We have used two new dye sets for automated dye-labeled terminator DNA sequencing. One set consists of four, 4,7-dichlororhodamine dyes (d-rhodamines). The second set consists of energy-transfer dyes that use the 5-carboxy-d-rhodamine dyes as acceptor dyes and the 5- or 6-carboxy isomers of 4'-aminomethylfluorescein as the donor dye. Both dye sets utilize a new linker between the dye and the nucleotide, and both provide more even peak heights in terminator sequencing than the dye-terminators consisting of unsubstituted rhodamine dyes. The unsubstituted rhodamine terminators produced electropherograms in which weak G peaks are observed after A peaks and occasionally C peaks. The number of weak G peaks has been reduced or eliminated with the new dye terminators. The general improvement in peak evenness improves accuracy for the automated base-calling software. The improved signal-to-noise ratio of the energy-transfer dye-labeled terminators combined with more even peak heights results in successful sequencing of high molecular weight DNA templates such as bacterial artificial chromosome DNA.

Genotyping, as applied to linkage mapping, human identification, or mapping of genetic traits, mandates electrophoretic separation systems that enable a user to identify alleles with high precision to obtain a correct genotype. For 2-bp microsatellites or short tandem repeats (STRs), standard deviations of ±0.3 nucleotide are required to ensure with 99.7% probability the identity or dissimilarity of tested alleles. A complete system, consisting of commercially available laser-induced fluorescence capillary electrophoresis (ABI PRISM 310) and performance optimized polymer 4 (POP-4), was evaluated for microsatellite separations. POP-4 is a low viscosity polymer for use in uncoated fused microbore silica capillaries. It separates DNA fragments that differ in size by 1 nucleotide up to 250 nucleotides and that differ in size by 2 nucleotides for fragments up to at least 350 nucleotides in length in about 30 min. The presence of denaturants and, more importantly, operation at 60°C was mandatory for high-precision and high-resolution sizing operation. Reproducible separation performance was achieved in excess of 100 injections per capillary with resulting standard deviations in the range of 0.04 to 0.17 nucleotide. Comparative sizing of known CEPH (Centre d'Etudes du Polymorphisme Humaine) samples performed at 22 independent test sites showed the usefulness of the system for genotyping with standard deviations of 0.24 nucleotide, or better.

Automated fluorescence analysis of polymerase chain reaction (PCR)-amplified short tandem repeat (STR) systems by capillary electrophoresis (CE) is becoming an established tool both in forensic casework and in the implementation of both state and national convicted offender DNA databases. A new capillary electrophoresis instrument, the ABI Prism 310 Genetic Analyzer, along with the Performance Optimized Polymer 4 (POP-4) provides an automated and precise method for simultaneously analyzing ten fluorescently labeled STR loci from a single PCR amplification kit, which provides a power of discrimination of approximately one in five billion from a single PCR amplification. Data are presented on sizing precision, sizing accuracy, and resolution for the STR loci in the AmpFlSTR Profiler kit. Sizing accuracy is highly dependent on the electrophoresis system, and therefore the reporting of alleles based on the nucleotide size obtained from an electrophoresis system is not recommended for forensic work. The precision of the 310 capillary electrophoresis system, coupled with software developed for automated genotyping of alleles based on the use of an allelic ladder, allows for accurate genotyping of STR loci. Sizing precision of < or = 0.16 nucleotide standard deviation was obtained with this system, thus allowing for accurate genotyping of length variants that differ in length by a single nucleotide.

Interpolation algorithms can be developed to size unknown single-stranded (ss) DNA fragments based on their electrophoretic mobilities, when they are compared with the mobilities of standard fragments of known sizes; however, sequence-specific anomalous electrophoretic migration can affect the accuracy and precision of the called sizes of the fragments. We used the anomalous migration of ssDNA fragments to optimize denaturation conditions for capillary electrophoresis. The capillary electrophoretic system uses a refillable polymer that both coats the capillary wall to suppress electro-osmotic flow and acts as the sieving matrix. The addition of 8 M urea to the polymer solution, as in slab gel electrophoresis, is insufficient to fully denature some anomalously migrating ssDNA fragments in this capillary electrophoresis system. The sizing accuracy of these fragments is significantly improved by the addition of 2-pyrrolidinone, or increased capillary temperature (60 degrees C). the effect of these two denaturing strategies is additive, and the best accuracy and precision in sizing results are obtained with a combination of chemical and thermal denaturation.

A fertilization antigen, FA-1, was purified from either deoxycholate-or lithium diiodosalicylate-solubilized murine testes by immunoaffinity chromatography using a monoclonal antibody, MA-24, which inhibited fertilization in vitro. The FA-1 was recovered at high (11.4) or low (2.8) pH using stepwise elution procedures of the deoxycholate or lithium diiodosalicylate extracts, respectively. Both of these fractions showed a single band of 47 kDa when analyzed by NaDodSO4/PAGE and silver staining. Following removal of the detergent and extensive dialysis at pH 5.8 or treatment with 0.15 M NaCl, even in the presence of detergent, a monomer of 23 kDa was detected. Two-dimensional PAGE of FA-1 showed, four or five polypeptides in the 47-kDa or 23-kDa range. The dialyzed FA-1 contained a major 23-kDa and a minor 48-kDa band when separated on both sucrose and cesium chloride gradients. High performance size-exclusion chromatography showed a major peak at 23 kDa and a minor peak at 50 kDa. Further analysis of the 23-kDa peak by reverse-phase chromatography resolved the antigen into three peaks, which gave similar two-dimensional gel patterns as the native FA-1. Lectin affinity chromatography on a lens culinaris column demonstrated that a part of the antigen was bound to the lectin while the rest was not. The FA-1 revealed a positive reaction with periodic-Schiff reagent and contained glucose and mannose, which together constituted 18.8% of the total antigen mass. Amino acid analysis showed a high percentage of aspartic acid, glutamic acid, serine, and glycine. As a single injection of MA-24 significantly reduced fertilization rates in vivo, the purified FA-1 is an attractive candidate for the development of contraceptive vaccine.Immunization of male and female animals of various species with extracts of sperm or mature testes results in a significant inhibition of fertility (1, 2). The whole sperm or testes cannot be used for immunoregulation of fertility due to the presence of numerous antigens on the germ-cell surface that could be shared with other somatic tissues (3,4). The utility of any antigen as a contraceptive vaccine is contingent upon its tissue-specificity, its involvement in fertilization and fertility, and its homogeneity. A few sperm-specific antigens have been purified to homogeneity and characterized, and some of them are relevant to fertility. Immunization with lactate dehydrogenase-X (LDH-C4) resulted in inhibition of fertility (5, 6), and antiserum to rabbit sperm autoantigen (RSA-1) also showed a reduction in fertility (7).We reported a monoclonal antibody, MA-24, developed against human spermatozoa that inhibited in vitro fertilization of murine oocytes by mouse sperm and zona-free hamster ova penetration by human sperm without agglutinating or immobilizing spermatozoa (8). MA-24 was directed against the 23-kDa antigen of human germ-cell plasma membranes, and the fertilization antigen FA-1 was isolated using immunoaffinity chromatography from human testes and sperm. Interestingly, FA-1 cross-...