Pulsed-Field Gel Electrophoresis (PFGE) PFGE was developed in 1984 and has since become the gold standard for bacterial subtyping.  PFGE is the primary subtyping method used by PulseNet, a network of public health laboratories that perform PFGE on foodborne disease organisms such as Salmonella, E. coli O157:H7, Shigella (11), Listeria monocytogenes (7), and Campylobacter jejuni (12).  PulseNet has been responsible for detecting many foodborne disease outbreaks (3, 4, 13), and has been extremely successful.   In 1999, PulseNet won the Innovations in American Government Program and in 2002, PulseNet was recognized as one of the fifteen most significant government initiative programs to have won the Innovations in American Government Award. The first step of PFGE is to make a suspension of bacteria and buffer (Figure 1-step 1). Molten agarose is added to the bacterial suspension and the agarose/suspension mixture is added to a mold (Figure 1-step 2). The agarose is allowed to solidify then added to a lysis solution which breaks open the bacteria releasing the DNA. Subsequent washing steps remove extracellular debris leaving intact, circular genomic DNA in the agarose plug (Figure 1-step 3). A small piece of the plug is cut off and added to a restriction endonuclease mixture which cleaves DNA at a specific sequence (Figure 1-step 4) resulting in 10-20 DNA fragments of varying sizes. The large DNA fragments are then separated by size by pulsed-field gel electrophoresis (PFGE) (Figure 1-step 5). PFGE facilitates the migration of the large DNA fragments through the agarose gel by regularly changing the direction of the electrical field during electrophoresis, allowing the fragments to maneuver through the agarose. The smaller DNA fragments will move faster through the agarose than the larger fragments and the result is a pattern of DNA fragments (also called bands). The pattern from one isolate can be compared to other patterns to determine whether the samples may have originated from a common source. PFGE Advantages -Easily applied to different species -Patterns consistent within and between laboratories (strict adherence to standard conditions is necessary) -PFGE generally yields a high amount of pattern diversity PFGE Drawbacks -Labor-intensive -Relatively slow (approximately 24 hours to completion) -Complex patterns challenging for inter-laboratory pattern comparisons -One mutation can yield differences in several fragments (PFGE cannot determine phylogenic relationships) -Dependant on isolated bacteria Multiple-Locus Variable Number Tandem Repeat Analysis (MLVA) Multiple-locus variable number tandem repeat analysis (MLVA) is a subtyping method that utilizes short, repetitive regions of DNA called variable-number tandem repeats (VNTR) which may be present in different numbers of repeats between strains. These variable number tandem-repeat (VNTR) regions have been identified in many species of bacteria. Multiple VNTR regions can be analyzed in one typing method (MLVA) to differentiate bacteria within a species. Most VNTR regions used in MLVA are small, containing between 3 and 21 base pair repeats. There are several steps involved in MLVA (Figure 2). The first step involves amplifying the VNTR using polymerase chain reaction (PCR is a common method used in molecular biology to amplify a specific region). PCR is used to amplify the conserved DNA outside the VNTR in addition to the variable VNTR region. The fragment size is dependant on the number of repeats for a sample. The size of the resulting fragment is determined by either horizontal or capillary electrophoresis (2). Horizontal electrophoresis uses traditional agarose gels to estimate the size of DNA fragments while capillary electrophoresis uses a DNA sequencer for fragment size analysis. Capillary electrophoresis is a particularly attractive option because you can attain extremely precise measurement of the fragments’ size (often within one base pair), which is ideal for large databases and inter-laboratory comparison. Detection of candidate loci for MLVA is dependant on knowledge of the genome for a given species. Due to the explosion of bacterial genomes that have been fully sequenced over the last decade, MLVA is becoming a more common subtyping method. MLVA has been successfully used for many genera of bacteria, including E. coli O157:H7 (8), Salmonella Typhimurium (9), Salmonella Enteritidis, and Listeria monocytogenes (many references). MLVA is used by PulseNet to further differentiate groups of E. coli O157:H7 and S. Typhimurium isolates that have the same PFGE type. MLVA Advantages -Rapid (results completed in about 5 hours) -Relatively inexpensive to perform -Portable. Amenable to inter-laboratory comparisons -Not labor intensive -Amenable for high-throughput -Loci can be added or removed to adjust diversity MLVA Drawbacks -Significant development time to create a new MLVA system -Need sequences to identify possible VNTR candidates -Sequencer is expensive and not available to many laboratories (sequencers are needed for creation of large databases) Nucleic Acid Sequence Subtyping Bacteria and viruses can be differentiated within species by determining the sequence of nucleotides bases (adenine, cytosine, guanine, or thymine) for one or more loci of an organism (Figure 3). Sequencing of a single, diverse locus is necessary to create adequate discrimination for some organisms. Single locus sequence subtyping is used for genotyping norovirus (15) and hepatitis C (14). Combined sequence of multiple regions increase subtype diversity and may yield more information about the evolutionary history of a group of strains. The most common example of subtyping an organism by sequencing multiple regions is multiple locus sequence typing (MLST). MLST is the sequencing of multiple housekeeping genes (genes necessary for organism survival) for an organism. MLST methods have been created for numerous organisms, including Streptococcus pneumonia (6), Staphylococcus aureas (10), Campylobacter jejuni (5), and Neisseria meningitides (1). Housekeeping regions generally have limited diversity so techniques have been developed to sequence the more genetically diverse virulence genes, a method called multiple virulence locus sequence typing (MVLST). MVLST has been used for subtyping Listeria monocytogenes among other organisms. Sequence Subtyping Advantages -Can be used for bacteria or virus and fungus -Portable – information can be readily shared between laboratories -Can be high throughput -Can be automated -Can be used to investigate evolutionary relationships Sequence Drawbacks -Relatively expensive -Need sequence information for organism to sequence -Lacks the discriminatory power of differentiate some species (MLST) -Labor intensive if not automated -Most current methods are slow Single nucleotide polymorphisms (SNP, pronounced snip) are mutations that occur at a single nucleotide in the genome. The fifth nucleotide in the following sequences (ATAGCA, ATAGGA) is an example of a SNP. Most SNPs have only two alleles. Identification of SNPs is dependent on having the sequence of multiple organisms within a species. Analysis of multiple SNPs for an organism has recently been utilized to differentiate bacteria. The combination of multiple SNPs can be compared and used to determine the evolutionary relationships between organisms. Since such little sequence is needed to identify the SNPs, it is a method that is more amenable to rapid, high-through-put sequence methods such as pyrosequencing. SNP Advantages -Can be used for bacteria or virus and fungus -Portable-Information can be readily shared between laboratories -Can be high throughput -Can be automated -Can be used to investigate evolutionary relationships -Only small amount of sequence needed, amenable to more rapid sequence methods SNP Drawbacks -Need sequence data to identify SNPs -Many SNPs needed to increase diversity -Need automated sequencer which may not be available in many laboratories -More diversity may be needed Dictionary of Terms

  • Agarose-a substance obtained from agar which is used as a medium for electrophoresis procedures
  • Genome-an organisms genetic material
  • Restriction endonuclease-an enzyme the cleaves DNA at a specific site to produce fragments
  • Electrophoresis-a technique to migrate proteins or nucleic acid through a medium
  • Locus-the chromosomal position of a gene


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  • Jennifer S.

    PFGE: how do you see the DNA fragments in your results? When did you add the loading buffer? I don’t know much about this, just comparing it to the conventional elecrophoresis for a class. Thanks

  • It is a question. I would like to know if in the group of MLVA and VNTR methodologies are included those known as Rep-PCR or ERIC or BOX PCR
    Thank you for your answer