Developmental validation of Applied Biosystems YFiler Platinum Casework PCR Amplification Kit.

The YFiler Platinum Casework PCR Amplification Kit is a 6-dye multiplex assay that simultaneously amplifies a set of 38 male-specific, Y-chromosome Short Tandem Repeat (YSTR) markers (DYS576, DYS389I, DYS635, DYS389II, DYS627, DYS549, DYS593, DYS645, DYS460, DYS458, DYS19, YGATAH4, DYS448, DYS391, DYS557, DYS522, DYS456, DYS390, DYS438, DYS392, DYS518, DYS444, DYS533, DYS570, DYS437, DYS385, DYS449, DYS643, DYS596, DYS393, DYS439, DYS481, DYF387S1, DYS527, DYS447), three insertion/deletion polymorphic markers (Yindels: rs771783753, rs759551978, rs199815934), and an internal quality control (IQC) system. When compared to the YFiler Platinum PCR Amplification kit for database samples, YFiler Platinum Casework kit was developed to include an improved Primer Mix incorporating a brighter TED dye, an updated internal quality control system, better resolution of large DNA fragments in Applied Biosystems POP-4 Polymer, and reduced female DNA cross-reactivity. Here, we report the results of the developmental validation study which followed the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines and includes data for PCR-based studies, sensitivity, species specificity, stability, precision, reproducibility and repeatability, population concordance, stutter, DNA mixtures, and performance on mock casework samples. The results validate the multiplex design as well as demonstrate the kit's robustness, reliability, and suitability as an assay for human identification with casework DNA samples.

Developmental validation of VeriFiler™ Plus PCR Amplification Kit: A 6-dye multiplex assay designed for casework samples.

The VeriFiler™ Plus PCR Amplification Kit is a 6-dye multiplex assay that simultaneously amplifies a set of 23 autosomal markers (D3S1358, vWA, D16S539, CSF1PO, D6S1043, D8S1179, D21S11, D18S51, D5S818, D2S441, D19S433, FGA, D10S1248, D22S1045, D1S1656, D13S317, D7S820, Penta E, Penta D, TH01, D12S391, D2S1338, and TPOX), a quality indicator system, and two sex-identification markers. Combined, the markers satisfy the requirements of the Chinese National autosomal DNA database as well as expanded CODIS (Combined DNA Index System). The VeriFiler Plus kit was developed with an improved Master Mix which incorporates the brighter TED™ dye, and accommodates a higher sample loading volume thus allowing for increased sensitivity and enabling maximum information recovery from challenging casework samples including touch, degraded, and inhibited samples. Here, we report the results of the developmental validation study which followed the SWGDAM (Scientific Working Group on DNA Analysis Methods) guidelines and includes data for PCR-based studies, sensitivity, species specificity, stability, precision, reproducibility and repeatability, concordance, stutter, DNA mixtures, and performance on mock casework samples. The results validate the multiplex design as well as demonstrate the kit's robustness, reliability, and suitability as an assay for human identification with casework DNA samples.

Developmental validation of the Huaxia™ Platinum PCR amplification kit: A 6-dye multiplex direct amplification assay designed for Chinese reference samples.

The Huaxia™ Platinum Kit for short tandem repeat (STR) amplification was designed to meet the needs of the rapidly growing Chinese forensic database. This PCR multiplex allows simultaneous amplification of the following autosomal loci: D3S1358, vWA, D16S539, CSF1PO, TPOX, D8S1179, D21S11, D18S51, Penta E, D2S441, D19S433, TH01, FGA, D22S1045, D5S818, D13S317, D7S820, D6S1043, D10S1248, D1S1656, D12S391, D2S1338, Penta D and the gender-identification markers Yindel, and AMEL. The Huaxia™ Platinum Kit enables direct amplification from blood and buccal samples stored on treated and untreated paper, and features an optimized PCR protocol that yields time to results in less than 45 min. Developmental validation testing followed SWGDAM guidelines and demonstrated that this assay produces reproducible and accurate results. Studies on 798 individuals in 4 major Chinese ethnic groups produced highly concordant results with other commercially available STR genotyping kits. The validation results demonstrate that the Huaxia™ Platinum Kit is a robust and reliable identification system for forensic DNA databasing applications.

Developmental validation of the GlobalFiler(®) Express PCR Amplification Kit: A 6-dye multiplex assay for the direct amplification of reference samples.

In order to increase the power of discrimination, reduce the possibility of adventitious matches, and expand global data sharing, the CODIS Core Loci Working Group made a recommendation to expand the CODIS core loci from the "required" 13 loci to 20 plus three additional "highly recommended" loci. The GlobalFiler(®) Express Kit was designed to incorporate all 20 required and 3 highly recommended loci along with a novel male-specific Y insertion/deletion marker. The GlobalFiler(®) Express Kit allows simultaneous amplification of the following loci: D3S1358, vWA, D16S539, CSF1PO, TPOX, Yindel, AMEL, D8S1179, D21S11, D18S51, DYS391, D2S441, D19S433, TH01, FGA, D22S1045, D5S818, D13S317, D7S820, SE33, D10S1248, D1S1656, D12S391, and D2S1338. The kit enables direct amplification from blood and buccal samples stored on paper or swab and the chemistry features an optimized PCR protocol that yields time to results in less than an hour. Developmental validation testing followed SWGDAM guidelines and demonstrated the quality and robustness of the GlobalFiler(®) Express Kit over a number of variables. The validation results demonstrate that the 24-locus multiplex kit is a robust and reliable identification assay as required for forensic DNA typing and databasing.

Synthesis and hepatic transport of strongly fluorescent cholephilic dipyrrinones.

A new class of highly fluorescent (phi(F) 0.3-0.8) low molecular weight water-soluble cholephilic compounds has been synthesized in two steps from dipyrrinones. The dipyrrinone nitrogens are first bridged by reaction with 1,1'-carbonyldiimidazole to form an N,N'-carbonyldipyrrinone (3H,5H-dipyrrolo[1,2-c:2',1'-f]pyrimidine-3,5-dione) nucleus, and a sulfonic acid group is then introduced at C(8) by reaction with concd H(2)SO(4). The resulting sulfonated N,N'-carbonyl-bridged dipyrrinones ("sulfoglows") are isolated as their sodium salts. When the alkyl substituents of the lactam ring are lengthened from ethyl to decyl, sulfoglows become increasingly lipophilic while maintaining water solubility. Low molecular weight sulfoglows were rapidly excreted intact in both bile and urine after intravenous infusion into rats, but higher molecular weight sulfoglows were excreted more selectively in bile. Hepatobiliary excretion of sulfoglows was partially, but not completely, blocked in mutant rats deficient in the multidrug-resistance associated transport protein Mrp2 (ABCC2). These observations point to the feasibility of developing simple sulfoglows with clinical diagnostic potential that are normally excreted in bile but appear in urine when hepatic elimination is impaired by cholestatic liver disease.

Lifelong elimination of hyperbilirubinemia in the Gunn rat with a single injection of helper-dependent adenoviral vector.

Crigler-Najjar syndrome is a recessively inherited disorder characterized by severe unconjugated hyperbilirubinemia caused by a deficiency of uridine diphospho-glucuronosyl transferase 1A1. Current therapy relies on phototherapy to prevent kernicterus, but liver transplantation presently is the only permanent cure. Gene therapy is a potential alternative, and recent work has shown that helper-dependent adenoviral (HD-Ad) vectors, devoid of all viral coding sequences, induce prolonged transgene expression and exhibit significantly less chronic toxicity than early-generation Ad vectors. We used a HD-Ad vector to achieve liver-restricted expression of human uridine diphospho-glucuronosyl transferase 1A1 in the Gunn rat, a model of the human disorder. Total plasma bilirubin levels were reduced from >5.0 mg/dl to <<1.4 mg/dl for >2 yr after a single i.v. administration of vector expressing the therapeutic transgene at a dose of 3 x 10(12) viral particles per kg. HPLC analysis of bile from treated rats showed the presence of bilirubin glucuronides at normal WT levels >2 yr after one injection of vector, and i.v. injection of bilirubins IIIalpha and XIIIalpha in the same animals revealed excess bilirubin-conjugating capacity. There was no significant elevation of liver enzymes (alanine aminotransferase) and only transient, moderate thrombocytopenia after injection of the vector. A clinically significant reduction in serum bilirubin was observed with a dose as low as 6 x 10(11) viral particles per kg. We conclude that complete, long-term correction of hyperbilirubinemia in the Gunn rat model of Crigler-Najjar syndrome can be achieved with one injection of HD-Ad vector and negligible chronic toxicity.

Hepatobiliary excretion of dipyrrinone sulfonates in Mrp2-deficient (TR(-)) rats.

The biliary excretion of the sodium salts of 8-(2-ethanesulfonic acid)-3-ethyl-2,7,9-trimethyl-1,10-dihydro-11H-dipyrrin-1-one (xanthosulfonic acid) and a fluorescent analogue (8-desethyl-N,N'-carbonyl-kryptopyrromethenone-8-sulfonic acid) was compared in Mrp2-deficient (TR(-)) and normal rats. Both organic anions were excreted rapidly in bile in Mrp2-deficient rats, but the biliary excretion of the fluorescent sulfonate was impaired relative to normal controls. The rat clearly has efficient Mrp2-independent mechanisms for biliary efflux of these anions that are not used by bilirubin or its mono- and diglucuronides.

Hepatobiliary excretion of biliverdin isomers and C10-substituted biliverdins in Mrp2-deficient (TR(-)) rats.

Multidrug resistance protein 2 (Mrp2) is considered the major mammalian membrane transporter of non-bile salt organic anions from liver to bile. Using Mrp2-deficient rats, we show that the protein is not essential for biliary excretion of biliverdin, its IIIalpha and XIIIalpha isomers, mesobiliverdin XIIIalpha or biliverdins bearing bulky lipophilic groups that are not reduced by biliverdin reductase in vivo. Yet, Mrp2 deficiency does retard the biliary excretion of these verdins to different degrees. The data indicate that there are Mrp2-independent mechanisms in the rat for biliary excretion of dicarboxylate organic anions related to biliverdin.