Fluorescent Short Tandem Repeat Research Articles

Developmental validation of the EX16+22Y system

The EX16+22Y system is a polymerase chain reaction (PCR)-based amplification kit that enables typing of 15 autosomal short tandem repeat (STR) loci (i.e., D3S1358, D13S317, D7S820, D16S539, TPOX, TH01, D2S1338, CSF1PO, D19S433, vWA, D18S51, D21S11, D8S1179, D5S818, and FGA) and 22 widely used Y chromosome STR (Y-STR) loci (DYS391, DYS527a/b, DYS635, DYS458, DYS456, DYS385a/b, DYS438, DYS448, DYS437, DYS19, DYS576, DYS533, DYS393, DYS389I/II, DYS439, DYS392, Y_GATA_H4, DYS390, and DYS481) which contains 20 core Y-STR recommended by the Ministry of Public Security and amelogenin. This multiplex system was designed for the simultaneous analysis of amelogenin-Y allele mutation, single-source searches, kinship (including familial searching), mixture profiles, international data sharing, and other forensic applications. In this study, the multiplex system was validated for sensitivity of detection, species specificity, DNA mixtures, stability, sizing precision, stutter, reproducibility, and PCR-based conditions according to the Scientific Working Group on DNA Analysis Methods developmental validation guidelines and Chinese criteria for the human fluorescent STR multiplex PCR reagent. The results show that the EX16+22Y system is a robust and reliable amplification kit which can be used for human identification testing.

MG-143 Age is significantly associated with the bone marrow engraftment in patients with allogeneic stem cell transplantation

Objectives Allogeneic stem cell transplantation is a curative therapy for patients with haematological malignancies and nonmalignant haematological disorders. Measuring the bone marrow engraftment via donor chimerism following transplantation is often used as a predictor for the overall survival. However, prognostic factors associated with bone marrow engraftment are poorly understood. The objective of this study is to evaluate the effect of age, gender and the recipient-donor relationship on the extent of bone marrow engraftment following transplantation and thereby provide more precise counselling to patients. Methods The bone marrow engraftment was routinely monitored using the multiplex fluorescent short tandem repeat analysis. Complete engraftment is defined as ≥95% of both donor-derived white blood cells and T cells. Data from a cohort of 194 patients with various conditions and up to 10 years of follow up was retrospectively analysed. Logistic regression and survival analyses were performed using the R statistic software. Results 64.9% of patients reached complete engraftment, ˜70% out of which achieved this milestone within 2 years post-transplantation; but 6.3% of these patients relapsed. Gender and recipient-donor relationship (related or not, same sex or not) did not have significant effect on the probability and the time for reaching complete engraftment. Age was inversely related to the probability of complete engraftment. The older the patient was, the less likely he/she achieved complete engraftment (p value 0.0015), and the longer time took for reaching complete engraftment (p value 0.0009). Conclusion Age is significantly associated with the likelihood and time for reaching complete engraftment.

Developmental validation of the EX20+4 system.

The EX20+4Y System is a polymerase chain reaction (PCR)-based amplification kit that enables typing of 19 autosomal short tandem repeat (STR) loci (i.e., CSF1PO, D13S317, D16S539, D18S51, D21S11, D3S1358, D5S818, D7S820, D8S1179, FGA, TH01, TPOX, vWA, Penta D, Penta E, D2S1338, D19S433, D12S391, D6S1043), four widely used Y chromosome-specific STR (Y-STR) loci (DYS458, DYS456, DYS391, DYS635), and amelogenin. In this study, this multiplex system was validated for sensitivity of detection, DNA mixtures, inhibitor tolerance, species specificity based on the Scientific Working Group on DNA Analysis methods (SWGDAM) developmental validation guidelines, and the Chinese criteria for the human fluorescent STR multiplex PCR reagent. The results show that the EX20+4 System is a robust and reliable amplification kit which can be used for human identification testing.

Donor and Recipient STR Analysis Before Allogeneic Haematopoietic Stem Cell Transplantation: Possible Correlation With Post-Transplant Outcome

Background The chimerism analysis after allogeneic haematopoietic stem cell transplantation (HSCT) is a useful tool to monitor the engraftment of donor cells and to early predict graft failure or disease relapse. Among several available methods to perform chimerism study, multiplex fluorescent short tandem repeat (STR) analysis represent a sensitive, accurate and reproducible technique. It provide a high rate of discrimination between donor and recipient cells since that fluorescent signal is proportional to the cells number, obtaining a quantitative assessment of chimerism. Aims Evaluation of a possible correlation between basal donor/recipient allelic discrepancies and post-transplant outcome in terms of graft versus host disease (GvHD) development, neutrophil engraftment, relapse, DFS and OS. Methods We enrolled 111 patients (pts) affected by onco-haematological diseases, consecutively submitted to HSCT at our division between February 2005 and December 2012. Pts were 67 males and 44 females with a median age of 51 years (range 14-70). Underlying diseases were: 58 AML, 10 MDS, 16 ALL, 8 CLL, 6 MM, 2 HL, 9 NHL, 1 IMF, 1 MPD. Myeloablative conditioning regimen was used in 26 pts while a reduced conditioning regimen was used for the other 85 pts. Graft was obtained from sibling donor and unrelated donor in 65 and 46 pts, respectively. GvHD prophylaxis was performed with cyclosporine (CSA) and methotrexate in 61 pts, CSA and mycophenolate mofetil in 43 pts and CSA in 7 pts. Donor and recipient allelic status was performed using a multiplex PCR amplification of ten STR loci: Amelogenin alleles X/Y, D3S1358, FGA, D8S1179, D18S51, D13S317, vWA, D21S11, D5S818, D7S820. We evaluated the donor/recipient (D/R) match or mismatch for each locus and the following variables: GvHD development and onset time, time to neutrophil engraftment, relapse, DFS and OS. Statistical analysis was performed by Kaplan Meier analysis using IBM SPSS Statistics 20 Core System. Results Pts with D/R mismatch for D8S1179 locus achieved neutrophil engraftment (ANC>1* 109/L) at a median time of 18 days (CI 95% 17.202-18.798) compared with 21 days (CI 95% 17.328-24.672) of pts with D/R match for the same locus (p=0.007) (Figure 1A). Pts with D/R mismatch for D3S1358 locus developed acute GvHD at a median time of 20 days (CI 95% 15.781-24.219) compared with 29 days (CI 95% 15.421-42.579) of pts with D/R match for the same locus (p=0.031) (Figure 1B). Pts with D/R mismatch for D3S1358 locus showed an OS of 16 months (CI 95% 11.016-20.984) compared with 41 months (CI 95% 25.420-56.580) of pts with D/R match for the same locus (p=0.025) (Figure 1C). Pts with D/R mismatch for D8S1179 locus showed an OS of 16 months (CI 95% 8.528-23.472) compared with 56 months (CI 95% 11.254-78.057) of pts with D/R match for the same locus (p=0.012) (Figure 1D). No relationships were found with relapse, DFS neither for the other loci. Summary Our results highlight that pts who presented a D/R mismatch for D8S1179 locus showed an earlier neutrophil engraftment but a worse OS compared with the others. Moreover, for D3S1358 locus, D/R mismatch was associated with an earlier onset of acute GvHD after HSCT and with a shorter OS than the D/R match ones. Conclusion STRs are highly polymorphic di-, tri- and tetra-nucleotide repeat non-coding sequences, which are interspersed throughout the genome. Whether or not discrepancies between donor and recipient basal allelic status could be considered useful in predicting post-transplant outcome will require validation in a large sample of pts. Disclosures: No relevant conflicts of interest to declare.

Molecular sexing of tigers, Panthera tigris

We report the development of a fast and reliable PCR-based method for sex identification of tiger DNA designed to be incorporated into fluorescent short tandem repeat (STR) profiling. A single primer pair, consisting of a fluorescently-labelled forward primer and an unlabelled reverse primer, is used to co-amplify homologous fragments of a zinc finger (ZF) protein intron which exhibits size polymorphism between the X and Y chromosomes. The ZFX and ZFY amplicons differ in size by 12 bp and can thus be differentiated by capillary electrophoresis.

Molecular sexing of African rhinoceros

We report the development of a fast and reliable PCR-based method for sex identification of African Rhinoceros (Ceratotherium simum and Diceros bicornis) that could easily be incorporated into fluorescent short tandem repeat (STR) profiling. A single primer pair, consisting of a fluorescently labelled forward primer and an unlabelled reverse primer, is used to co-amplify homologous fragments of a zinc finger (ZF) protein intron which exhibits size polymorphism between the X and Y chromosomes. In both species, the amplified ZFX and ZFY amplicons differ in size by 7 bp and can thus be differentiated by capillary electrophoresis. Blood, tissue, horn, and faecal samples were correctly sexed using this method. Cross species testing also demonstrated that this method could be used to sex Indian rhinoceros (Rhinoceros unicornis) samples.

Large Pathogenic Expansions in the SCA2 and SCA7 Genes Can Be Detected by Fluorescent Repeat-Primed Polymerase Chain Reaction Assay

Large expansions in the SCA2 and SCA7 genes (>100 CAG repeats) have been associated with juvenile and infantile forms of cerebellar ataxias that cannot be detected using standard polymerase chain reaction (PCR). Here, we describe a successful application of the fluorescent short tandem repeat-primed PCR method for accurate identification of these expanded repeats. The test is robust, reliable, and inexpensive and can be used to screen large series of patients, although it cannot give a precise evaluation of the size of the expansion. This test may be of practical value in prenatal diagnoses offered to affected or pre-symptomatic at-risk parents, in which a very large expansion inherited from one of the parents can be missed in the fetus by standard PCR.

Publishers note to: Validation of a male-specific, 12-locus fluorescent short tandem repeat (STR) multiplex

Publishers note to: Validation of a male-specific, 12-locus fluorescent short tandem repeat (STR) multiplex

Precision and accuracy in fluorescent short tandem repeat DNA typing: Assessment of benefits imparted by the use of allelic ladders with the AmpFlSTR®Profiler Plus™ kit

Base-calling precision of short tandem repeat (STR) allelic bands on dynamic slab-gel electrophoresis systems was evaluated. Data was collected from over 6000 population database allele peaks generated from 468 population database samples amplified with the AmpF/STR Profiler Plus (PP) kit and electrophoresed on ABD 377 DNA sequencers. Precision was measured by way of standard deviations and was shown to be essentially the same, whether using fixed or floating bin genotyping. However, the allelic ladders have proven more sensitive to electrophoretic variations than database samples, which have caused some floating bins of D18S51 to shift on occasion. This observation prompted the investigation of polyacrylamide gel formulations in order to stabilize allelic ladder migration. The results demonstrate that, although alleles comprised in allelic ladders and questioned samples run on the same gel should migrate in an identical manner, this premise needs to be verified for any given electrophoresis platform and gel formulation. We show that the compilation of base-calling data is a very informative and useful tool for assessing the performance stability of dynamic gel electrophoresis systems, stability on which depends genotyping result quality.

Identification of HepG2 variant cell lines by short tandem repeat (STR) analysis.

In the past years, in our laboratory, several cell lines have been generated starting from a human liver (H7). Some of them have been used successfully in studies of the infection with and propagation of Hepatitis B and Hepatitis C viruses. Recently, several lines of evidence indicated that the origin of these cell lines was uncertain. Therefore, we now have determined the genetic characteristics of these cell lines in comparison to HepG2 cells received from ATCC and to HepG2 isolates grown at other laboratories. Quadruplex fluorescent short tandem repeat (STR) typing and karyotyping were performed. In addition, some biochemical characteristics of selected clones were studied. Genetically, all H7-derived cell lines were identical to HepG2 cells. However, some liver-specific functions varied between the different sub-cloned lines. The H7-derived cell lines that were generated proved to be sub-cloned lines of HepG2. The problem of cross-contamination during cloning of cell lines appears to be not uncommon. We found that two out of six HepG2 isolates obtained from other laboratories were not derived from the same individual as the original HepG2 cells. Therefore, STR typing should be applied as a rapid and sensitive technique to determine and monitor the origin of cell lines and to safeguard against contamination.

Allele frequencies of eight STR loci in a Japanese population detected by the fluorescent image analyzer

Population studies on eight short tandem repeat (STR) loci D16S539, D7S820, D13S317, D5S818, CSF1PO, TPOX, TH01, and vWA were carried out in a sample of 460 unrelated Japanese individuals living in the Gifu Prefecture (central region of Japan). The eight loci were co-amplified using the GenePrintk PowerPlexk Fluorescent STR system (Promega). The amplified products were electrophoresed using 4% denaturing polyacrylamide. After the electrophoresis, the gels were scanned on a FMBIO II MultiView fluorescent image analyzer (Hitachi Software Engineering) and the fluorescent DNA bands of the eight loci were detected simultaneously. The fragment sizes of the bands were determined using FMBIO Analysis Software (Hitachi Software Engineering) and typed using Excel STaRCall Genotyping Software (Hitachi Software Engineering). The following allele frequency ranges were found: D16S539: seven alleles with frequencies ranging from 0.002 (allele 8) to 0.329 (allele 9); D7S820: eight alleles with frequencies ranging from 0.003 (allele 14) to 0.316 (allele 11); D13S317: eight alleles with frequencies ranging from 0.001 (allele 7) to 0.265 (allele 8); D5S818: 10 alleles with frequencies ranging from 0.003 (alleles 6, 7, and 15) to 0.263 (allele 11); CSF1PO: eight alleles with frequencies ranging from 0.003 (allele 15) to 0.396 (allele 12); TPOX: six alleles with frequencies ranging from 0.003 (allele 14) to 0.463 (allele 8); TH01: six alleles with frequencies ranging from 0.005 (allele 10) to 0.399 (allele 9); vWA: nine alleles with frequencies ranging from 0.002 (allele 21) to 0.279 (allele 17). The combined power of discrimination (PD) for the eight loci was 0.999999992 and the mean exclusion chance (MEC) 0.998. For all loci, no deviations from Hardy–Weinberg expectations were detected using the homozygosity, the likelihood ratio, and the exact tests. The results indicate that these eight loci are useful genetic markers for forensic personal identification and paternity testing in the Japanese population.

Standardisation of multiplex fluorescent short tandem repeat analysis for chimerism testing.

To evaluate the origin of cells after allogeneic haematopoietic stem cell transplantation we optimised and evaluated two commercially available systems (AmpFlSTR Profiler Plus and GenePrint Powerplex-16) which are based on multiplex fluorescent short tandem repeat (STR) analysis. A standard procedure for interpretation of electropherographs was found essential to obtain reproducible results. On the basis of the relative length of donor and recipient alleles, TYPE-I (no shared alleles are used to calculate chimerism), TYPE-II (one shared and one unshared allele is used to calculate chimerism) or TYPE-III (not informative) allelic distribution types were distinguished. Also, stutter peaks were recognised as an important criterion to exclude a marker for analysis. Intralaboratory and multicentre evaluation of the AmpFlSTR Profiler Plus system showed that mixed blood samples could be determined with an absolute deviation of less than 2%. A sensitivity threshold was set at 5% for TYPE-I and 10% for TYPE-II markers since relative imprecision increases at low chimerism values. No significant difference of calculated chimerism values was observed between STR markers shared between both systems. By monitoring 26 allogeneic peripheral blood stem cell transplants, the applicability of the proposed method was demonstrated.

Sequence variation in humans and other primates at six short tandem repeat loci used in forensic identity testing

A large number of alleles from the six different short tandem repeat (STR) loci FGA, D3S1358, vWA, CSF1PO, TPOX and TH01, used in human identity testing were sequenced to provide support for the robustness of fluorescent STR DNA typing by allele size. Sequence information for some of these loci (FGA, vWA, TH01) is an extension of published work, whereas no extensive sequence information is available with respect to the D3S1358, CSF1PO, and TPOX loci. Sequencing of alleles at each locus has provided quantitative data with respect to the true nucleotide length of common alleles, and of alleles that vary in length from the common alleles. All alleles that were identified as “off-ladder” alleles through fluorescent typing at these STR loci have proven to be true length variant alleles. Sequencing at the D3S1358 and CSF1PO loci allowed for the establishment of a common nomenclature for these loci. A correlation between percent stutter and the length of the core tandem repeat is demonstrated at the FGA locus. Alleles in which the core tandem repeat is interrupted by a repeat unit of different sequence have a reduced percent stutter. DNA samples from three non-human primates (chimpanzee, orangutan, and gorilla) were compared to the human sequences, and shown to differ markedly across loci with respect to their homology. The effects of primer binding site mutations on the amplification efficiency at a particular locus, and methods used to interpret amplification imbalance of heterozygous alleles at a locus is also addressed.

Fingerprint Enhancement Revisited and the Effects of Blood Enhancement Chemicals on Subsequent Profiler Plus™ Fluorescent Short Tandem Repeat DNA Analysis of Fresh and Aged Bloody Fingerprints

This study was aimed at determining the effect of seven blood enhancement reagents on the subsequent Profiler Plus fluorescent STR DNA analysis of fresh or aged bloody fingerprints deposited on various porous and nonporous surfaces. Amido Black, Crowle's Double Stain. 1,8-diazafluoren-9-one (DFO), Hungarian Red, leucomalachite green, luminol and ninhydrin were tested on linoleum, glass, metal, wood (pine, painted white), clothing (85% polyester/15% cotton, 65% polyester/35% cotton, and blue denim) and paper (Scott 2-ply and Xerox-grade). Preliminary experiments were designed to determine the optimal blood dilutions to use to ensure a DNA typing result following chemical enhancement. A 1:200 blood dilution deposited on linoleum and enhanced with Crowle's Double Stain generated enough DNA for one to two rounds of Profiler Plus PCR amplification. A comparative study of the DNA yields before and after treatment indicated that the quantity of DNA recovered from bloody fingerprints following enhancement was reduced by a factor of 2 to 12. Such a reduction in the DNA yields could potentially compromise DNA typing analysis in the case of small stains. The blood enhancement chemicals selected were also evaluated for their capability to reveal bloodmarks on the various porous and nonporous surfaces chosen in this study. Luminol. Amido Black and Crowle's Double Stain showed the highest sensitivity of all seven chemicals tested and revealed highly diluted (1:200) bloody fingerprints. Both luminol and Amido Black produced excellent results on both porous and nonporous surfaces, but Crowle's Double Stain failed to produce any results on porous substrates. Hungarian Red, DFO, leucomalachite green and ninhydrin showed lower sensitivities. Enhancement of bloodmarks using any of the chemicals selected, and short-term exposure to these same chemicals (i.e., less than 54 days), had no adverse effects on the PCR amplification of the nine STR systems surveyed (D3S 1358, HumvWA, HumFGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820) or of the gender determination marker Amelogenin. The intensity of the fluorescent signals was very similar and the allele size measurements remained constant and identical to those of untreated bloody fingerprints. No additional background fluorescence was noted. Continuous exposure (for 54 days) to two of the seven enhancement chemicals selected (i.e., Crowle's Double Stain and Hungarian Red) slightly reduced the amplification efficiency of the longer STR loci in profiles of fresh and 7 to 14-day-old bloodprints. This suggests that long-term exposure to these chemicals possibly affects the integrity of the DNA molecules. This study indicates that significant evidence can be obtained from fresh or aged bloody fingerprints applied to a variety of absorbent and nonabsorbent surfaces which are exposed to different enhancement chemicals for short or long periods of time. It also reaffirms that PCR STR DNA typing procedures are robust and provide excellent results when used in concert with fluorescence-based detection assays after fingerprint identification has taken place.