Single nucleotide polymorphism profiling assay to confirm the identity of human tissues

Abstract

To identify issues of sample mix-ups, various molecular techniques are currently used. These techniques, however, are time consuming and require experience and/or DNA sequencing equipment or have a relatively high risk of errors because of contamination. Therefore, a quick and straightforward single nucleotide polymorphism (SNP) profiling assay was developed to link human tissues to a source. SNPs are common sequence variations in the human genome, and each individual has a unique combination of these nucleotide variations. Using potentially mislabeled paraffin-embedded tissues, DNA was extracted and SNP profiles were determined by real-time polymerase chain reaction analysis of the purified DNA using a selection of 10 commercially available SNP amplification assays. These profiles were compared with profiles of the supposed owners. All issues (34 in total) of potential sample mix-ups during the last 3 years were adequately solved, with six cases described here. The SNP profiling assay provides a quick (within 24 hours), easy, and reliable way to link human samples to a source, without polymerase chain reaction postprocessing. The chance for two randomly chosen individuals to have an identical profile is 1 in 18,000. Solving potential sample mix-ups will secure downstream evaluations and critical decisions concerning the patients involved.

Figure 1

SNP detection with MGB probes. A: Principle of SNP detection using hydrolysis probes. When the probe is intact, the quencher quenches the fluorescent signal from the reporter. A/C SNP: in the presence of A, the VIC probe, complementary to A, is hydrolyzed during the PCR by the Taq polymerase. The VIC reporter is no longer quenched by the close proximity of the quencher and can be measured. In the presence of C, the VIC probe is removed from the DNA strand without being hydrolyzed. The FAM probe is complementary to C and, according to the same principle, generates a fluorescent signal only in the presence of C. B: Fluorescent signals accompanying the different genotypes (SNP10; genotype is indicated in top left corner).

Figure 2

Fluorescent signals (light gray line, VIC; black line, FAM) of three SNP assays performed on DNA isolated from blood of four patients.

Figure 3

Paraffin-embedded mammary tissue. A: Tissue assigned 1-A-I, an infiltrative ductal carcinoma. B: Tissue assigned 1-A-II, a benign, apocrine metaplasia. C: Infiltrative adenocarcinoma. H&E stain. Original magnifications, ×100.

Figure 4

Fluorescent signals (light gray line, VIC; black line, FAM) of three SNP assays performed on DNA isolated from two mammary tissues (1-A-I and 1-A-II) from patients 1 and 2 and a cervical polyp from patient 1 (1-B).

Figure 5

Diminished fluorescent signal of SNP14 probably attributable to LOH. Fluorescent signal (light gray line, VIC; black line, FAM) of the SNP assays performed on DNA isolated from the lymph node biopsy (2-B), the floater of lung tumor tissue (2-A), and a skin sample from another patient (for SNP14 homozygous, genotype T/T). The near absence of the VIC signal for SNP14 in the homozygous individuals demonstrates that the VIC signals in the floater are not because of cross-reaction of the VIC probe with the other allele. Thus, the diminished VIC signal for SNP14 in the floater is likely attributable to LOH. The fluorescent signals of SNP13 show no indication of LOH.