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View additional product information for Quantifiler™ Duo DNA Quantification Kit - FAQs (4387746)
12 product FAQs found
Knowing the ratio of male to female DNA present in a mixed sample can help the analyst to decide whether autosomal or Y-STR analysis will provide the maximum amount of information on the male component. Generally, for samples with a male:female DNA ratio up to 1:10, both male and female components can usually be visualized and interpreted using an autosomal STR system such as the Quantifiler Duo DNA Quantification Kit. For samples with a male:female DNA ratio above 1:10, the minor component may be below the level of detection of the instrument platform if analyzed using an autosomal STR kit, and therefore the male component may best be analyzed using a Y-STR system such as the Yfiler Plus kit. Each laboratory should determine the minimum level of detection for the male component of a mixed sample using an autosomal amplification system and identify the cut-off value at which Y-STR analysis may prove more successful. This can be achieved as part of an internal validation study of the Quantifiler Duo DNA Quantification Kit.
The Quantifiler Duo DNA Quantification Kit quantitates the amount of total human and human male DNA in biological samples. The ratio of male to female DNA (Male DNA : Female DNA Ratio) can be calculated using the following equation:
Male DNA (Human DNA - Male DNA)
------------ : ---------------------------------
Male DNA Male DNA
(All quantities in the above equation are ng/µL). For example, assuming:
Male DNA concentration = 2 ng/µL
Human DNA concentration = 8 ng/µL
Then the Male DNA : Female DNA Ratio = 2/2 : (8-2)/2 = 1:3
The dynamic range of the Quantifiler Duo assay is 23 pg-50 ng/µL and is the same as for the previous Quantifiler DNA quantification kits. The Quantifiler Duo DNA Quantification Kit will detect samples containing a DNA concentration of less than 23 pg/µL. Sensitivity studies have demonstrated the ability to detect samples with a DNA concentration as low as 6 pg/µL. However, detection of <100 pg/µL using any amplification-based quantification method is subject to stochastic effects which may result in some uncertainty in quantification results and prediction of amplification success. Any samples yielding quantification values of 100 pg/µL or less will require the maximum volume of DNA for STR amplifications targeting 1 ng regardless of the DNA concentration.
Laboratories can use either the HID Real-Time PCR Analysis Software, designed specifically for the forensic workflow, or SDS software v1.2.3. The HID Real-Time PCR Analysis Software provides Quantifiler Duo specific M:F flags and other advanced data analysis functionality not available with the SDS software v1.2.3.
The Quantifiler Duo DNA Quantification Kit has been optimized and validated for use on the Applied Biosystems 7500 Real-Time PCR System and the Applied Biosystems Human Identification (HID) QuantStudio 5 Real-Time PCR System.
The DNA standard for the Quantifiler Duo DNA Quantification Kit consists of pooled human male genomic DNA for improved stability and correlation with forensic samples. This is in contrast to the cell line DNA used in the previous Quantifiler kits.
The sizes of the amplicons were chosen to improve the estimation of DNA quantity, thereby delivering a more realistic estimate of the amount of DNA present in degraded samples and providing better correlation between the quantitation values and the performance of the AmpFℓSTR kits.
The amplicon size for the Ribonuclease P RNA component H1 (RPPH1) human target is 140 bp. The amplicon size for the SRY human male target is 130 bp. The amplicon size for the IPC (internal PCR control) artificial template is 130 bp.
The human target is located in the RPPH1 gene (Ribonuclease P RNA component H1) on chromosome 14 (location: 14q11.2) and the male target is located in the SRY gene on the Y chromosome (location: Yp11.3).
Real-time PCR is the ability to monitor the accumulation of PCR product as it occurs (i.e., in real-time). Real-time PCR reactions are characterized by the point in time during cycling when amplification of a specific target is first detected, rather than the amount of target accumulated after a fixed number of cycles. The higher the starting copy number of the target, the sooner a significant increase in fluorescence is observed. Through comparison to a DNA standard of known quantity, the concentration of unknown DNA samples can be estimated reliably. Using a quantitation technique based on PCR helps to identify the amount of amplifiable DNA present in a sample and provides an effective prediction of the way each sample will perform in a downstream PCR assay. The Quantifiler Duo DNA Quantification kit takes advantage of the ability of multiplexing different targets in a single real-time PCR assay and delivers effective estimations of total human and human male DNA quantity.
The Quantifiler Duo kit is a multiplexed TaqMan real-time PCR assay for DNA quantitation which amplifies a total human and human male target simultaneously.
There is an additional large autosomal (LA) target that has been added as part of the degradation index feature in the Quantifiler Trio DNA Quantification kit. This marker plus the improved small autosomal (SA) and male targets make three different quantification targets in the Quantifiler Trio kit, whereas the Quantifiler Duo kit has only two targets: one autosomal and one male. The new degradation index functionality in the Quantifiler Trio kit enables a more comprehensive view of sample quality (both degradation and inhibition), and the kit also has increased sensitivity and robustness to better correlate with more recently released Applied Biosystems STR chemistries, such as those in the Identifiler Plus, NGM SElect, and GlobalFiler PCR Amplification Kits.