Development of two new Mini-STR multiplex assay for typing archival Bouin's fluid-fixed paraffin-embedded tissues

1. Introduction 

Paternity testing is generally ascertained using buccal swab or blood samples, but other biological materials such as biopsy paraffin-embedded tissues are potential samples for DNA extraction and genetic testing. Degradation and low quantity of high molecular weight DNA, produced by intrinsic and extrinsic characteristics of samples, fail often to yield reproducible results using commercial multiplex STR systems. The loss of signal is specially observed with larger-sized STR products. The need to obtain a genetic profile from degraded DNA has led to redesigned STR primers that generate shorter amplicons to increase the number of template molecules available for the PCR [1].

In this study the primer pairs of eight conventional STR markers, were redesigned in order to obtain amplicons less than 120bp in size and were assembled in two new Mini-STR PCR-multiplexes that, after validation, were employed in a paternity testing case to get nuclear DNA profile from archival postmortem Bouin's fluid-fixed paraffin-embedded tissue.

2. Materials and methods 

Eight conventional STR markers (D8S1179, D3S1358, TPOX, TH01, D5S818, CSF1P0, D13S317, D16S539) included in multiplex PCR kits commercially available, were redesigned and converted into Mini-STR, in order to reduce or eliminate the polymorphism's flanking regions. The eight STRs were amplified in two quadruplex: D8S1179, D3S1358, TPOX, D16S539 and CSF1P0, TH01, D13S317, D5S818 (see Table 1).

Table 1. Primer sequences and PCR product sizes of the two Mini-STR quadruplex systems.

	Locus	Mini-STR primers (5′–3′)	Allele range	Mini-STR size (bp)
	D8S1179	F [6-FAM] GTATTTCATGTGTACATTCG R GATTATTTTCACTGTGGGG	8–19	68–112
	D3S1358	F [VIC] CAGAGCAAGACCCTGTCT R GAAATCAACAGAGGCTTGC	8–19	73–117
	TPOX	F [NED] AGGCACTTAGGGAACCCT R GTCAGCGTTTATTTGCCC	6–13	64–92
	D16S539	F [PET] CAGACAGACAGACAGGTG R GTATCTATCATCCATCTCTG	8–15	86–114
	CSF1P0	F [6-FAM] CAGTAACTGCCTTCATAGATAG R GACCCTGTTCTAAGTACTTCC	6–15	82–118
	TH01	F [VIC] ATTCCCATTGGCCTGTTC R GTCACAGGGAACACAGACTC	4–13.3	69–115
	D13S317	F [NED] GCCTATCTGTATTTACAAATAC R CCCAAAAAGACAGAAAGAA	8–15	92–120
	D5S818	F [PET] CCTCTTTGGTATCCTTATGT R CTTTATCTGTATCCTTATTTATACC	7–16	84–120

DNA amplification of the two quadruplex was performed in a reaction volume of 12.5μl using Qiagen® Multiplex PCR kit (Qiagen, Hilden, Germany) following the manufacturer's recommendations in terms of primers and DNA concentrations. Thermal cycling was performed with a GeneAmp 9700 (Applied Biosystem) using following conditions: 95°C for 15min, 28 cycles at 94°C for 30s, 57°C for 1.30min, 72°C for 1min, 60°C for 30min and 4°C forever.

Allelic ladders for Mini-STRs were created using 1:1000 dilution of allelic ladder from Identifiler kit®. 2μl of these diluted ladders were amplified in the two quadruplex sets using the thermal cycling parameters outlined for the PCR above, except amplified for 15 cycles instead of the standard 28 cycles [1]. The cell line samples K562 and 9947A (Promega, Milan, Italy) were used as control DNA for calibrating allelic ladders.

DNA extracted from 100 buccal swab provided by healthy donors and previously typed with Identifiler® and Minifiler™, were analyzed to validate the new PCR-multiplexes. Furthermore, DNA extraction from postmortem Bouin's fluid-fixed paraffin-embedded tissue samples was performed by QIAamp DNA Investigator kit (Qiagen), amplified by Amp-FlSTR Identifiler® PCR Amplification Kit (Applied Biosystems) and subsequently by Amp-FlSTR®MiniFiler™ PCR Amplification Kit (Applied Biosystems) according to manifacturer's recommendations.

All PCR amplifications, together with positive and negative control samples, were performed on Gene Amp PCR System 9700 (Applied Biosystem) and amplification products were separated on the ABI Prism 3100 Avant Genetic Analyzer (Applied Biosystems, Foster City, CA) and ABI Prism 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA). Electrophoresis data was automatically analyzed using Genescan v.3.7 with Genotyper software and GeneMapper ID-X v. 1.1 (Applied Biosystems).

3. Results and discussion 

The forensic usefulness of Mini-STRs and the significant improvements in terms of number of loci amplified with MiniFiler™ kit on degraded DNA compared to Identifiler® kit, is widely demonstrated [2]. Thus, new Mini-STR PCR-multiplexes were designed in order to obtain PCR products with length less than 120bp in size. Not all conventional STRs included in multiplex amplification kits can be reduced less than 120bp, but only those that are characterized by a limited number of repetitive units. Three of eight Mini-STRs (CSF1P0, D8S1179 and D13S317) described in this work, were previously validated and tested in singleplex on archival Bouin's fluid-fixed paraffin-embedded tissues by Turrina et al. [3]. Moreover these three markers are already included in the MiniFiler™ kit, but in this work the primer sizes were further reduced. The two new PCR-multiplexes were successfully employed in our degraded samples.

The comparison of typing results between the two new Mini-STRs and conventional STRs (AmpFlSTR Identifiler®, ABI) on a sample of 100 healthy donors showed no genotype differences with good balance between alleles, no double peaks due to +A/−A and stutter products higher less than 15% of the main peak; confirming that changes in the dimensions of the STRs do not influence the profile.

DNA extracted from archival Bouin's fluid-fixed paraffin-embedded tissue specimens showed a higher degree of degradation so that PCR amplification with Identifiler® kit produced only 3 of 15 STRs with the lowest molecular size (D8S1179, D19S433, D3S1358), plus amelogenin marker. Typing with MiniFiler™ kit and the two new Mini-STR quadruplex systems was successful for all STR loci yielded a complete profile.

The two new multiplexes could provide additional information on Bouin's fluid-fixed paraffin-embedded tissue samples. Considering all STR markers employed in this work a genetic profile of 14 STR markers was obtained (D8S1179, D3S1358 and D19S433 from Identifiler® kit, CSF1P0, FGA, D2S1338, D7S820, D13S317, D16S539, D18S51, and D21S11 from MiniFiler and TPOX, TH01 and D5S818 from new Mini-STR multiplexes performed in this work).

In conclusion, analysis using the present two Mini-STR PCR-multiplexes combined with commercial kits is clearly more effective for forensic applications and confirmed their usefulness in analyzing degraded DNA samples.

Conflict of interest 

None.