Biology Reference
In-Depth Information
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Figure 6.6
Internal-lane size standards are used to precisely size the PCR products. Two commonly
used internal-lane size standards are (a) the GeneScan
-500 (Applied Biosystems) and (b) the
ILS600 (Promega Corporation)
Because the internal-lane size standard is analysed along with each PCR, any
differences between runs that could affect the migration rates during electrophoresis,
such as temperature, do not impact significantly on the analysis [41]. The software
generates a size calling curve from the internal-lane size standards; the data point of
the unknown fragments are compared with 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 [42] (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 [6].
The loci used in forensic casework have been well characterized and multiple
alleles have been sequenced to determine the allelic structure and verify that the size
of the peaks is a good indicator of the alleles they represent. However, because the
migration of PCR products and internal-lane size standard varies slightly with factors
such as temperature and the electrophoretic conditions, and because some STR alleles
