Rapid individual identification by minisatellite variant repeat (MVR)-PCR at D1S8 locus using "exponential law".

We describe an efficient and simple minisatellite variant repeat mapping by PCR (MVR-PCR) method based on the assignment of the tandem array of 29 bp repeating units into a-type, t-type and 0-type (a rarely appearing unamplified unit), from the first repeat unit position (code position 0) of 293 bp in D1S8 locus. After microchip electrophoresis of PCR product amplified from the target DNA of a human hair root, each rung of the ladder at the position of 293 + 29 n bp (n: code position) was detected and the type of repeating unit was determined, i.e., aa, a0, tt, t0, at and 00. The peak area of the rungs from PCR product decreased with increase in code position. We found for the first time that the logarithmic plots of the peak area against the code position showed a linear relationship, which implied that peak areas decrease exponentially. The present method was successfully applied to identify 37 individuals using only a hair root as a biological specimen. This "exponential law" is expected to be an effective tool in forensic science.     

Analysis of five rare alleles at the STR loci D1S1656, D12S391, D13S317, Penta D,and D2S441

Short tandem repeat (STR) automatic typing technology is extensively used in forensic laboratories with commercial kits, in rare cases genotyping misinterpretations or mislabeling may occur due to unexpected rare alleles. This study refers to the investigation of several rare alleles observed from routine cases. Besides cross-kit verification with Goldeneye 25A (Beijing PeopleSpot Inc, China) and Huaxia platinum (Thermo Fisher Scientific, USA) kits, the next-generation sequencing technology by MiSeq FGx System (Illumina, USA) was applied to further validation. To solve the inconsistent outcomes reached by the above mentioned approaches at D2S441 locus, single gene amplification, gene cloning, and genetic sequencing was also performed. As a result, five rare alleles were detected. Two novel alleles of allele 3 at the D13S317 locus and allele 5 at the D2S441 locus were found; three previously reported alleles of allele 9 at D1S1656 locus, allele 19 at Penta D locus, and allele 28 at D12S391 locus in STRBase were initially supplemented with sequence information. We, therefore, propose that such uncommon observations with rare events should be carefully investigated and interpreted.     

Mutation at minisatellite locus DYF155S1: allele length mutation rate is affected by age of progenitor

A father/son material consisting of 1071 pairs was screened for de novo allele length mutation in locus DYF155S1. Six hundred of these pairs were also analyzed in locus DYF155S1 to detect de novo mutations in the minisatellite variant repeat (MVR)-code not resulting in a length change ("boundary switch" mutations). A modified MVR-polymerase chain reaction (PCR) method was used for this purpose. Twenty-seven de novo allele length mutations and eight "boundary switch" mutations were detected indicating mutation frequencies of approximately 2.5% and 1.3%, respectively. The combined mutation rate for MVR-code mutation is approximately 3.8%. There is a significant increase in mutation rate with paternal age (p = 0.049) in allele length mutations. In the present material, the mutation rate in the oldest age group is three times that of the youngest age group. A similar age relationship is not observed in "boundary switch" mutations. A comparison between progenitors and the other fathers in the material revealed no obvious association between mutation rate and allele length or modular structure (variation in repeat sequence). More than 75% of the length mutations involved the gain or loss of one repeat only. This finding as well as the observed paternal age influence on mutation rate, suggests replication slippage to be the major mutation mechanism in length mutations. However, in one particular case, an allele length mutant revealed rearrangements with direct duplication of repeats at distant sites within the repeat array, and with both loss and gain of repeats. Such complex structural changes could indicate that some of the mutants might arise from sister chromatide exchange. The mutation rate of "boundary switch" mutations is by far higher than would be expected if these mutations are two independent one-step allele length mutations. A different age distribution of "boundary switch" mutations than of allele length mutations also argue against such a hypothesis. Together this could indicate that "boundary switches" are products of another mutation mechanism than the one-step allele length mutations.     

Application of electrophoresis technology to DNA analysis

We used the variable number tandem repeat (VNTR) polymorphism and the ten short tandem repeat (STR) polymorphisms to study a number of disputed paternity cases in the Japanese population. For the determination of VNTR locus (D1S80) and the ten STR loci (vWA, F13B, TH01, TPOX, CSF1PO, F13A01, LPL, D3S1744, D12S1090, D18S849) we used polymerase chain reaction (PCR) amplification and the vertical polyacrylamide gel electrophoresis technique followed by SYBR green I staining. The irregular repeats were analyzed by sequencing from bands of vertical polyacrylamide gel electrophoresis using the latest gene analyzing equipment, the ABI PRISM 310 Genetic Analyzer. The probable genotypes of the deceased putative father were deduced by Komatu's method from the genotypes of the widow and the genotypes of their children. The calculation of paternity probability used the Essen-Moller formula and Bayes's theorem. Calculated in eleven loci, the distinguishing probabilities (DP) and the mean exclusion chance (MEC) were 0.9999 and 0.9989, respectively. Therefore, information obtained from eleven DNA polymorphisms is enough to determine paternity plausibility.     

Characterization of a highly variable short tandem repeat polymorphism at the D2S1242 locus

We report the evaluation of short tandem repeat (STR) locus D2S1242 (GDB ID G00-309-429) for forensic purposes, investigated by polymerase chain reaction (PCR) amplification and both native and denaturating polyacrylamide gel electrophoresis in 147 unrelated Austrians. No deviations from Hardy-Weinberg expectations were observed. The mean exclusion chance (MEC) was 0.669, the discriminating power (DP) was 0.947, and the observed heterozygosity rate was 0.856. An allelic ladder consisting of eight sequenced alleles (141-167 and 175 bp) was constructed. Sequence analysis revealed that the locus comprised two repeat motifs varying in number between alleles GAAA and GAAG. According to the number of tetranucleotide repeats the smallest allele was designated as 10 and the largest allele as 18.     

Prestaining method as a useful tool for the agarose gel electrophoretic detection of polymerase chain reaction products with a fluorescent dye SYBR gold nucleic acid gel stain.

Three staining methods using SYBR Gold Nucleic Acid Gel Stain (SYBR Gold) as a fluorescent dye were evaluated for the agarose gel electrophoretic detection of DNA. The methods involve prestain, in-gel stain, and poststain methods. DNA markers and polymerase chain reaction (PCR) products obtained by minisatellite variant repeat-PCR (MVR-PCR) amplification in a D1S8 locus were used as model DNA and practical samples, respectively. Among the three methods tested under the usual electrophoretic conditions, a prestain method using a 10000-fold diluted SYBR Gold solution showed most excellent features regarding cost and rapidity to use with good stainability and resolution over loaded DNA amounts of about 98 ng to 300 ng. The prestain method was found to be applicable to the analysis of DNA in MVR-PCR products from a human hair root.     

Haplotype studies support slippage as the mechanism of germline mutations in short tandem repeats

Germline mutations of human short tandem repeat (STR) loci are expansions or contractions of repeat arrays which are not well understood in terms of the mechanism(s) underlying such mutations. Although polymerase slippage is generally accepted as a mechanism capable to explain most features of such mutations, it is still possible that unequal crossing over plays some role in those events, as most studies in humans could not exclude unequal crossing over (UCO). Crossing over can be studied by analyzing haplotypes using flanking markers. To check for UCO in mutations, we have analyzed 150 paternity cases for which more than the usual trio (mother, child, and father) were available for testing by analyzing 16 STR loci. In a total of 4900 parent-child allele transfers four mutations were observed at different loci (D8S1179, D18S51, D21S11, and SE33/ACTBP2). To identify the mutated allele and to check for UCO, we typed at least four informative loci flanking the mutated locus and used the pedigree data to establish haplotypes. By doing so we were able to exclude UCO in each case. Moreover, we were able to identify the mutations as one-repeat contractions/expansions. Our data thus support slippage as the mechanism of germline mutations in STRs.     

Rapid sizing of microsatellite alleles by gel electrophoresis on microfabricated channels: application to the D19S394 tetranucleotide repeat for cosegregation study of familial hypercholesterolemia.

This study evaluated the applicability of microchip electrophoresis to the sizing of microsatellites suitable to genetic, clinical and forensic applications. The evaluation was performed with the D19S394 tetranucleotide (AAAG) repeat characterized by a wide variation in the repeat number (1-17) and a short recombination distance from the low-density lipoprotein (LDL)-receptor gene that makes it suitable to cosegregation analysis of familial hypercholesterolemia (FH). The study was performed with 70 carriers of two LDL-receptor mutations common in northern Italy (i.e., the 4 bp insertion in exon 10 known as FH-Savona and the D200G missense mutation in the exon 4, known as FH-Padova 1) and 100 healthy controls. The polymerase chain reaction (PCR) amplification products prepared with a cosolvent PCR protocol and an antibody-protected polymerase were directly analyzed with an apparatus for high-voltage capillary electrophoresis on microchips and laser-induced fluorescence detection equipped with chips for the analysis of 25-500 bp dsDNA fragments. The test could not be extended to dinucleotide repeats due to the resolution characteristics of the available microchip. This novel approach was able to distinguish 17 microsatellite alleles varying from 0 to 17 repeats. Many of these alleles were quite rare, but the seven more abundant accounted for over the 70% of allele distribution in control population. The standard deviation in the sizing of the most abundant alleles ranged from +0.60 to +/- 0.75 bp. This indicated that the size attribution to a conventional allele using the +/- 1 bp range around it allowed a confidence limit above the 80 %. The sizing of D19S394 obtained this way allowed the cosegregation analysis with both the FH mutations tested. Therefore, this innovative approach to microsatellite sizing was much simpler, but equally effective as traditional capillary electrophoresis, at least with tetranucleotide repeats.     

Imperfect units of an extended microsatellite structure involving single nucleotide changes

Short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs) are widely used as markers in human genome studies. We have characterized a highly polymorphic STR locus (D20S85) with (AAAG)n repeats, by a combination of direct DNA sequencing and single-strand conformation polymorphism (SSCP) analysis. Eight STR alleles were first identified on denaturing gels, and SSCP gels were then used to demonstrate the existence of previously indistinguishable multiple alleles at the locus on the basis of variable allelic flanking sequences. This was confirmed by direct sequencing of the alleles. Four transitions, two G to A and two A to G in the 5'-flanking region of the locus at positions 14, 22, 24, and 26 effectively subdivided the STR alleles into two groups, with frequencies of 0.431 and 0.569, respectively. The mutational processes that generated the polymorphisms involved both simple changes in the number of AAAG repeats and single nucleotide mutations in the region flanking the repeat. The findings have potential application in the avoidance of false linkage and association. A composite locus of this nature, with separate STR and SNP evolutionary histories and resulting from different mutational processes, also could have wide application in studies of selection, drift, migration and inbreeding.     

PCR-free digital minisatellite tandem repeat genotyping

We demonstrated a proof‐of‐concept for novel minisatellite tandem repeat typing, called PCR‐free digital VNTR (variable number tandem repeat) typing, which is composed of three steps: a ligation reaction instead of PCR thermal cycling, magnetic bead‐based solid‐phase capture for purification, and an elongated sample stacking microcapillary electrophoresis (μCE) for sensitive digital coding of repeat number. We designed a 16‐bp fluorescently labeled ligation probe which is complementary to a repeat unit of a biotinylated synthetic template mimicking the human D1S80 VNTR locus and is randomly hybridized with the minisatellite tandem repeats. A quick isothermal ligation reaction was followed to link the adjacent ligation probes on the DNA templates, and then the ligated products were purified by streptavidin‐coated magnetic beads. After a denaturing step, a large amount of ligated products whose size difference was equivalent to the repeat unit were released and recovered. Through the elongated sample stacking μCE separation on a microdevice, the fluorescence signal of the ligated products was generated in the electropherogram and the peak number was directly counted which was exactly matched with the repeat number of VNTR locus. We could successfully identify the minisatellite tandem repeat number with only 5 fmol of DNA template in 30 min.     

The determination for the three genotypes of D16S539 locus based on near-infrared spectroscopy and chemical pattern recognition

The paper has established an approach of typing short tandem repeats (STRs) based on the near-infrared spectroscopy (NIRS)-chemical pattern recognition. Taking the three genotypes 9-9, 9-11 and 11-11 of D16S539 locus as example, which have a middle degree of difference, DNA fragments containing the polymorphism sites were amplified by a pair of primers to obtain three genotypes samples; these samples were tested by the NIRS directly; using their spectra as recognition variables, the chemical pattern recognition models of the three genotypes were respectively established by using the principal discriminant variate (PDV) and support vector machine (SVM). The two models have a good fitting ability and strong prediction (i.e. the predicting accuracy was 100%). They are robust for these strong collinear spectra and the small number of the calibration samples. Without any preprocessing for the analyzed samples after PCR, the three genotypes of D16S539 locus could be indirectly determined by using the NIRS-s of the samples with the help of the models. This method is simple, rapid and low cost.     

Massively parallel sequencing of STRs using a 29-plex panel reveals stutter sequence characteristics

Massively parallel sequencing of forensic STRs simultaneously provides length-based genotypes and core repeat sequences as well as flanking sequence variations. Here, we report primer sequences and concentrations of a next-generation sequencing (NGS)-based in-house panel covering 28 autosomal STR loci (CSF1PO, D1GATA113, D1S1627, D1S1656, D1S1677, D2S441, D2S1776, D3S3053, D5S818, D6S474, D6S1017, D6S1043, D8S1179, D9S2157, D10S1435, D11S4463, D13S317, D14S1434, D16S539, D18S51, D18S853, D20S482, D20S1082, D22S1045, FGA, TH01, TPOX, and vWA) and the sex determinant locus Amelogenin. Preliminary evaluation experiments showed that the panel yielded intralocus- and interlocus-balanced sequencing data with a sensitivity as low as 62.5 pg input DNA. A total of 203 individuals from Yunnan Bai population were sequenced with this panel. Comparative forensic genetic analyses showed that sequence-based matching probability of this 29-plex panel reached 2.37 × 10⁻²⁹, which was 23 times lower than the length-based data. Compound stutter sequences of eight STRs were compared with parental alleles. For seven loci, repeat motif insertions or deletions occurred in the longest uninterrupted repeat sequences (LUS). However, LUS and non-LUS stutters co-existed in the locus D6S474 with different sequencing depth ratios. These results supplemented our current knowledge of forensic STR stutters, and provided a sound basis for DNA mixture deconvolution.     

Developmental validation of the Microreader™ 20A ID system

The Microreader? 20A ID system is designed for forensic applications such as personal identification, parentage testing, and research. It includes 13 combined DNA index system (CODIS) short tandem repeat (STR) loci (CSF1PO, FGA, TH01, TPOX, vWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, and D21S11), three expanded CODIS STR loci (D12S391, D19S433, and D2S1338), three non‐CODIS STR loci (D6S1043, Penta D, and Penta E), and the amelogenin locus in one reaction with a six‐dye fluorescent (FAM, HEX, TAMAR, ROX, PUR, and QD550) analysis system. In this study, the Microreader? 20A ID system was validated according to the Scientific Working Group on DNA Analysis Methods validation guidelines for forensic DNA Analysis methods and Chinese national standard, including PCR‐based studies, sensitivity study, precision, and accuracy evaluation, stutter calculation, inhibitor tests, species specificity, and DNA mixture studies. Our results suggest that the Microreader? 20A ID system is a useful tool for personal identification and parentage testing.     

Genetic polymorphisms of 20 short tandem repeat loci from the Han population in Henan,China

The aim of this study was to investigate the genetic polymorphism of 20 short tandem repeat (STR) loci including D1S1656, D2S1338, D3S1358, D5S818, D6S1043, D7S820, D8S1179, D12S391, D13S317, D16S539, D18S51, D19S433, D21S11, CSF1PO, FGA, Penta D, Penta E, TH01, TPOX, and vWA in Han population of Henan, China and to assess its value in forensic science. Genomic DNA was extracted from 274 blood samples of unrelated healthy individuals in the Henan Han population. Alleles were amplified with PowerPlex® 21 system kit and PCR products were detected with ABI3130 genetic analyzer (Applied Biosystems) and the data were analyzed with modified PowerStats v1.2. A total of 229 alleles were observed in this Han population and the allelic frequencies ranged from 0.0020 to 0.5090 in the present study. Observed genotype distributions for each locus do not show deviations from Hardy–Weinberg equilibrium expectations (p < 0.05). The combined power of discrimination, combined power of exclusion, and combined matching probability of this 20 STR loci were 0.999999999, 0.999999994603, and 4.0433 × 10^?24, respectively. The 20 STR loci are highly polymorphic in the Han population of Henan, China and they may be of great value in forensic science and human population genetics.     

Sequence data of six unusual alleles at SE33 and D1S1656 STR Loci

When profiling a reference dataset of 500 DNA samples for the population of Saudi Arabia, using the GlobalFiler® PCR amplification kit, six unusual alleles were detected. At the SE33 locus, four novel alleles were found: 2, 14.3, 20.3, and 38; two alleles at the D1S1656 locus: 7 and 8 had been previously reported, but no published sequence data was available. The D1S1656 alleles were sequenced using ForenSeq™ DNA Signature Prep with the MiSeq FGx System (Illumina, USA). As the SE33 is not reported by available Massively Parallel Sequencing (MPS) systems, samples that exhibited the unreported alleles were sequenced using BigDye™ Terminator v3.1 Cycle Sequencing Kit. Here we present the sequence and structure of the previously uncharacterized alleles.     

Preparation of glucagon-like peptide-1 loaded PLGA microspheres: characterizations, release studies and bioactivities in vitro/in vivo

The gut hormone glucagon-like peptide-1 (GLP-1) is proposed for treatment of Type II diabetes mellitus. However, the short half life of GLP-1 in vivo is a major limitation for its application due to the frequent invasive administrations. To provide a optimal formulation to overcome this limitation, we developed a GLP-1 entrapped microspheres to achieve sustained release GLP-1 for 4-week. GLP-1 was stabilized by GLP-1-zinc complexation with zinc carbonate and encapsulated in poly(D,L-lactic-co-glycolic acid) (PLGA) with S/O/O solvent extraction to obtain GLP-1 loaded PLGA microspheres (MS). The characteristics of MS were evaluated as follows: The surface morphology was assessed by scanning electron microscopy (SEM); The drug encapsulation efficiency and GLP-1 controlled release profile was tested by HPLC; The sustained release of GLP-1 MS in vivo and pharmacological efficacy were studied in normal mice and streptozotocin (STZ)-induced diabetic mice model after subcutaneous administration of GLP-1 MS. GLP-1-zinc complexation significantly reduced initial burst release from 37.2 to 7.5%. The controlled release bioactive GLP-1 in vitro was achieved for 4-week period by zinc complexation and addition of ZnCO(3). The optimal and complete cumulative release of GLP-1 from MS was increased from 23 to 63% in 28 d by using low MW PLGA (MW 14000). The in vivo testing in normal mice and diabetic mice suggest that this zinc-stabilized technique combined with S/O/O method in the presence of water insoluble antacid additive ZnCO(3) preserve the biological activity of GLP-1. GLP-1 MS formulation achieved controlled released in vivo for 28 d and exhibit sustained long term pharmacological efficacy to decrease blood glucose level in diabetic mice. This GLP-1 MS formulation provides a practical formulation for long-term sustained delivery of GLP-1 to treat Type II diabetes.     

Fifty-nine bp repeat polymorphism in the uncommon intron 36 of the human mucin gene MUC5B

A variable number of tandem repeat (VNTR) polymorphism within the intron 36 of the human mucin gene MUC5B, which is mapped to chromosome 11 band p15.5, have been identified using Southern blotting experiments. This polymorphism can be easily assayed by polymerase chain reaction (PCR) to detect linkage of inherited disorder. Five alleles were observed in 86 unrelated individuals due to 3-8 direct perfect repeats of 59 bp. This repeat has the particularity to begin at the end of the preceding exon. Southern blot experiments revealed the locus specificity of the repeat. The sequence of the repeat unit does not match the consensus sequence of Chi-related minisatellites.     

Complete assignment of the 1H and 13C NMR spectra of garciniaphenone and keto-enol equilibrium statements for prenylated benzophenones

This article reports the structural elucidation by IR, UV and MS spectroscopic data along with 1H and 13C NMR chemical shift assignments of two benzophenones isolated from the fruit pericarp of Garcinia brasiliensis Mart. (Clusiaceae): garciniaphenone, (1R,5S,7S)-3-benzoyl-4-hydroxy-6,6-dimethyl-5,7-di(3-methyl-2-butenyl)bicyclo[3.3.1]non-3-ene-2,9-dione, a novel triprenylated benzophenone; and 7-epi-clusianone, a tetraprenylated benzophenone that has already been extracted from another species of the same family. Furthermore, the keto-enol tautomeric equilibrium at solution-state was described for these compounds by 1D and 2D NMR spectral methods and one attempt to rationalize the different ratios between the noted tautomers was based on stereochemical features.     

Standardized genotyping of HLA STR by CE as surrogate for HLA class I and II markers and for identification of HLA identical siblings

Linkage disequilibria (LD) between alleles and haplotypes of human leucocyte antigen, locus A (HLA) and STR loci located in the human major histocompatibility complex were analyzed in order to investigate whether or not HLA alleles and haplotypes are predictable by alleles or haplotypes of HLA STRs. Standardized genotyping of eight STR loci (D6S2972, D6S2906, D6S2691, D6S2678, D6S2792, D6S2789, D6S273, and DQIV) was performed by CE on 600 individuals from 150 Austrian Caucasoid families with known HLA‐A,‐B,‐C and –DRB1 typing. From those, 576 full haplotypes of four HLA and eight STR loci were obtained. Haplotypes of two flanking STRs predicted HLA alleles and two‐locus HLA haplotypes better than single STR alleles, except HLA‐DRB1 alleles (92% were in LD with DQIV alleles only). A percentage of 65–86% of three and four‐locus HLA haplotypes were in LD with haplotypes of three, four, and eight of their flanking STR loci including numerous clear‐cut predictions (20–61%). All eight and a set of the four most informative STR loci D6S2972, D6S2678, D6S2792, and DQIV could identify all HLA identical and nonidentical siblings in 138 pairs of siblings. The results of this proof of concept study in Austrian Caucasoids show, that HLA STRs can aid the definition of HLA‐A,‐B,‐C,‐DRB1 haplotypes and the selection of sibling donors for stem cell transplantation.     

Structural varieties of selectively mixed G‐ and C‐rich short DNA sequences studied with electrospray ionization mass spectrometry

Short guanine(G)‐repeat and cytosine(C)‐repeat DNA strands can self‐assemble to form four‐stranded G‐quadruplexes and i‐motifs, respectively. Herein, G‐rich and C‐rich strands with non‐G or non‐C terminal bases and different lengths of G‐ or C‐repeats are mixed selectively in pH 4.5 and 6.7 ammonium acetate buffer solutions and studied by electrospray ionization mass spectrometry (ESI‐MS). Various strand associations corresponding to bi‐, tri‐ and tetramolecular ions are observed in mass spectra, indicating that the formation of quadruplex structures is a random strand by strand association process. However, with increasing incubation time for the mixtures, initially associated hybrid tetramers will transform into self‐assembled conformations, which is mainly driven by the structural stability. The melting temperature values of self‐assembled quadruplexes suggest that the length of G‐repeats or C‐repeats shows more significant effect on the stability of quadruplex structures than that of terminal residues. Accordingly, we can obtain the self‐associated tetrameric species generated from the mixtures of various homologous G‐ or C‐strands efficiently by altering the length of G‐ or C‐repeats. Our studies demonstrate that ESI‐MS is a very direct, fast and sensitive tool to provide significant information on DNA strand associations and stoichiometric transitions, particularly for complex mixtures. Copyright © 2016 John Wiley & Sons, Ltd.