Interpretation of STR profiles
The spectra of the dyes used to label the PCR products overlap and the raw data contains peaks that are composed of more than one dye colour.
After data collection the GeneScan® or GeneMapperTM ID software removes spectral overlap in the profile and calculates the sizes of the amplified DNA fragments. The software calculates how muchspectraloverlapthereisbetweeneachdyeandsubtractsthisfromthepeakswithin the profile (Figure 6.5). A good matrix file, which contains information on the amount of overlap in the spectra, will produce peaks within the profile that are composed of only one colour.
The height of the peaks is measured in relative fluorescent units (rfu) – the height is proportional to the amount of PCR product that is detected. To be able to size the PCR products an internal-lane size standard is used. The internal-lanesizestandardscontainfragmentsofDNAofknownlengthsthatarelabelled
Figure 6.5 The application of a matrix file, using the GeneScan® or GeneMapperTM ID software removes the spectral overlap from the raw data (a) to produce peaks within the profile that are composed of only one colour (b) (see plate section for full-colour version of this figure). The scale of the X-axis is relative fluorescent units (rfu)
Figure 6.6 Internal-lane size standards are used to size the PCR products precisely. Two commonly used internal-lane size standards are (a) the GeneScanTM-500 (Applied Biosystems) and (b) the ILS600 (Promega) (see plate section for full-colour version of this figure) with a fluorescent dye, and the fragments are detected along with the amplified PCR products during capillary electrophoresis . Commonly used commercial internal- lane size standards are the GeneScanTM-500 standards that can be labelled with either ROXTM or LIZTM dyes (Applied Biosystems) and the ILS600 (Promega Corporation) (Figure 6.6) Because the internal-lane size standard is analysed along with each PCR any differ- ences between runs that could affect the migration rates during electrophoresis, such as temperature, do not impact significantly on the analysis . The software gener- ates a size calling curve from the internal-lane size standards – the data point of the unknown fragments are compared to the size calling curve. Different algorithms have been developed to measure the size of DNA molecules, the most common one is the local Southern method  (Figure 6.7). After analysing the raw data with the software, the end result is an electropherogram with a series of peaks that represent different alleles: the size, peak height and peak area is also measured by the software (Figure 6.8). The final stage of generating a STR profile is to assign specific alleles to the amplified PCR products. Each peak in the profile is given a number that is a description of the structure of that allele – this is straightforward when naming simple repeats but is more problematic with complex repeat sequences .