Quantitative Information From Fluorescence Measurements

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The ability to obtain quantitative information from peaks in an electrophero-gram, either using the ABI 310, 377, or 3100 platform or a fluorescence scanner such as the FMBIO II (see Chapter 14), now permits relative peak heights or areas of STR alleles to be measured. This peak information can then be used to decipher the possible genotypes of the contributors to the mixed sample. Due to peak shape variation, peak areas have been advocated as being superior to peak heights when comparing allele peak information (Gill et al. 1998a). However, peak heights are successfully used in many laboratories for mixture interpretation.

Figure 7.3 illustrates how typical single-source samples differ from mixed samples in their STR profiles. STR allele peak patterns for heterozygous samples will generally have stutter products that are less than 15% of the associated allele peak height/area. In addition, the peak height ratio, as measured by dividing the height of the lower quantity peak in relative fluorescence units by the height of the higher quantity allele peak, should be greater than approximately

Figure 7.3

Illustration of typical single-source (a) versus mixed sample (b) heterozygote peak patterns. The relative peak areas due to the measured fluorescent signal are useful indicators to decipher the presence of a sample mixture. If the highest peak at a locus is set at 100%, then heterozygous alleles should have peak areas and peak heights that are greater than 70% of the highest alleles. Stutter products are typically less than 15% of their corresponding allele peak and shorter by four base pairs for tetranucleotide repeats.

100%

Heterozygous peak region

85%

>70%

MIXTURE REGION

<15%

Stutter region

9%. A.

Wrong side of allele to be typical stutter product

70% in a single-source sample (Gill et al. 1997, Applied Biosystems 1998). Thus, if peaks fall in the region between 15% and 70% of the highest peak at a particular STR locus, a mixed sample that has resulted from two or more contributors is probable. The observation of three or more alleles at multiple loci is also a strong indicator of the presence of a mixture.

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