Human sample identification can be defined as three applications: human cell line authentication, sample authentication, and mixed sample analysis. Short tandem repeat (STR) genotyping is an important tool in verifying the authenticity of human cell lines, quality control of stored human tissues and fluids, and assessing the nature of known mixtures.

Cell line authentication

When cell lines are misidentified, misleading results can ensue, which is why many journals and funding agencies are now requiring scientists take measures to verify their cell lines. These measures often involve analysis of alleles of several loci to make sure they match expected alleles. This can be done in a number of ways, but analysis of highly variable STR markers has become known as a simple, inexpensive way to get a highly specific genetic "fingerprint" of a cell line.

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Video: Importance of Human Cell Line Authentication

Sample authentication

Sample authentication is carried out when storing or processing biological samples of human origin. Establishing the STR genotype of a sample upon receipt allows one to confirm its identity when the sample is accessed from storage or at any point in the workflow.

Video: Importance of Human Sample Authentication

Mixed sample analysis

Mixed sample analysis (MSA) is the use of an STR chemistry to evaluate the percent mixture between two or three known DNA samples by semi-quantitative endpoint PCR. By increasing the amount of template DNA and decreasing the number of amplification cycles, a balanced profile from both the major and minor contributors can be examined. When the level of a mixture or contamination is critical to a downstream process, MSA is performed to identify the level of mixture or contamination in a sample.

Human sample identification workflow

Typically, analysis of STRs is performed by capillary electrophoresis (CE) of fragments amplified from microsatellite loci with varying number of repeats. We have established the gold standard for CE instrumentation by developing and offering instruments that are optimized for researchers’ needs in sensitivity and throughput. Furthermore, the Applied Biosystems product portfolio has several different kits for PCR-based STR fingerprinting for use on CE instruments. The Identifiler Plus PCR Amplification Kit has been optimized to analyze 16 highly variant human STRs over a wide range of purified gDNA preparations. The Identifiler Direct PCR Amplification Kit was first developed to analyze the same 16 STR loci, starting from dried blood or buccal spots (for example, on NUCLEIC-CARD devices) or buccal swabs. For the NUCLEIC-CARD device, a 1.2-mm punch from the card is placed directly into a PCR tube or well and amplified without any further purification. Finally, GeneMapper Software 5 facilitates analysis of STRs by making use of pre-established allelic ladders and sizing bin sets for the various STR alleles covered by the Identifiler kits.

An illustration of the complete workflows for human sample identification is shown in Figure 1.

Figure 1. Workflows for cell line authentication. Two methods are available for cell line authentication. (A) Cells can be spotted onto NUCLEIC-CARD devices, punches of the cards amplified directly using the Identifiler Direct kit, and fragments analyzed on Applied Biosystems™ CE instruments using GeneMapper Software 5. (B) Alternatively, gDNA can be purified from cell lines, amplified using the Identifiler Plus kit, and fragments analyzed by capillary electrophoresis and GeneMapper Software 5.

Technical specifications

Specification GlobalFiler kit Identifiler Plus kit Identifiler Direct kit
Markers 24 (21 autosomal and 3 sex determination markers) 16 (15 autosomal and amelogenin) 16 (15 autosomal and amelogenin)
Applied Biosystems instrument compatibility 3500 series, 3130 series, and 3730 (GFE) SeqStudio Genetic Analyzer, 3500 series, 3130 series, 3730, and 310 SeqStudio, 3500 series, 3130 series, 3730, and 310
Polymer/array POP-4, POP-7, 36 cm, and 50 cm POP-4, POP-7, 36 cm, and 50 cm POP-4, POP-7, 36 cm, and 50 cm
Applied Biosystems software compatibility GeneMapper ID-X and GeneMapper 5.0
Applied Biosystems thermal cycler compatibility GeneAmp PCR System 9700 (gold or silver blocks only), Veriti 96-well, and ProFlex PCR System (96-well, 2 x 96-well, and 3 x 32-well)
Kit size 200 and 1000 50, 100, 200, 1000 50, 100, 200, 1000
Reaction volume (µL) 25 25 25
Dye label 6-dye chemistry (FAM, VIC, NED, TAZ, LIZ, and SID) 5-dye chemistry (FAM, VIC, NED, PET,  and LIZ) 5-dye chemistry (FAM, VIC, NED, PET,  and LIZ)
Amplicon size ≤400 bp; SE33 is under 450 bp ≤360 bp ≤360 bp
Amplification time <90 min 2.5–3 hr 2.5–3 hr
Mini loci–250 bp 12 full, 3 partial, Y indel, and amelogenin 10 10
Sample input or sample type 5 µL; total 1 ng but also optimized for 2.5–5 ng Up to 10 µL; total 1 ng but also optimized for 2.5–5 ng 1.2 mm punch from a treated paper or 2–3 μL of Applied Biosystems Prep-n-Go Buffer-treated swabs or 1.2-mm punch from an untreated paper

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