Capillary Electrophoresis Platform Research Articles

Genetic characterization evaluation of a novel multiple system containing 57 deletion/insertion polymorphic loci with short amplicons in Hunan Han population and its intercontinental populations analyses.

Deletion/insertion polymorphism (DIP) is a promising genetic marker of DNA length polymorphism. However, there are relatively few studies on the exploration of DIP genetic polymorphisms and investigation of population genetic data at present, which limits its application in forensic identification. In this study, the genetic polymorphisms of 57 autosomal DIPs and forensic application evaluations of the novel panel were analyzed in Chinese Hunan Han population using capillary electrophoresis platform, andthe differences of genetic polymorphic distributions at these loci were compared among the Hunan Han and 26 reference populations. The present results showed the combination of total 57 DIPs could be a robust tool for forensic individual identification and paternity testing. Due to the different allele frequency distributions in the different continental populations, the system could also effectively distinguish among East Asian, European and African populations.

Use of Microfluidic Capillary Electrophoresis for the Determination of Multi-Component Protein Adsorption Isotherms: Application to High-Throughput Analysis for Hydrophobic Interaction Chromatography.

Chromatography is a widely used separation process for purification of biopharmaceuticals that is able to obtain high purities and concentrations. The phenomena that occur during separation, mass transfer and adsorption are quite complex. To better understand these phenomena and their mechanisms, multi-component adsorption isotherms must be investigated. High-throughput methodologies are a very powerful tool to determine adsorption isotherms and they waste very small amounts of sample and chemicals, but the quantification of component concentrations is a real bottleneck in multi-component isotherm determination. The behavior of bovine serum albumin, Corynebacterium diphtheriae CRM197 protein and lysozyme, selected as model proteins in binary mixtures with hydrophobic resin, is investigated here. In this work we propose a new method for determining multi-component adsorption isotherms using high-throughput experiments with filter plates, by exploiting microfluidic capillary electrophoresis. The precision and accuracy of the microfluidic capillary electrophoresis platform were evaluated in order to assess the procedure; they were both found to be high and the procedure is thus reliable in determining adsorption isotherms for binary mixtures. Multi-component adsorption isotherms were determined with a totally high-throughput procedure that turned out to be a very fast and powerful tool. The same procedure can be applied to every kind of high-throughput screening.

Forensic Application Evaluation of a Novel Canine STR System in Pembroke Welsh Corgi and Shiba Inu Groups.

Aim To evaluate the forensic application values of 19 autosomal short tandem repeat (STR) loci in canines. Methods The 19 STR loci in two canine groups (Pembroke Welsh Corgis, n = 200; Shiba Inus, n = 175) were analysed by the capillary electrophoresis platform. The allele frequencies and forensic parameters were calculated, and the genetic relationships between these two canine groups and a previously reported Labrador group were analysed. Results These two canine groups conformed to the Hardy-Weinberg equilibrium at all STRs except for locus VGL3438 in the Pembroke Welsh Corgi group, and there was no linkage disequilibrium among pairwise loci at the 19 STRs. All STRs were polymorphic in the Pembroke Welsh Corgi and Shiba Inu groups, of which the locus C38 had the highest polymorphism. And it was found that the genetic relationship between the Pembroke Welsh Corgi and Labrador groups were closer in the three canine groups (Pembroke Welsh Corgi, Shiba Inu and Labrador). Conclusion The 19 STR loci had high genetic polymorphisms and forensic application values in Pembroke Welsh Corgi and Shiba Inu groups.

A Ge.F.I. – ISFG European collaborative study on DNA identification of Cannabis sativa samples using a 13-locus multiplex STR method

Cannabis sativa is the most used controlled substance in Europe. With the advent of new and less restrictive European laws on cannabis sale for recreational use (including in Italy), an increase in indoor cannabis crops were observed. This increase was possible due to the availability of cannabis seeds through the internet market. Genetic identification of cannabis can link seizures and if in possession then might aid in an investigation. A 13-locus multiplex STR method was previously developed and validated by Houston et al. A collaborative exercise was organized by the Italian Forensic Geneticists – International Society of Forensic Genetics (Ge.F.I. – ISFG) Working Group with the aim to test the reproducibility, reliability and robustness of this multiplex cannabis STR kit. Twenty-one laboratories from three European countries participated in the collaborative exercise and were asked to perform STR typing of two cannabis samples. Cannabis DNA samples and the multiplex STR kit were provided by the University of Barcelona and Sam Houston State University. Different platforms for PCR amplification, capillary electrophoresis (CE) and genotyping software were selected at the discretion of the participating laboratories. Although the participating laboratories used different PCR equipment, CE platforms and genotyping software, concordant results were obtained from the majority of the samples. The overall genotyping success ratio was 96%. Only minor artifacts were observed. The mean peak height ratio was estimated to be 76.3% and 78.1% for sample 1 and sample 2, respectively. The lowest amount of −1 / + 1 stutter percentage produced, when the height of the parent allele was higher than 8000 RFU, resulted to be less than 10% of the parent allele height. Few common issues were observed such as a minor peak imbalance in some heterozygous loci, some artifact peaks and few instances of allelic drop-out. The results of this collaborative exercise demonstrated the robustness and applicability of the 13-locus system for cannabis DNA profiling for forensic purposes.

Development of an In-Line Enzyme Reactor Integrated into a Capillary Electrophoresis System.

The goal of this paper was to develop an in-line immobilized enzyme reactor (IMER) integrated into a capillary electrophoresis platform. In our research, we created the IMER by adsorbing trypsin onto the inner surface of a capillary in a short section. Enzyme immobilization was possible due to the electrostatic attraction between the oppositely charged fused silica capillary surface and trypsin. The reactor was formed by simply injecting and removing trypsin solution from the capillary inlet (~1–2 cms). We investigated the factors affecting the efficiency of the reactor. The main advantages of the proposed method are the fast, cheap, and easy formation of an IMER with in-line protein digestion capability. Human tear samples were used to test the efficiency of the digestion in the microreactor.

Native fluorescence detection with a laser driven light source for protein analysis in capillary electrophoresis

While ultraviolet light (UV) absorbance detection is the most widely used detection mode in capillary electrophoresis (CE), it can yield poor concentration sensitivity and has tendencies to exhibit baseline fluctuations. In order to overcome these challenges, alternative detection strategies, including the use of dedicated wavelength lasers, have been applied, resulting in enhancements of concentration sensitivity as well as decreased baseline disturbance. In this work, using a laser driven light source for excitation, we reported a native fluorescence detection (NFD) scheme for use in a commercial CE platform, PA 800 Plus Pharmaceutical Analysis System, for protein analysis. The CE-NFD system was characterized using tryptophan and a reduced IgG. We compared NFD with UV absorbance detection as applied to sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) and capillary isoelectric focusing (cIEF). In SDS-CGE, with the reported NFD a non-reduced IgG standard sample yielded a signal-to-noise ratio which was 14.6 times higher than with UV absorbance detection at 214 nm. In cIEF analysis of NISTmAb, Humanized IgG1k, with NFD ∼170 times less sample mass was needed to obtain similar profile quality to that with UV absorbance detection at 280 nm. NFD also eliminated baseline anomalies observed with UV absorbance detection and showed less interference by other absorbing species. These results suggest that CE-NFD is a practical and powerful tool for protein characterization in the biopharmaceutical industry.

A Novel System for Newborn Screening of Thalassemia and Hemoglobinopathies Using Capillary Electrophoresis Is Superior Than Isoelectric Focusing

Introduction:Thalassemia (thal) and hemoglobinopathies (Hb variants) is highly prevalent in Thailand and nearly all Southeast Asian countries. In developed countries such as US and European countries, a newborn screening program for thal and hemoglobinopathies (NBS-Hb) has been incorporated in a routine healthcare to detect those with abnormal Hb in particular Hb S (Sickle cell disease) to provide early intervention for affected patients. Application of detecting patients and carriers could be useful for many developing countries such as Thailand as a part of our prevention and control program at a national level. At presence, isoelectric focusing (IEF) is a widely used platform for NBS-Hb, however a new capillary electrophoresis (CE) has recently been developed. It is of interest to evaluate whether which system provides a better efficiency under a circumstance with various types of thal and Hb variants.Objectives:To evaluate whether IEF or CE, is more effective for Hb screening at neonatal period and to develop new diagnostic criterion for different types of Hb variants in ThailandMethods:After informed consent by parent, the dried blood spot (DBS) samples were collected by heel prick puncture from 2-day old babies and processed in two Hb screening methods; IEF & ISOSCAN RESOLVE® SYSTEM Neonatal Hemoglobin Kits (Perkin Elmer, Turku, Finland) and Capillarys 2 NEONAT FAST® (SEBIA, Évry, France). These DBS were also extracted gDNA used for gold standard molecular method covering >98% of common globin mutations found in Thailand; 7-deletional α-thal GAP-PCR, 6-non-deletional α-thal ARMS-PCR, 16 mutations of β-thal ARMS-PCR, PCR-RFLP by Mnl I restriction cleavage for Hb E disorder and/or DNA sequencing. The statistics analysis was performed by PASW 18 Software (SPSS Inc, 2015) to analyze the detection efficiency of IEF vs. CE. Cut-off levels of Hb variants were also determined by using receiver operating characteristic (ROC) curve to set up diagnostic criterion.Results: The study recruited 1,213 healthy newborns (48.4% male and 51.6% female) with average 38 weeks of gestational age (GA). The profiles of IEF pattern and CE histogram were analyzed. Hb E was the most common Hb variants of β-globin gene found and Hb Bart's (γ4) was indicative of α-thal, both were confirmed by molecular methods. Comparison between the efficiency of IEF vs. CE on detecting Hb variants showed a higher efficiency of CE on Hb Bart's detection for most α-thal carriers and on Hb E detection in Hb E trait than those of IEF for sensitivity, specificity, and accuracy as shown in Table 1. There was no difference between two platforms on Hb H disease, homozygous Hb E and β-thal/Hb E. In order to set up cut-off levels of Hb Bart's, Hb E and Hb A for each globin genotypes by ROC curve analysis, the cut-off levels provided from CE seemed to be more clear and discriminative than those of IEF as shown in Table 2. The cut-off levels of Hb Bart's of ≥7.40%, ≥0.75%, ≥0.45%, and ≥0.10% were suggested different α-globin abnormalities; Hb H, αº-, non-deletional and deletional α+-thal carrier, respectively. Detection of Hb E of ≥0.25%, ≥4.85%, and ≥0.95% were suggestive of Hb E carrier, Hb E homozygote, and β-thal/Hb E, respectively. Interestingly, we can screen for β-thal carriers using a lower Hb A level (≤10.35%) and Hb A/Hb F ratio (≤0.115). This data was analyzed from 11 β-thal carrier newborns confirmed by DNA testing compared to 148 GA-sex matched newborn controls with normal β-globin genotype. These cut-off levels based on CE were powerful on detecting thal diseases (Hb H and β-thal/Hb E) and carriers of Hb E, αº- and non-deletional α+-thal. However, our screening criterions still have a limitation on fully detecting deletional α+-thal and β-thal traits.Conclusions:Our data suggested that neonatal CE platform is more effective system for Hb screening with excellent efficiency and precision to detect Hb variants compared to conventional IEF. Using NBS-Hb by CE could successfully screen for all important thal carriers in one simple test. Implementing our NBS-Hb to the national level would be simplest way to generate a national registry that can be linked to our national health database of severe diseases and carriers. By applying this approach into a national scale might be the only measure to effectively control the health burden of thal and hemoglobinopathies in Thailand where provides high prevalence of defective globin genes. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Novel SNP-STR System Based on a Capillary Electrophoresis Platform.

Various compound markers encompassing two or more variants within a small region can be regarded as generalized microhaplotypes. Many of these markers have been investigated for various forensic purposes, such as individual identification, deconvolution of DNA mixtures, or forensic ancestry inference. SNP-STR is a compound biomarker composed of a single nucleotide polymorphism (SNP) and a closely linked short tandem repeat polymorphism (STR), and possess the advantages of both SNPs and STRs. In addition, in conjunction with a polymerase chain reaction (PCR) technique based on the amplification refractory mutation system (ARMS), SNP-STRs can be used for forensic unbalanced DNA mixture analysis based on capillary electrophoresis (CE), which is the most commonly used platform in worldwide forensic laboratories. Our previous research reported 11 SNP-STRs, but few of them are derived from the commonly used STR loci, for which existing STR databases can be used as a reference. For maximum compatibility with existing DNA databases, in this study, we screened 18 SNP-STR loci, of which 14 were derived from the expanded CODIS core loci set. Stable and sensitive SNP-STR multiplex PCR panels based on the CE platform were established. Assays on simulated two-person DNA mixtures showed that all allele-specific primers could detect minor DNA components in 1:500 mixtures. Population data based on 113 unrelated Chengdu Han individuals were investigated. A Bayesian framework was developed for the likelihood ratio (LR) evaluation of SNP-STR profiling results obtained from two-person mixtures. Furthermore, we report on the first use of SNP-STRs in casework to show the advantages and limitations for use in practice. Compared to 2.86 × 103 for autosomal STR kits, the combined LR reached 7.14 × 107 using the SNP-STR method in this casework example.

Multi-Indel: A Microhaplotype Marker Can Be Typed Using Capillary Electrophoresis Platforms.

Since the concept of microhaplotypes was proposed by Kidd in 2013, various microhaplotype markers have been investigated for various forensic purposes, such as individual identification, deconvolution of DNA mixtures, or forensic ancestry inference. In our opinion, various compound markers are also regarded as generalized microhaplotypes, encompassing two or more variants in a short segment of DNA (e.g., 200 bp). That is, a set of variants (referred to herein as multi-variants) within a certain length includes single nucleotide polymorphisms (SNP), insertion/deletion polymorphisms (Indels), or short tandem repeat polymorphisms (STRs). At present, multi-variant is mainly aimed at multi-SNPs. However, the haplotype genotyping of multi-variants relies on single-strand analysis, mainly using massively parallel sequencing (MPS). Here, we describe a method based on a capillary electrophoresis (CE) platform that can directly obtain haplotypes of individuals. Several microhaplotypes consisting of three or more Indels with different insertion or deletion lengths in the range of less than 200 bp were screened out, each of which had at least three haplotypes. As a result, the haplotype of an individual was reflected by the length of its polymorphism. Finally, we established a multiplex amplification system containing 18 multi-Indel markers that could identify haplotypes on each chromosome of an individual. The combined power of discrimination (CPD) and the cumulative probability of exclusion (CPE) were 0.999999999997234 and 0.9984, respectively.

Application of affinity capillary electrophoresis in the study of protein-DNA interactions

Protein-DNA interactions play essential roles in various biological events that determine the cell fate. Research on the molecular mechanism of protein-DNA interactions has helped elucidate diverse fundamental life processes, thereby providing theoretical guidance for establishing clinical treatment and screening potential drug of target diseases. Furthermore, well-known protein-DNA interactions have been utilized to develop advanced bioengineering and bioanalytical techniques, therefore providing robust technical support for related research. Hence, it is important to establish sensitive and rapid analytical methods to study protein-DNA interactions. High-performance capillary electrophoresis (CE) has been widely used in many research fields such as chemistry, life sciences, and environmental sciences, mainly due to its advantages including ultra-high separation efficiency, extremely low sample consumption, and short analysis time. For instance, affinity capillary electrophoresis (ACE) has become an important analytical tool for investigating molecular interactions. In this paper, we review the applications of ACE in studying protein-DNA interactions since it was first proposed in 1992, addressing previous significant work in this field. Three major aspects have been summarized in this review: (1) brief introduction to the development of ACE technique; (2) applications of ACE in the fundamental research on the molecular mechanism of protein-DNA interactions; and (3) applications of well-known protein-DNA interactions in CE-based detection of target molecules and reactions. In the first aspect, along with the concept and separation modes of ACE, general strategies to enhance the analytical ability of ACE are briefly introduced. In the second aspect, the applications of ACE in studying several important protein-DNA interactions involving transcription factors (e.g., GCN4), DNA repair proteins (e.g., UvrA, UvrB, and RecA), and methylated DNA-binding proteins (MBDs) are reviewed. In the third aspect, the applications of well-known molecular interactions (e.g., antigen-antibody, aptamer-target, etc.) to facilitate CE-based detection of target molecules (e.g., DNA adducts, DNA methylation, microRNA, single nucleotide polymorphism, etc.) and target reactions (e.g., DNA strand exchange) are addressed. Finally, we prospect and discuss the advancements of ACE that can be established in future studies. The following two aspects should be improved in future ACE analysis: (1) the advantages of extremely low volume consumption and short analysis time should be fully utilized to develop sensitive and high-throughput CE platforms for the assessment of rare biological samples and massive uncertain samples, respectively; (2) ACE should be combined with other advanced techniques, such as DNA sequencing and mass spectrometry, to rapidly screen and identify the precise interacting sites of unknown protein-DNA interactions.

A capillary electrophoresis based approach for the identification of anti-drug antibodies against camelid VHH biologics (Nanobodies®)

Undesired immune responses against protein therapeutics may adversely affect the pharmacokinetics, efficacy, and safety of the product. The presence of anti-drug-antibodies (ADA) has been the key determinant of immunogenic responses. Here we describe the use of a capillary electrophoresis platform for the identification of ADAs against several experimental camelid VHH biologics (Nanobodies®). Hereafter, we refer to this assay as WESADA. We modified the Wes platform by ProteinSimple to screen serum samples for ADA against covalently linked multi-modular Nanobodies and compared it to standard ADA methodologies. We were able to identify ADA positive samples and determine which individual VHH module in a multivalent Nanobody construct stimulated the predominant ADA response. WESADA requires denaturation of the experimental immobilized drug, which could affect recognition of the immunogenic epitope and alter ADA signal. To address this issue, we demonstrated that signal can be immunodepleted by pre-incubation of serum samples with native Nanobody. This capillary electrophoresis based approach allows for rapid analysis without the need for individually tailored assay optimization or reagent labeling, while consuming small amounts of sample and drug. It also allows for the simultaneous ADA analysis of multiple targets of different molecular size in the same experimental sample. WESADA is not intended to replace traditional ADA assay formats, but it facilitates the expedient immunogenic assessment of a large number of experimental drug candidates in the early developmental space.

Revisiting the genetic background and phylogenetic structure of five Sino-Tibetan-speaking populations: insights from autosomal InDels.

The Tibetan-Yi Corridor, located on the eastern edge of the Tibetan Plateau, is the main route of the people of the plateau. Human settlement and diffusion along the corridor have played a pivotal role in shaping the genetic architecture of Sino-Tibetan-speaking (STs) populations in China. In this study, five STs groups (Chengdu Tibetan, Chengdu Han, Muli Tibetan, Lugu Lake Mosuo and Xichang Yi) settling in the Tibetan-Yi Corridor were genotyped via AGCU InDel 50 kit on the capillary electrophoresis platform to decrypt the genetic landscape and phylogenetic relationship of STs populations and investigate the forensic characteristics. Allele frequency distributions of all autosomal insertion/deletion polymorphisms (InDels) in studied groups comply with Hardy-Weinberg equilibrium. The combined power of discrimination values are 0.9999999999999999998, 0.9999999999999999995, 0.9999999999999999999, 0.999999999999999993 and 0.99999999999999999994, respectively, and all the combined probability of exclusion values exceed 0.9990. Forensically relevant statistics implied that these InDels could be used for individual identification and as a promising alternative to STR profiling in paternity testing. Typical population comparisons showed strikingly high homogeneity among studied STs people, indicating complicated genetic admixture among populations in the Tibetan-Yi Corridor. The STs groups in the Tibetan-Yi Corridor keep close genetic affinity with geographically or linguistically close populations, and the genetic components of investigated populations arose from a mixture of multiple ancestral gene pools (resulting from the admixture from the ancestral Highland Tibetans and ancestral Lowland indigenous populations).

Comparison of CE- and MPS-based analyses of forensic markers in a single cell after whole genome amplification

Whole genome amplification (WGA) allows for multiple genetic analyses with low template DNA, such as DNA derived from a single cell. WGA could increase the amount of input DNA from the pg to the μg level. However, there are no studies comparing the performance of forensic markers with DNA from a single cell after WGA evaluated on both capillary electrophoresis (CE) and massively parallel sequencing (MPS) platforms. In this study, cell lines consisting of female cultured B-lymphoblastoid cells and karyocytes from male venous blood were segregated into one, two, three and five cells. Including the references with the bulk cells, all samples were generated by WGA with the multiple displacement amplification (MDA) strategy in triplicate and genotyped on CE and MPS platforms. Allele balance, stutter ratio, accuracy, repeatability and concordance of short tandem repeat (STR) markers were used to evaluate the genotyping performance on both platforms. Additionally, the sequence coverage ratio (SCR) and SNP genotypes were evaluated for sequence information generated from the MPS. Heterozygous loci showed high allele balance, with an overall average allele balance ratio larger than 0.79 on the CE and 0.75 on the MPS platforms for the venous blood cell samples; the cultured B-lymphoblastoid cell samples had ratios of 0.62 and 0.70, respectively. The stutter ratio of every source and cell number from both cell line samples were very close, ranging from 5.3%–7.2% for autosomal STRs and approximately 10 % of Y chromosomal STRs on the CE platform. The average stutter, allele, and sequence-based and length-based noise ratios were 6.6 %, 88 %, 4.7 % and 0.7 %, respectively, in the single male cell sample. SNPs also showed high consistency and intralocus balance. Our study indicated that WGA with MDA strategy works relatively well of STR and SNP genotyping with low copy number samples on CE and MPS, even with one-cell sample.

Preliminary exploration of a novel method for the deconvolution of DNA mixtures by pyrosequencing

The deconvolution of DNA mixtures has been a thorny issue. Several kinds of genetic markers, such as STR, SNP, SNP/DIP-STR and microhaplotypes etc., as well as capillary electrophoresis, NGS and other technical platforms are used to solve this problem. In this study, we propose a novel method for the analysis of DNA mixtures. This method employed multiplex pyrosequencing to sequence several selected microhaplotypes. Employing a sparse representation-based AdvISER-MH-PYRO algorithm, the original pyrosequencing (PSQ) signals of the DNA mixtures were deciphered to determine the contributors of the mixtures. Our preliminary data show that this method can resolve the number of contributors to a DNA mixture composed of 2–4 individuals.

Validating the consistency of cSNPs analysis results between DNA and RNA using SNaPshot method

The use of coding region single nucleotide polymorphisms (cSNPs) was recently proposed as a potential method for individual identification because it allows mRNA profiling and DNA typing to be performed concurrently. Nevertheless, availability of this approach still needs some further validation in different aspect. In this study, we have initially selected several SNP loci located in mRNA molecules that were confirmed to be highly expressed in blood. Both coding regions (CDRs) and untranslated regions (UTRs) were taken into consideration during the screening. Genomic DNA (gDNA) and total RNA from venous blood samples were isolated, followed by the synthesis of first-strand complementary DNA (cDNA) using purified RNA samples. Subsequently, the genotypes of these SNPs were respectively determined with gDNA and cDNA by using SNaPshot method. The PCR primers for cDNA were designed to span an intron in order to ensure that the amplification products were not due to the presence of potential DNA contamination. In summary, our study revealed a high consistency of cSNP analysis results between DNA and RNA on capillary electrophoresis platform, which highlighted the potential use of cSNP in forensic investigation.

Development and validation of a multiplex insertion/deletion marker panel, SifaInDel 45plex system

In addition to commonly used short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs), insertion and deletion polymorphisms (InDels) have considerable potential in the field of forensic genetics because they combine desirable characteristics of both STRs and SNPs. In the present study, the SifaInDel 45plex system was designed to amplify 45 InDel markers, including 27 autosomal InDels (A-InDels), 16 X chromosome InDels (X-InDels) and two Y chromosome InDels (Y-InDels), simultaneously in a single PCR procedure and then detect products by capillary electrophoresis (CE). We also optimized the PCR conditions for the novel panel and performed several validation studies including repeatability/reproducibility, concordance, accuracy, sensitivity, stability, species specificity and population genetics.The results confirmed that full profiles could be obtained from ≥62.5 pg of input DNA and from a series of challenging samples encountered in routine casework. The SifaInDel 45plex panel could tolerate different concentrations of inhibitors, such as ≤50 μM hematin, ≤20 ng/μL nigrosine and ≤8000 ng/μL urea. In a population investigation, for the 27 A-InDels, the combined power of discrimination (CPD) exceeded 0.999999, and the combined power of exclusion in duos (CPED) and trios (CPET) was 0.955118 and 0.997754, respectively. For the 16 X-InDels, the combined PDMale and PDFemale was computed as 0.999845 and 0.999998, respectively, and the combined mean exclusion chance in father/daughter or mother/son duos (MECDuo) and mean exclusion chance in standard trios involving daughters (MECTrio) was 0.976220 and 0.998163, respectively. In addition, the two Y-InDels could play a role in correctly determining gender. Overall, the established SifaInDel 45plex panel is a well-performing, reliable and robust multiplex system that stands out for combining a considerable number of A-indels, X-indels and Y-indels based on a CE platform. The population study results also demonstrated that the SifaInDel 45plex panel could be a valid complementary approach for human identification and complex kinship analysis.

Design of an automated capillary electrophoresis platform for single-cell analysis.

Single-cell analysis of cellular contents by highly sensitive analytical instruments is known as chemical cytometry. A chemical cytometer typically samples one cell at a time, quantifies the cellular contents of interest, and then processes and reports that data. Automation adds the potential to perform this entire sequence of events with minimal intervention, increasing throughput and repeatability. In this chapter, we discuss the design considerations for an automated capillary electrophoresis-based instrument for assay of enzymatic activity within single cells. We describe the key requirements of the microscope base and capillary electrophoresis platforms. We also provide detailed protocols and schematic designs of our cell isolation, lysis, sampling, and detection strategies. Additionally, we describe our signal processing and instrument automation workflows. The described automated system has demonstrated single-cell throughput at rates above 100cells/h and analyte limits of detection as low as 10-20mol.

The establishment of a DNA fingerprinting database for 73 varieties of Lactuca sativa capitate L. using SSR molecular markers

Head lettuce originating near the Mediterranean coast was introduced to China in the fifth century. It is difficult to identify different varieties of head lettuce due to their close genetic relationships. Simple sequence repeats (SSR) technology is a practical method that has been widely used in variety identification, fingerprint construction and genetic diversity analysis. In this paper, 23 pairs of SSR primers were selected to identify and analyse 73 varieties of head lettuce. The results identified a total of 117 mutated alleles detected in 23 loci, with the number of each loci ranging from 2 to 11, with an average of 5.1 mutated alleles per locus. Additionally, 152 genotypes were detected in 23 loci, with an average of 6.6 genotypes per locus derived from a range of 2–13. The polymorphism information content ranged from 0.071 to 0.702, with an average of 0.499. The selected primer pairs could clearly reflect the genotypic diversity level of the 73 head lettuce varieties tested, and can be used in the detection and analysis of the head lettuce varieties. The results of our sequence analysis also showed that these 73 varieties of head lettuce could be effectively identified by a combination of 19 primer pairs even though the genetic similarity among the 73 varieties ranged from 0.647 to 0.991, with an average of 0.798. Based on a capillary electrophoresis platform, a DNA fingerprinting database of 73 head lettuce varieties was established.

Development and Use of Clinical Pharmacodynamic Assays to Demonstrate Target Engagement for AZD4573, a Selective and Potent CDK9 Inhibitor Currently in Phase I Clinical Trials

CDK9 (cyclin-dependent kinase 9) regulates RNA transcription through its phosphorylation of RNA polymerase II and subsequent release from a “paused” elongation state, resulting in transcriptional activity. A transient inhibition of CDK9 activity will significantly impact transcripts with a short half-life, which includes the transcript for MCL1, an anti-apoptotic protein. AZD4573 is a highly potent and selective CDK9 inhibitor. We have previously reported that AZD4573 treatment results in dose-dependent inhibition of pSer2 of RNA polymerase II, reductions in MCL1 mRNA and protein, and an increase in cleaved caspase 3, resulting in cell death of hematological tumor cell lines and regressions in AML/DLBCL in vivo models [Cidado et. al., AACR Annual Meeting, Abstract 310 (2018)].AZD4573 is currently being evaluated in a Phase I all-comers relapsed/refractory hematological malignancy trial (NCT03263637). To generate clinical evidence that the compound is inhibiting its target and having the expected downstream effects, we have developed pharmacodynamic biomarker (PD) assays for clinical use to measure the changes in pSer2/RNApol II, MCL1 protein, cleaved Caspase 3 and cleaved PARP. We have chosen a capillary electrophoresis platform which allows quantification and multiplexing while requiring lower sample quantities (<20ng/capillary) than Western blotting. Data from AZD4573-treated MV4,11 AML cells using the clinical assays were similar to what had been shown previously with Western blotting, i.e. similar dose-dependence across 0.1nM to 1uM and >80% inhibition of both pSer2 and MCL1 at 100nM (in 10% FCS). Next, we validated the use of these assays with human peripheral blood mononuclear cells (PBMCs) since we expected that some patients from the trial are likely to have significant numbers of circulating tumor cells (i.e. AML/CLL). After incubation of human whole blood with 1uM AZD4573 for different times, PBMCs were isolated and analyzed using the capillary electrophoresis assays. These results showed at least ~80% inhibition of pSer2 at 2h, consistent with preclinical efficacy modeling and predicted clinically achievable levels. In addition, we optimized the PBMC isolation protocol for the clinical sites and showed that compound washout during the procedure did not diminish AZD4573-mediated effects. In another study, we mimicked potential clinical conditions (e.g. 30 min, 1h, and 2h between blood collection and PBMC isolation, ambient vs 37°C storage conditions) and demonstrated that significant ex vivo apoptosis (i.e. activation of cleaved caspase 3) was unlikely to occur if PBMCs were isolated within 1-2h of blood collection. Finally, by showing that AZD4573 target engagement and pathway inhibition can be measured in normal PBMCs, these assays will be informative for all patients, including those without circulating tumor cells (e.g. many lymphomas).Here we describe the development and characterization of a suite of pharmacodynamic assays for clinical use with blood samples for our CDK9 program. Preliminary data from our Phase I study demonstrate successful implementation of these assays and support that AZD4573 inhibits CDK9 with the expected downstream effects, in the clinical setting. Updated results will be presented at the meeting. DisclosuresYeh:AstraZeneca: Employment, Equity Ownership. O'Connor:AstraZeneca: Employment, Equity Ownership. Cidado:AstraZeneca: Employment, Equity Ownership. Barrett:AstraZeneca: Employment, Equity Ownership.

Two-person DNA mixture interpretation based on a novel set of SNP-STR markers

Autosomal short tandem repeats (STR) markers analysed by PCR and capillary electrophoresis (CE) represent the gold-standard for forensic DNA analysis. With the improved sensitivity of detection equipment, a larger number of mixed DNA profiles can be obtained from trace amounts of DNA that conventionally used to appear as a single source. More specifically, two-source DNA mixtures, comprising the victim’s and the perpetrator’s DNAs, are often encountered in forensic casework, where the victim’s DNA represents a major component of the mixture. Unfortunately, unbalanced two-person DNA mixtures with a ratio larger than 20:1 (here we have named this kind of mixture extremely unbalanced DNA mixture) provide limited information on the minor component. Although the development of probabilistic software has made interpretation of results from mixed DNA easier, high mixture ratios lead to an uninformative likelihood ratio (LR), considering the minor component. Therefore, a technique that can be performed on the conventional CE platform, while enhancing the ability to detect minor DNA in extremely unbalanced DNA mixtures, may be very useful in forensic casework. Our previous research has reported that SNP-STRs, in conjunction with a PCR technique based on amplification refractory mutation system (ARMS), can be used to resolve extremely unbalanced two-person DNA mixtures. To further explore the capacity of SNP-STR markers to help analyse such DNA mixtures, we developed 11 novel SNP-STR markers. The ARMS-based PCR was then used to design allele-specific primers, where each primer targeted one SNP allele located in the flanking region of the tandem repeats. This method allowed primers to specifically and selectively amplify minor DNA without interference from DNA of the major component because the selected SNP allele was not shared with the major contributor. A survey of the selected 11 SNP-STRs in a southwest Chinese Han population showed high levels of polymorphism. Assays on two-person DNA mixtures showed that all the allele-specific primers could target minor DNA even when the amount of major DNA was a 100-fold higher. Therefore, this novel set of SNP-STR markers provides an alternative method for the analysis of extremely unbalanced two-person DNA mixtures.