Q: What is the Quantifiler® Duo kit?
A: 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.
Q: What is real-time PCR and what advantages does a DNA quantitation assay based on this technology offer forensic analysis?
A: 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 starting copy number, 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 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.
Q: Which human and male targets does the Quantifiler® Duo kit use?
A: 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).
Q:What are the lengths of the new amplicons?
|Human||Ribonuclease P RNA component H1 (RPPH1)||140 bp|
|Human Male||SRY||130 bp|
|IPC||Artificial Template||130 bp|
Q: Why have the amplicon lengths for the Quantifiler® Duo kit been increased compared to the current Quantifiler® Y and Quantifiler® Human kits?
A: 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.
Q: What is the nature of the DNA standard contained in the Quantifiler® Duo kit?
A: The DNA standard for the Quantifiler® Duo 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 current Quantifiler® kits.
Q: On which real-time PCR instruments will the Quantifiler® Duo kit be supported?
A: The Quantifiler® Duo kit has been optimized and validated for use on the Applied Biosystems 7500 Real-Time PCR System only.
Q: What software version should be used when running Quantifiler® Duo on the Biosystems 7500 Real-Time PCR System?
A: Laboratories can choose either the new HID Real-Time PCR Analysis Software v1.0, designed specifically for the forensic workflow, or SDS software v1.2.3. The HID Real-Time PCR Analysis Software v1.0 provides Quantifiler® Duo specific M:F flags and other advanced data analysis functionality not available with the SDS software v1.2.3.
Q: Will the existing Quantifiler® kits be discontinued when the Quantifiler® Duo kit is released?
A: The current Quantifiler® kits will continue to be available upon release of the Quantifiler® Duo kit.
Q: If I am a current Quantifiler® kit user, what level of validation will be required to switch to the Quantifiler® Duo kit?
A: All laboratories will be required to perform an internal validation to determine the optimal DNA input amount for STR amplification based on the results of the Quantifiler® Duo assay.
Q: What is the dynamic range of the Quantifiler® Duo kit and can the kit quantify DNA present at very low levels?
A: The dynamic range of the Quantifiler® Duo assay is 23pg-50ng/µL and is the same as for the existing Quantifiler® kits. The Quantifiler® Duo kit will detect samples containing a DNA concentration of less than 23pg/µL; sensitivity studies have demonstrated the ability to detect samples with a DNA concentration as low as 6pg/µL. However, detection of <100pg/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 100pg/µL or less will require the maximum volume of DNA for STR amplifications targeting 1 ng regardless of the DNA concentration.
Q: How can results from the Quantifiler® Duo kit be used to calculate the ratio of male to female DNA present in a mixed source sample?
A: The Quantifiler® Duo kit quantitates the amount of total human and human male DNA in biological samples. The ratio of male to female DNA can be calculated using the following equation:
|Male DNA : Female DNA Ratio||=||
For example, assuming:
|Male DNA concentration||= 2 ng/µl|
|Human DNA concentration||= 8 ng/µl|
Male DNA : Female DNA Ratio = 2/2 : (8-2)/2 = 1:3
Q: How can knowledge of the ratio of male to female DNA present in a mixed sample be used to determine the best analysis route for the sample?
A: 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. As a general rule, for samples showing 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 Identifiler® kit; for samples showing 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™ 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 kit.