric field.
• The problem of this is that its migration is inversely proportional to the log of its size whereby small fragments move through the gel matrix faster than larger ones, and very large molecules express the same mobility, resulting in poor resolution of bands.
• PFGE overcome the problem encounter by conventional gel electrophoresis by applying the idea that once the electric field has been removed the DNA returns to its relaxed state, thus charging the orientation of the electric field at regular intervals.
• This forces the DNA molecules ion the gel to relax on removal of the first field and elongate to align with a new field, a process that is size-dependent.
• This technique of PFGE involves embedding the organism in an agarose plug, thus reducing shearing of the DNA, lysing the organism in situ, and digesting the chromosomal DNA with an appropriate restriction enzyme,

Type of electrophoresis systems commercially available:

• contour-clamped homoge-nous electric field (CHEF),
• transverse alternating field electrophoresis (TAFE)
• field-inversion gel electrophoresis (FIGE)
• orthogonal-field alternation gel electrophoresis.

Interpretation of PFGE Gel:

Chromosomal DNA restriction patterns (Banding pattern) produced by PFGE are interpreted based on the Tenover et al. criteria for bacterial strain typing. The banding pattern is affected by the genetic events. By comparing the banding pattern to that of the outbreak strain, each isolate is assigned to one of four categories:

- Indistinguishable: Restriction patterns had the same number of bands and the corresponding bands were the same apparent size from the outbreak strain.
- Closely related: Restriction patterns differed from the outbreak strain by a single genetic event that explains by a point mutation or an insertion or delete of DNA.
- Possibly related: Restriction patterns differed from the outbreak strain by two independent genetic events giving rise to 4-6 band differences that explains by a point mutation or simple insertions or deletions of DNA.
- Unrelated: Restriction patterns differed from the outbreak strain by three or more independent genetic events giving rise to seven or more band differences.
Statistical Analysis of PFGE Gel interpretation:
- Uses the ‘Dice coefficient’,
- Expressed algebraically as: 2* n/ (a +b).
Where n is the number of restriction fragments that both isolates have ion common;
a is the number of restriction fragments observed for the outbreak strain;
b is the number of restriction fragments observed for the isolates being compared to the outbreak strain.
- The closer the Dice coefficient is to 1, the greater the similarity between the Salmonella strain.


Reference: Rachel Gorman and Catherine C. Adley. 2006. Pulsed-Field Gel Electrophoresis As a Molecular Technique in Salmonella Epidemiological Studies. In: Catherine C. Adley. 21. Place of publication: Human Press Inc. 81 – 90.