Forensic Science International: Genetics 13 (2014) 10–19
Contents lists available at ScienceDirect
Forensic Science International: Genetics journal homepage: www.elsevier.com/locate/fsig
Direct qPCR quantification using the Quantifiler1 Trio DNA quantification kit Jason Yingjie Liu * Human Identification, Thermo Fisher Scientific, 180 Oyster Point Boulevard, South San Francisco, CA 94080, USA
A R T I C L E I N F O
A B S T R A C T
Article history: Received 29 March 2014 Received in revised form 25 June 2014 Accepted 26 June 2014
The effectiveness of a direct quantification assay is essential to the adoption of the combined direct quantification/direct STR workflow. In this paper, the feasibility of using the Quantifiler1 Trio DNA quantification kit for the direct quantification of forensic casework samples was investigated. Both lowlevel touch DNA samples and blood samples were collected on PE swabs and quantified directly. The increased sensitivity of the Quantifiler1 Trio kit enabled the detection of less than 10 pg of DNA in unprocessed touch samples and also minimizes the stochastic effect experienced by different targets in the same sample. The DNA quantity information obtained from a direct quantification assay using the Quantifiler1 Trio kit can also be used to accurately estimate the optimal input DNA quantity for a direct STR amplification reaction. The correlation between the direct quantification results (Quantifiler1 Trio kit) and the direct STR results (GlobalFilerTM PCR amplification kit*) for low-level touch DNA samples indicates that direct quantification using the Quantifiler1 Trio DNA quantification kit is more reliable than the Quantifiler1 Duo DNA quantification kit for predicting the STR results of unprocessed touch DNA samples containing less than 10 pg of DNA. ß 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Forensic science Direct DNA quantification Direct PCR amplification Case work samples PE-Swab Quantifiler1 Trio
1. Introduction The current short tandem repeat (STR) typing workflow for forensic casework samples involves sample collection, sample screening, DNA extraction, DNA quantification, STR amplification, fragment separation and allele detection. Although very effective and powerful, this workflow is time consuming and still has room for improvements. First, the current screening assays [1–5] do not provide DNA-related information and do not work with touch DNA samples, which constitute the majority of property crime samples. The current lack of an effective screening tool and the increasing number of casework samples collected and submitted, particularly those obtained from property crimes, have made it difficult to process all of the samples in a timely manner [6]. As a result, a DNA backlog has been created. Some laboratories also limit the number and type of samples that can be submitted in each case. Law enforcement officers have to make difficult decisions on which DNA samples to submit, and this may result in more probative samples left un-submitted. Second, the current STR workflow
* Tel.: +1 650 872 7283; fax: +1 650 266 3063. E-mail addresses: Jason.Liu@thermofisher.com, [email protected] http://dx.doi.org/10.1016/j.fsigen.2014.06.016 1872-4973/ß 2014 Elsevier Ireland Ltd. All rights reserved.
requires DNA extraction and purification before STR typing. Although serving the purpose of inhibitor removal, such sample preparation operations could result in DNA loss and therefore contribute to the reduced STR success rate with low-level touch DNA samples [7]. Third, the DNA Quality Assurance Standards (QAS) require that all evidentiary samples must be quantified prior to amplification [8]. Until recently, only reference standards were eligible for direct amplification because of this requirement. The ability to use direct PCR for the analysis of an evidentiary sample following a direct quantification step would be a way to streamline the forensic DNA workflow without violating the QAS. To meet the forensic community’s demand for a simpler, faster, and more efficient STR typing workflow, technologies that enable direct qPCR DNA quantification [9] and direct STR amplification [10] of unprocessed forensic casework samples (no DNA extraction and purification) were recently developed. The feasibility of direct quantification technology was first demonstrated using the Quantifiler1 Duo DNA quantification kit [9]. After collecting the forensic casework sample on a PE-Swab, one can generate a paper punch containing the unprocessed DNA sample and place it directly in a Quantifiler1 Duo assay to assess the gender and DNA quantity of the sample. By optimizing the size of the paper punch and the baseline setting for Ct determination, the presence of a
J.Y. Liu / Forensic Science International: Genetics 13 (2014) 10–19
0.5-mm paper punch in the Quantifiler1 Duo assay has a minimal effect on DNA quantification. Because direct qPCR quantification is correlated to the post-extraction qPCR quantification and postextraction STR typing results, direct qPCR can be used to prioritize both touch DNA samples and blood stains such that crime laboratories have the choice to process those samples with a better chance of obtaining probative STR profiles first. However, because of its limited sensitivity, the Quantifiler1 Duo assay often produces false-negative quantification results, i.e., samples with no DNA detected in a direct quantification assay produce informative STR profiles [9]. Recently, Thermo Fisher Scientific introduced the Quantifiler1 Trio DNA quantification kit. Compared to the Quantifiler1 Duo assay, the Quantifiler1 Trio assay is faster (1 h) and more sensitive and offers better inhibitor tolerance [11]. These improvements are desirable attributes for a direct quantification assay that is intended to screen unprocessed forensic casework samples quickly and reliably. In this paper, the performance comparison between the Quantifiler1 Trio assay and Quantifiler1 Duo assay for the direct quantification of low-level touch DNA samples is reported. The effectiveness of using the DNA quantity obtained from the direct Quantifiler1 Trio assay for establishing the direct STR reaction was demonstrated with 24 low-level touch DNA samples. The correlation between the direct quantification results (Quantifiler1 Trio assay) and direct STR results was examined to evaluate the effectiveness of the direct quantification assay as a screening tool for prioritizing low-level touch DNA samples. An effective method for estimating the DNA quantity from heavily inhibited blood samples was reported such that an accurate input DNA quantity can be obtained for direct STR amplification of unprocessed blood stain samples.
11
Inc. Redding, CA, USA), punches of the desired size are generated from the active sampling area of the filter paper for direct qPCR assay or direct STR amplification. 2.2. Touch DNA sample collection and swabbing using a 5-mm PESwab Touch DNA samples were collected on transparency films. There were eight donors (4 males and 4 females), and each donor contributed three fingerprint samples. The touch samples were stored in paper envelopes to prevent contamination before testing. Ten microliters of ethanol was applied to each fingerprint sample before it was swabbed using a 5-mm PE-Swab. 2.3. Preparation of liquid blood samples and dry blood stain samples and procedures for the collection of blood samples onto PE-Swabs Liquid blood samples: Forty microliters of liquid blood from a male donor was diluted with water to create a two-fold dilution series down to 32-fold. Four-microliter aliquots pipetted from each blood dilution was spread onto a piece of transparency film, and the film was immediately swabbed with a 5-mm PE-Swab while the blood was still in liquid form. Mixed male/female dry blood stain samples: Mixed male/female blood samples were first prepared in 1.5-mL tubes according to the volume and mixture ratio information shown in Table 1. Fourmicroliter aliquots from each mixed sample were spread onto a piece of transparency film and left to dry overnight. Before swabbing, 10 mL of water was spread over the dry blood stain. The liquefied blood was then collected onto a 5-mm PE-Swab. 2.4. Direct quantification of touch DNA samples and blood samples collected on a PE-Swab
2. Materials and methods 2.1. PE-Swab assembly A PE-Swab consists of three components: a filter paper stripe, a holder and a clip. A PE-Swab was assembled by wrapping a filter paper stripe around the holder and then securing the paper stripe on the holder using the clip at the end of the PE-Swab handle. An example of a functional 5-mm PE-swab is shown in Fig. 1. The prefix before the PE-Swab indicates the height of the active sampling area. The width of the active sampling area is defined by the angled fold of the holder. Additional description regarding the PE-Swab can be found in previous publications [9,10]. PE-Swabs were used to swab objects of interest that contained touch DNA samples or blood samples. When a swabbing liquid is used, it is applied to the object of interest before swabbing. After swabbing, the filter paper stripe is detached from the swab holder and air dried before punching. Using a Harris Uni-CoreTM punch (Ted Pella,
To prepare a direct qPCR reaction plate, a punch generated from a PE-Swab was placed directly into a well of a MicroAmp1 Optical 96-Well Reaction Plate. The Quantifiler1 Duo DNA quantification kit (Thermo Fisher Scientific) was used for the direct qPCR assay. A 25-mL qPCR reaction mix (10.5 mL of Quantifiler1 Duo Primer Mix, 12.5 mL of Quantifiler1 Duo PCR reaction Mix and 2 mL of deionized water) was then added to the well containing a blank or a sample punch. The Quantifiler1 Trio DNA quantification kit (Thermo Fisher Scientific) was used for the direct qPCR assay. A 20-mL qPCR reaction mix (8 mL of Quantifiler1 Trio Primer Mix, 10 mL of Quantifiler1 Trio PCR reaction Mix and 2 mL of de-ionized water) was then added to the well containing a blank or a sample punch. The quantification reactions were performed on the Applied Biosystems 7500 Real-Time PCR System** (Thermo Fisher Scientific) using the manufacturer’s recommended protocol. The quantification results were analyzed using the SDS Software v2.0.6** (Thermo Fisher Scientific). 2.5. Direct STR amplification using the GlobalFilerTM PCR amplification kit and CE analysis Based on the direct qPCR results, the desirable input DNA quantity was obtained for each STR reaction by adjusting the punch size and the number of punches generated from a PE-Swab and released into a well of a MicroAmp1 Optical 96-Well Reaction Table 1 Volume (mL) and ratio used in blood mixture preparation.
Fig. 1. A 5-mm PE-Swab.
Male blood Female blood
M1F39
M1F19
M1F9
M1F3
M1F1
M3F1
M9F1
1.25 48.75
2.5 47.5
5 45
12.5 37.5
25 25
37.5 12.5
45 5
12
J.Y. Liu / Forensic Science International: Genetics 13 (2014) 10–19
Plate. To increase the sensitivity of the STR amplification of lowlevel touch DNA samples, a smaller 7-mL PCR reaction mix (2.1 mL of GlobalFilerTM Master Mix, 0.7 mL of GlobalFilerTM Primer Mix and 4.2 mL of water) was added to each well containing the punches. The standard 25-mL PCR reaction mix volume (7.5 mL of GlobalFilerTM Master Mix, 2.5 mL of GlobalFilerTM Primer Mix and 15 mL of water) was used for the amplification of blood samples. The thermal cycling conditions were 95 8C/1 m, 30 (touch samples) or 28 (blood samples) cycles of 94 8C/10 s and 59 8C/90 s, 60 8C/ 10 min and 4 8C/hold. After thermal cycling, 1 mL of the PCR product from each sample was mixed with 9 mL of GeneScanTM 600 LIZ1 size standard and deionized formamide. The CE was run on an ABI 3130xl capillary electrophoresis instrument under the following conditions: oven temperature, 60 8C; prerun, 15 kV for 180 s; injection, 3 kV for 10 s; run, 15 kV for 1500 s; capillary length, 36 cm; separation polymer, POP4TM polymer; and dye set, G6. The resulting STR electropherograms were analyzed using the GeneMapper1 ID-X software** (Thermo Fisher Scientific) with an analytical detection threshold of 50 RFU. 3. Results and discussion 3.1. Effect of the punch size and baseline setting for qPCR using the Quantifiler1 Trio DNA quantification kit As previously reported, the presence of a filter paper punch in the reaction well of a Quantifiler1 Duo assay affects the shape of the amplification curves in the FAMTM (male target), VIC1 (human target) and NEDTM (IPC) channels. The magnitude of this effect was
also dependent on the size of the filter paper punch [9]. In the Quantifiler1 Trio assay, the same TaqMan1 probe dyes were used for the detection of the male target (T.Y) and the small autosomal target (T.SA). However, the large autosomal target (T.LA) was detected with TaqMan1 probe dye ABY1, the internal PCR control (IPC) was detected with TaqMan1 probe dye JUN1, and the dye used for the passive reference was MPTM. The effect of a filter paper punch on the fluorescent background of the different dye channels in the Quantifiler1 Trio assay is shown in Fig. 2. Similar to what was observed using the Quantifiler1 Duo assay, the presence of a filter paper punch in the reaction well results in elevated background fluorescence signals in the FAMTM (T.Y) and VIC1 (T.SA) channels, and the magnitude of the background elevation is positively correlated with the size of the filter paper punch. In addition, the background fluorescence signal increases slowly after each thermal cycle, and the rate of the increase in the background fluorescence signal is also positively correlated to the size of the filter paper punch. The fluorescence background in the ABY1 (T.LA), JUN1 (IPC) and MPTM channels (passive reference) is not affected by the presence of a filter paper punch, regardless of its size. However, because of the effect of the filter paper punch on the signals in the FAMTM and VIC1 channels, a 0.5-mm punch is preferred and used in the direct Quantifiler1 Trio assay. The effect of a 0.5-mm filter paper punch on the Quantifiler1 Trio DNA quantification assay was further investigated by placing a 0.5-mm filter paper punch in the Quantifiler1 Trio reaction containing a known concentration of DNA from the DNA standard in a Quantifiler1 Trio kit. Eight reactions were performed at each DNA input concentration. Because a 0.5-mm filter paper punch had
Fig. 2. The fluorescence signals collected from different dye channels during a qPCR reaction (Quantifiler1 Trio) with or without a filter paper punch of indicated size in a NTC reaction well.
J.Y. Liu / Forensic Science International: Genetics 13 (2014) 10–19
a minimal effect on the fluorescent backgrounds of the T.LA target (ABY1) and the IPC target (JUN1), the automatic baseline settings determined by the SDS software were used for these two targets. Due to the sloped baseline, the current SDS software was not able to determine the correct baseline setting for the T.SA (VIC1) and T.Y (FAMTM) targets on a consistent basis. Thus, the baseline settings for these two targets were defined manually. In addition, because the slopes of the baseline for the T.SA (VIC1) and T.Y (FAMTM) targets tended to be higher at the early cycle numbers compared to the later cycle numbers, the baseline at early cycle numbers (
Contents lists available at ScienceDirect
Forensic Science International: Genetics journal homepage: www.elsevier.com/locate/fsig
Direct qPCR quantification using the Quantifiler1 Trio DNA quantification kit Jason Yingjie Liu * Human Identification, Thermo Fisher Scientific, 180 Oyster Point Boulevard, South San Francisco, CA 94080, USA
A R T I C L E I N F O
A B S T R A C T
Article history: Received 29 March 2014 Received in revised form 25 June 2014 Accepted 26 June 2014
The effectiveness of a direct quantification assay is essential to the adoption of the combined direct quantification/direct STR workflow. In this paper, the feasibility of using the Quantifiler1 Trio DNA quantification kit for the direct quantification of forensic casework samples was investigated. Both lowlevel touch DNA samples and blood samples were collected on PE swabs and quantified directly. The increased sensitivity of the Quantifiler1 Trio kit enabled the detection of less than 10 pg of DNA in unprocessed touch samples and also minimizes the stochastic effect experienced by different targets in the same sample. The DNA quantity information obtained from a direct quantification assay using the Quantifiler1 Trio kit can also be used to accurately estimate the optimal input DNA quantity for a direct STR amplification reaction. The correlation between the direct quantification results (Quantifiler1 Trio kit) and the direct STR results (GlobalFilerTM PCR amplification kit*) for low-level touch DNA samples indicates that direct quantification using the Quantifiler1 Trio DNA quantification kit is more reliable than the Quantifiler1 Duo DNA quantification kit for predicting the STR results of unprocessed touch DNA samples containing less than 10 pg of DNA. ß 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Forensic science Direct DNA quantification Direct PCR amplification Case work samples PE-Swab Quantifiler1 Trio
1. Introduction The current short tandem repeat (STR) typing workflow for forensic casework samples involves sample collection, sample screening, DNA extraction, DNA quantification, STR amplification, fragment separation and allele detection. Although very effective and powerful, this workflow is time consuming and still has room for improvements. First, the current screening assays [1–5] do not provide DNA-related information and do not work with touch DNA samples, which constitute the majority of property crime samples. The current lack of an effective screening tool and the increasing number of casework samples collected and submitted, particularly those obtained from property crimes, have made it difficult to process all of the samples in a timely manner [6]. As a result, a DNA backlog has been created. Some laboratories also limit the number and type of samples that can be submitted in each case. Law enforcement officers have to make difficult decisions on which DNA samples to submit, and this may result in more probative samples left un-submitted. Second, the current STR workflow
* Tel.: +1 650 872 7283; fax: +1 650 266 3063. E-mail addresses: Jason.Liu@thermofisher.com, [email protected] http://dx.doi.org/10.1016/j.fsigen.2014.06.016 1872-4973/ß 2014 Elsevier Ireland Ltd. All rights reserved.
requires DNA extraction and purification before STR typing. Although serving the purpose of inhibitor removal, such sample preparation operations could result in DNA loss and therefore contribute to the reduced STR success rate with low-level touch DNA samples [7]. Third, the DNA Quality Assurance Standards (QAS) require that all evidentiary samples must be quantified prior to amplification [8]. Until recently, only reference standards were eligible for direct amplification because of this requirement. The ability to use direct PCR for the analysis of an evidentiary sample following a direct quantification step would be a way to streamline the forensic DNA workflow without violating the QAS. To meet the forensic community’s demand for a simpler, faster, and more efficient STR typing workflow, technologies that enable direct qPCR DNA quantification [9] and direct STR amplification [10] of unprocessed forensic casework samples (no DNA extraction and purification) were recently developed. The feasibility of direct quantification technology was first demonstrated using the Quantifiler1 Duo DNA quantification kit [9]. After collecting the forensic casework sample on a PE-Swab, one can generate a paper punch containing the unprocessed DNA sample and place it directly in a Quantifiler1 Duo assay to assess the gender and DNA quantity of the sample. By optimizing the size of the paper punch and the baseline setting for Ct determination, the presence of a
J.Y. Liu / Forensic Science International: Genetics 13 (2014) 10–19
0.5-mm paper punch in the Quantifiler1 Duo assay has a minimal effect on DNA quantification. Because direct qPCR quantification is correlated to the post-extraction qPCR quantification and postextraction STR typing results, direct qPCR can be used to prioritize both touch DNA samples and blood stains such that crime laboratories have the choice to process those samples with a better chance of obtaining probative STR profiles first. However, because of its limited sensitivity, the Quantifiler1 Duo assay often produces false-negative quantification results, i.e., samples with no DNA detected in a direct quantification assay produce informative STR profiles [9]. Recently, Thermo Fisher Scientific introduced the Quantifiler1 Trio DNA quantification kit. Compared to the Quantifiler1 Duo assay, the Quantifiler1 Trio assay is faster (1 h) and more sensitive and offers better inhibitor tolerance [11]. These improvements are desirable attributes for a direct quantification assay that is intended to screen unprocessed forensic casework samples quickly and reliably. In this paper, the performance comparison between the Quantifiler1 Trio assay and Quantifiler1 Duo assay for the direct quantification of low-level touch DNA samples is reported. The effectiveness of using the DNA quantity obtained from the direct Quantifiler1 Trio assay for establishing the direct STR reaction was demonstrated with 24 low-level touch DNA samples. The correlation between the direct quantification results (Quantifiler1 Trio assay) and direct STR results was examined to evaluate the effectiveness of the direct quantification assay as a screening tool for prioritizing low-level touch DNA samples. An effective method for estimating the DNA quantity from heavily inhibited blood samples was reported such that an accurate input DNA quantity can be obtained for direct STR amplification of unprocessed blood stain samples.
11
Inc. Redding, CA, USA), punches of the desired size are generated from the active sampling area of the filter paper for direct qPCR assay or direct STR amplification. 2.2. Touch DNA sample collection and swabbing using a 5-mm PESwab Touch DNA samples were collected on transparency films. There were eight donors (4 males and 4 females), and each donor contributed three fingerprint samples. The touch samples were stored in paper envelopes to prevent contamination before testing. Ten microliters of ethanol was applied to each fingerprint sample before it was swabbed using a 5-mm PE-Swab. 2.3. Preparation of liquid blood samples and dry blood stain samples and procedures for the collection of blood samples onto PE-Swabs Liquid blood samples: Forty microliters of liquid blood from a male donor was diluted with water to create a two-fold dilution series down to 32-fold. Four-microliter aliquots pipetted from each blood dilution was spread onto a piece of transparency film, and the film was immediately swabbed with a 5-mm PE-Swab while the blood was still in liquid form. Mixed male/female dry blood stain samples: Mixed male/female blood samples were first prepared in 1.5-mL tubes according to the volume and mixture ratio information shown in Table 1. Fourmicroliter aliquots from each mixed sample were spread onto a piece of transparency film and left to dry overnight. Before swabbing, 10 mL of water was spread over the dry blood stain. The liquefied blood was then collected onto a 5-mm PE-Swab. 2.4. Direct quantification of touch DNA samples and blood samples collected on a PE-Swab
2. Materials and methods 2.1. PE-Swab assembly A PE-Swab consists of three components: a filter paper stripe, a holder and a clip. A PE-Swab was assembled by wrapping a filter paper stripe around the holder and then securing the paper stripe on the holder using the clip at the end of the PE-Swab handle. An example of a functional 5-mm PE-swab is shown in Fig. 1. The prefix before the PE-Swab indicates the height of the active sampling area. The width of the active sampling area is defined by the angled fold of the holder. Additional description regarding the PE-Swab can be found in previous publications [9,10]. PE-Swabs were used to swab objects of interest that contained touch DNA samples or blood samples. When a swabbing liquid is used, it is applied to the object of interest before swabbing. After swabbing, the filter paper stripe is detached from the swab holder and air dried before punching. Using a Harris Uni-CoreTM punch (Ted Pella,
To prepare a direct qPCR reaction plate, a punch generated from a PE-Swab was placed directly into a well of a MicroAmp1 Optical 96-Well Reaction Plate. The Quantifiler1 Duo DNA quantification kit (Thermo Fisher Scientific) was used for the direct qPCR assay. A 25-mL qPCR reaction mix (10.5 mL of Quantifiler1 Duo Primer Mix, 12.5 mL of Quantifiler1 Duo PCR reaction Mix and 2 mL of deionized water) was then added to the well containing a blank or a sample punch. The Quantifiler1 Trio DNA quantification kit (Thermo Fisher Scientific) was used for the direct qPCR assay. A 20-mL qPCR reaction mix (8 mL of Quantifiler1 Trio Primer Mix, 10 mL of Quantifiler1 Trio PCR reaction Mix and 2 mL of de-ionized water) was then added to the well containing a blank or a sample punch. The quantification reactions were performed on the Applied Biosystems 7500 Real-Time PCR System** (Thermo Fisher Scientific) using the manufacturer’s recommended protocol. The quantification results were analyzed using the SDS Software v2.0.6** (Thermo Fisher Scientific). 2.5. Direct STR amplification using the GlobalFilerTM PCR amplification kit and CE analysis Based on the direct qPCR results, the desirable input DNA quantity was obtained for each STR reaction by adjusting the punch size and the number of punches generated from a PE-Swab and released into a well of a MicroAmp1 Optical 96-Well Reaction Table 1 Volume (mL) and ratio used in blood mixture preparation.
Fig. 1. A 5-mm PE-Swab.
Male blood Female blood
M1F39
M1F19
M1F9
M1F3
M1F1
M3F1
M9F1
1.25 48.75
2.5 47.5
5 45
12.5 37.5
25 25
37.5 12.5
45 5
12
J.Y. Liu / Forensic Science International: Genetics 13 (2014) 10–19
Plate. To increase the sensitivity of the STR amplification of lowlevel touch DNA samples, a smaller 7-mL PCR reaction mix (2.1 mL of GlobalFilerTM Master Mix, 0.7 mL of GlobalFilerTM Primer Mix and 4.2 mL of water) was added to each well containing the punches. The standard 25-mL PCR reaction mix volume (7.5 mL of GlobalFilerTM Master Mix, 2.5 mL of GlobalFilerTM Primer Mix and 15 mL of water) was used for the amplification of blood samples. The thermal cycling conditions were 95 8C/1 m, 30 (touch samples) or 28 (blood samples) cycles of 94 8C/10 s and 59 8C/90 s, 60 8C/ 10 min and 4 8C/hold. After thermal cycling, 1 mL of the PCR product from each sample was mixed with 9 mL of GeneScanTM 600 LIZ1 size standard and deionized formamide. The CE was run on an ABI 3130xl capillary electrophoresis instrument under the following conditions: oven temperature, 60 8C; prerun, 15 kV for 180 s; injection, 3 kV for 10 s; run, 15 kV for 1500 s; capillary length, 36 cm; separation polymer, POP4TM polymer; and dye set, G6. The resulting STR electropherograms were analyzed using the GeneMapper1 ID-X software** (Thermo Fisher Scientific) with an analytical detection threshold of 50 RFU. 3. Results and discussion 3.1. Effect of the punch size and baseline setting for qPCR using the Quantifiler1 Trio DNA quantification kit As previously reported, the presence of a filter paper punch in the reaction well of a Quantifiler1 Duo assay affects the shape of the amplification curves in the FAMTM (male target), VIC1 (human target) and NEDTM (IPC) channels. The magnitude of this effect was
also dependent on the size of the filter paper punch [9]. In the Quantifiler1 Trio assay, the same TaqMan1 probe dyes were used for the detection of the male target (T.Y) and the small autosomal target (T.SA). However, the large autosomal target (T.LA) was detected with TaqMan1 probe dye ABY1, the internal PCR control (IPC) was detected with TaqMan1 probe dye JUN1, and the dye used for the passive reference was MPTM. The effect of a filter paper punch on the fluorescent background of the different dye channels in the Quantifiler1 Trio assay is shown in Fig. 2. Similar to what was observed using the Quantifiler1 Duo assay, the presence of a filter paper punch in the reaction well results in elevated background fluorescence signals in the FAMTM (T.Y) and VIC1 (T.SA) channels, and the magnitude of the background elevation is positively correlated with the size of the filter paper punch. In addition, the background fluorescence signal increases slowly after each thermal cycle, and the rate of the increase in the background fluorescence signal is also positively correlated to the size of the filter paper punch. The fluorescence background in the ABY1 (T.LA), JUN1 (IPC) and MPTM channels (passive reference) is not affected by the presence of a filter paper punch, regardless of its size. However, because of the effect of the filter paper punch on the signals in the FAMTM and VIC1 channels, a 0.5-mm punch is preferred and used in the direct Quantifiler1 Trio assay. The effect of a 0.5-mm filter paper punch on the Quantifiler1 Trio DNA quantification assay was further investigated by placing a 0.5-mm filter paper punch in the Quantifiler1 Trio reaction containing a known concentration of DNA from the DNA standard in a Quantifiler1 Trio kit. Eight reactions were performed at each DNA input concentration. Because a 0.5-mm filter paper punch had
Fig. 2. The fluorescence signals collected from different dye channels during a qPCR reaction (Quantifiler1 Trio) with or without a filter paper punch of indicated size in a NTC reaction well.
J.Y. Liu / Forensic Science International: Genetics 13 (2014) 10–19
a minimal effect on the fluorescent backgrounds of the T.LA target (ABY1) and the IPC target (JUN1), the automatic baseline settings determined by the SDS software were used for these two targets. Due to the sloped baseline, the current SDS software was not able to determine the correct baseline setting for the T.SA (VIC1) and T.Y (FAMTM) targets on a consistent basis. Thus, the baseline settings for these two targets were defined manually. In addition, because the slopes of the baseline for the T.SA (VIC1) and T.Y (FAMTM) targets tended to be higher at the early cycle numbers compared to the later cycle numbers, the baseline at early cycle numbers (
