Genotyping – Fragment Analysis

Microsatellites, also referred as STR (Short Tandem Repeats) are small regions, widely dispersed on a given genome, that contain a repeated sequence of 2 to 9 bases (like GTGTGTGTGTGT). Due to polymerase slippage, the length of the motif can vary between individuals. Microsatellites are amplified by PCR with a couple of specific primers, whose one is fluorescent, and the size of the fragments obtained is analyzed on a capillary sequencer. Diploid organisms may be homozygote or heterozygote at a particular locus that leads to one or two peak profile on the respective electropherogram.

AFLP (Amplified Fragment Length Polymorphism) imply the digestion of bacterial DNA by two restriction enzymes, followed by the ligation of two double-stranded DNA adaptors. Then, a subset of the fragments obtained is amplified with specific fluorescent PCR primers with selective 3-prime ends. The resulting PCR product is finally separated by length on a capillary sequencer leading a species-specific profile.

For each sample, one of the primer is labelled with a specific fluorophor (6-FAM, HEX, ROX or NED). So that you can mix up to four (4) sample on a tube (384 for a 96 wells plate) to save cost.

ARISA (Automated Ribosomal Intergenic Spacer Analysis) is a method used for the characterization of complex microbial communities. A specific couple of primer (with a fluorescent label) is used to amplify the 16S-23S spacer region of complex samples. Depending of the organism this region is very variable in size. So, the PCR product obtained is a complex mix of fragments of different size generally comprised between 50 to 1200 bp. This leads to profiles specific of these communities when separated on a capillary sequencer. Each peak (band) corresponds to a bacterial population or a group of populations of different importance correlated to the variation of intensities of the peak.

VNTR (Variable Number of Tandem Repeats) are minisatellites, that is, nucleotide patterns ranging from 10 to 100 bases typically repeated 5-50 times and located in certain regions of a given genome. Specific primers are used to PCR-amplify fragment that contain VNTRs. Their size of the fragments are measured on a capillary sequencer and by hence the number of repeats are deducted. Each variation of length represents an allele.

MLVA (Multiple Loci VNTR Analysis) use the length polymorphism of the VNTRs as a molecular method for the typing of organisms

MIRU (Mycobacterial Interspersed Repetitive Units) is a specific name for the MLVA analysis of the Mycobacterium tuberculosis complex. Similarly, the term SIRU is used for Staphylococcus aureus typing.