Deep dental phenotyping and a novel FAM20A variant in patients with amelogenesis imperfecta type IG.

OBJECTIVES: To identify etiologic variants and perform deep dental phenotyping in patients with amelogenesis imperfecta (AI). METHODS: Three patients of two unrelated families were evaluated. Genetic variants were investigated by exome and Sanger sequencing. An unerupted permanent third molar (AI1) from Patient1 and a deciduous first molar (AI2) from Patient2, along with three tooth-type matched controls for each were characterized. RESULTS: All three patients harbored biallelic pathogenic variants in FAM20A, indicating AI1G. Of the four identified variants, one, c.1231C > T p.(Arg411Trp), was novel. Patient1 possessed the largest deletion, 7531 bp, ever identified in FAM20A. In addition to hypoplastic enamel, multiple impacted teeth, intrapulpal calcification, pericoronal radiolucencies, malocclusion, and periodontal infections were found in all three patients, gingival hyperplasia in Patient1 and Patient2, and alveolar bone exostosis in Patient3. Surface roughness was increased in AI1 but decreased in AI2. Decreased enamel mineral density, hardness, and elastic modulus were observed in AI1 enamel and dentin and AI2 dentin, along with decreased phosphorus, increased carbon, and increased calcium/phosphorus and carbon/oxygen ratios. Severely collapsed enamel rods and disorganized dentin-enamel junction were observed. CONCLUSIONS: We report a novel FAM20A variant and, for the first time, the defective mineral composition and physical/mechanical properties of AI1G teeth.

C/EBPß and YY1 bind and interact with Smad3 to modulate lipopolysaccharide-induced amelotin gene transcription in mouse gingival epithelial cells.

Junctional epithelium (JE) develops from reduced enamel epithelium during tooth formation and is critical for the maintenance of healthy periodontal tissue through ensuring appropriate immune responses and the rapid turnover of gingival epithelial cells. We have previously shown a relationship between inflammatory cytokines and expression of JE-specific genes, such as amelotin (AMTN), in gingival epithelial cells. Here, we elucidated the effects of Porphyromonas gingivalis-derived lipopolysaccharide (Pg LPS) on Amtn gene transcription and the interaction of transcription factors. To determine the molecular basis of transcriptional regulation of the Amtn gene by Pg LPS, we performed real-time PCR and carried out luciferase assays using a mouse Amtn gene promoter linked to a luciferase reporter gene in mouse gingival epithelial GE1 cells. Gel mobility shift and chromatin immunoprecipitation assays were performed to identify response elements bound to LPS-induced transcription factors. Next, we analyzed protein levels of the LPS-induced transcription factors and the interaction of transcription factors by western blotting and immunoprecipitation. LPS increased Amtn mRNA levels and elevated luciferase activities of constructs containing regions between -116 and -238 of the mouse Amtn gene promoter. CCAAT/enhancer-binding protein (C/EBP) 1-, C/EBP2- and Ying Yang 1 (YY1)-nuclear protein complexes were increased by LPS treatment. Furthermore, we identified LPS-modulated interactions with C/EBPß, YY1 and Smad3. These results demonstrate that Pg LPS regulates Amtn gene transcription via binding of C/EBPß-Smad3 and YY1-Smad3 complexes to C/EBP1, C/EBP2 and YY1 response elements in the mouse Amtn gene promoter.

IL-1ß and TNF-α regulate mouse amelotin gene transcription in gingival epithelial cells.

Amelotin (AMTN) is an enamel protein expressed in maturation-stage ameloblasts and junctional epithelium. To clarify the transcriptional regulation of the AMTN gene by interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α), we conducted real-time PCR, Western blotting, transient transfection analyses with luciferase constructs including various lengths of the mouse AMTN gene promoter, and gel shift and chromatin immunoprecipitation assays using mouse gingival epithelial GE1 cells. The levels of AMTN mRNA and protein in GE1 cells were increased after 6 h of stimulation with IL-1ß (1 ng/mL) and TNF-α (10 ng/mL). IL-1ß and TNF-α induced luciferase activities of the constructs between -116AMTN and -705AMTN including the mouse AMTN gene promoter. Transcriptional activation by IL-1ß and TNF-α was partially inhibited in -460AMTN including 3-bp mutations in the CCAAT-enhancer-binding protein 1 (C/EBP1), C/EBP2 and Yin Yang 1 (YY1) elements. Transcriptional activities induced by IL-1ß and TNF-α were inhibited by tyrosine kinase, MEK1/2 and PI3-kinase inhibitors. Results of ChIP assays showed that IL-1ß and TNF-α increased C/EBPß and YY1 binding to the C/EBP1, C/EBP2 and YY1 elements. These results demonstrate that IL-1ß and TNF-α increase AMTN gene transcription via the C/EBP1, C/EBP2 and YY1 elements in the mouse AMTN gene promoter.

Genes Involved in the Enamel Development Are Associated with Calcium and Phosphorus Level in Saliva.

Saliva components play a crucial role in the integrity of the dental enamel and in caries susceptibility. The saliva characteristics are controlled by many factors, including genetic factors. Therefore, this study aimed to evaluate the association between the genetic variations in genes expressed in enamel development with calcium and phosphorus levels in saliva. We collected 276 unrelated 12-year-old children from private and public schools. Saliva was collected for DNA extraction from oral cells and for measurement of calcium and phosphorus. Inductively coupled plasma-mass spectrometry determined calcium and phosphorus levels in whole saliva. Fifteen genetic variations in 9 genes were analyzed. The genotype was determined by real-time polymerase chain reactions. Data were analyzed using Plink with an alpha of 5%. Genetic variations in AMELX, AMNB and ESRRB were associated with the calcium level in saliva (p < 0.05). A borderline association was observed in ENAM allele distribution shown with phosphate level in saliva (p = 0.049). In conclusion, our results are the first to report that genetic variations contribute to calcium and phosphorus levels in saliva.

Amelogenesis imperfecta and other biomineralization defects in Fam20a and Fam20c null mice.

The FAM20 family of secreted proteins consists of three members (FAM20A, FAM20B, and FAM20C) recently linked to developmental disorders suggesting roles for FAM20 proteins in modulating biomineralization processes. The authors report here findings in knockout mice having null mutations affecting each of the three FAM20 proteins. Both Fam20a and Fam20c null mice survived to adulthood and showed biomineralization defects. Fam20b (-/-) embryos showed severe stunting and increased mortality at E13.5, although early lethality precluded detailed investigations. Physiologic calcification or biomineralization of extracellular matrices is a normal process in the development and functioning of various tissues (eg, bones and teeth). The lesions that developed in teeth, bones, or blood vessels after functional deletion of either Fam20a or Fam20c support a significant role for their encoded proteins in modulating biomineralization processes. Severe amelogenesis imperfecta (AI) was present in both Fam20a and Fam20c null mice. In addition, Fam20a (-/-) mice developed disseminated calcifications of muscular arteries and intrapulmonary calcifications, similar to those of fetuin-A deficient mice, although they were normocalcemic and normophosphatemic, with normal dentin and bone. Fam20a gene expression was detected in ameloblasts, odontoblasts, and the parathyroid gland, with local and systemic effects suggesting both local and/or systemic effects for FAM20A. In contrast, Fam20c (-/-) mice lacked ectopic calcifications but were severely hypophosphatemic and developed notable lesions in both dentin and bone to accompany the AI. The bone and dentin lesions, plus the marked hypophosphatemia and elevated serum alkaline phosphatase and FGF23 levels, are indicative of autosomal recessive hypophosphatemic rickets/osteomalacia in Fam20c (-/-) mice.

Expression of phosphoproteins and amelotin in teeth.

The organic material of our teeth consists of collagens and a number of calcium-binding phosphoproteins. Six of these phosphoproteins have recently been grouped in the family of the SIBLINGs (small integrin-binding ligand, N-linked glycoproteins), namely osteopontin, bone sialoprotein, dentin matrix protein (DMP1), dentin sialophosphoprotein (DSPP), matrix extracellular phosphoglycoprotein (MEPE) and enamelin. We prepared a cDNA library from rat incisors in order to identify the genes involved in tooth formation. The library was screened by subtractive hybridization with two probes; one specific for teeth, the other for bone. We found that the vast majority of the clones from our library were expressed at similar levels in bone and teeth, demonstrating the close relationship of the two tissues. Only 7% of all the clones were expressed in a tooth-specific fashion. These included clones for the enamel proteins; amelotin, amelogenin, ameloblastin and enamelin; for the dentin proteins DSPP and DMP1; and for the intermediate filament protein cytokeratin 13. Several typical bone proteins, including collagen I, osteocalcin, alkaline phosphatase and FATSO, were also expressed at significantly higher levels in teeth than in bone, probably due to the extreme growth rate of rat incisors. The amino acid sequence of rat amelotin showed 62% identity with the sequence from humans. It was expressed considerably later than the other enamel proteins, suggesting that amelotin may serve a function different from those of amelogenin, ameloblastin and enamelin.

Porcine SPARC: isolation from dentin, cDNA sequence, and computer model.

Genes encoding the major enamel matrix proteins and non-collagenous proteins of bone and dentin are members of the secretory calcium-binding phosphoprotein (SCPP) family, which originated from ancestral SPARC (secreted protein, acidic and rich in cysteine; BM-40/osteonectin). To better understand the role of SPARC in mineralizing systems, we isolated SPARC from developing pig teeth, deduced its primary structure from the cDNA sequence, and determined its quaternary structure by homology modelling with reference to human SPARC crystal structures. The guanidine/EDTA extract from porcine dentin was fractionated by anion-exchange and size-exclusion chromatography. Stains-all positive bands at 38 and 35 kDa gave the N-terminal sequences APQQEALPDETEV and DFEKNYNMYIFPV, which corresponded to the SPARC N terminus and an internal region of the protein. Porcine SPARC contains 300 amino acids, including the 17-amino acid signal peptide, and shares 96.2% amino acid sequence identity with human SPARC. Without post-translational modifications, the 283-amino acid secreted protein has a molecular mass of 32.3 kDa. The three-dimensional model revealed that porcine SPARC contains a single N-linked glycosylation at N113, seven intramolecular disulfide bridges, and assembles into dimers. SPARC is composed of three structural/functional domains: an acidic Ca2+-binding, a follistatin-like, and an extracellular calcium-binding domain.

Identification of secreted and membrane proteins in the rat incisor enamel organ using a signal-trap screening approach.

The secretome represents the subset of proteins that are targeted by signal peptides to the endoplasmic reticulum. Among those, secreted proteins play a pivotal role because they regulate determinant cell activities such as differentiation and intercellular communication. In calcified tissues, they also represent key players in extracellular mineralization. This study was carried out to establish a secretome profile of rat enamel organ (EO) cells. A functional genomic technology, based on the signal trap methodology, was applied, starting with a library of 5'-enriched cDNA fragments prepared from rat incisor EOs. A total of 2,592 clones were analyzed by means of macroarray hybridizations and DNA sequencing. Ninety-four unique clones encoding a signal peptide were retrieved. Among those were 84 matched known genes, many not previously reported to be expressed by the EO. Most importantly, 10 clones were classified as being novel, with EO-009 identified as the rat homolog of human APin protein. These data indicate that many secreted and membrane-embedded EO proteins still remain to be identified, some of which may play crucial roles in regulating processes that create an optimal environment for the formation and organization of apatite crystals into a complex three-dimensional calcified matrix.

Transcription factor sumoylation and factor YY1 serve to modulate mouse amelogenin gene expression.

Amelogenin proteins are essential in the control of enamel biomineralization and the amelogenin gene therefore is spatiotemporally regulated to ensure proper amelogenin protein expression. In this study, we examined the role of sumoylation to alter CCAAT/enhancer-binding protein alpha (C/EBPalpha) activity, and performed a search using a protein/DNA array system for other proteins that act co-operatively with C/EBPalpha to alter amelogenin expression. We observed that C/EBPalpha was modified by sumoylation, and that this modification played an indirect inhibitory role on the regulation of C/EBPalpha activity which appeared to act through other transcription factors. The protein/DNA array allowed us to single out the transcription factor, YY1, which acts in the absence of direct DNA binding to repress both the basal amelogenin promoter activity and C/EBPalpha-mediated transactivation. Taken together, these pathways may account for part of the physiological modulation of the amelogenin gene expression in accordance with tooth developmental and enamel biomineralization requirements.

FAM20: an evolutionarily conserved family of secreted proteins expressed in hematopoietic cells.

BACKGROUND: Hematopoiesis is a complex developmental process controlled by a large number of factors that regulate stem cell renewal, lineage commitment and differentiation. Secreted proteins, including the hematopoietic growth factors, play critical roles in these processes and have important biological and clinical significance. We have employed representational difference analysis to identify genes that are differentially expressed during experimentally induced myeloid differentiation in the murine EML hematopoietic stem cell line. RESULTS: One identified clone encoded a previously unidentified protein of 541 amino acids that contains an amino terminal signal sequence but no other characterized domains. This protein is a member of family of related proteins that has been named family with sequence similarity 20 (FAM20) with three members (FAM20A, FAM20B and FAM20C) in mammals. Evolutionary comparisons revealed the existence of a single FAM20 gene in the simple vertebrate Ciona intestinalis and the invertebrate worm Caenorhabditis elegans and two genes in two insect species, Drosophila melanogaster and Anopheles gambiae. Six FAM20 family members were identified in the genome of the pufferfish, Fugu rubripes and five members in the zebrafish, Danio rerio. The mouse Fam20a protein was ectopically expressed in a mammalian cell line and found to be a bona fide secreted protein and efficient secretion was dependent on the integrity of the signal sequence. Expression analysis revealed that the Fam20a gene was indeed differentially expressed during hematopoietic differentiation and that the other two family members (Fam20b and Fam20c) were also expressed during hematcpoiesis but that their mRNA levels did not vary significantly. Likewise FAM20A was expressed in more limited set of human tissues than the other two family members. CONCLUSIONS: The FAM20 family represents a new family of secreted proteins with potential functions in regulating differentiation and function of hematopoietic and other tissues. The Fam20a mRNA was only expressed during early stages of hematopoietic development and may play a role in lineage commitment or proliferation. The expansion in gene number in different species suggests that the family has evolved as a result of several gene duplication events that have occurred in both vertebrates and invertebrates.

Design of a multiplex PCR for genotyping 16 short tandem repeats in degraded DNA samples.

The molecular genotyping of individuals and reconstruction of kinship through short and high polymorphic DNA markers, so-called short tandem repeats (STR), has become an important and efficient method in anthropology and forensic science. The here introduced experimental design describes a multiplex PCR capable of simultaneously amplifying 16 STRs and the sex determinant locus amelogenin in a short fragment lengths range from 84 bp to 275 bp. Thus, the design depends predominantly on the routines for DNA typing of historical samples with highly degraded ancient DNA. It is shown, that the newly designed multiplex PCR is suitable for successful typing of both forensic and historical material.

Polypeptides translated from alternatively spliced transcripts of the amelogenin gene, devoid of the exon 6a, b, c region, have specific effects on tooth germ development in culture.

Recombinant proteins have been produced from cDNAs corresponding to alternatively spliced transcripts comprised from exons 2,3,4,5,6d,7 and 2,3,5,6d,7 of the rat amelogenin gene. These peptides, designated as [A + 4] and [A - 4], respectively, induce embryonic muscle fibroblasts in culture in vitro to express proteins characteristic of the chondrogenic and osteogenic phenotypes, and in matrix-supported implants into rat muscle, in vivo, induce typical bone matrix proteins. The aim of the present work was to examine the potential role of these proteins on the development of odontogenic tissue. The lower first molars were collected from Charles River CD-1 mice at postnatal days 1 and 2 and were grown on semisolid, serum-free medium supplemented with ascorbic and retinoic acids and transferin. The peptides were added to the serum-free media at 10 ng/ml. As controls, the medium was either 20% fetal bovine serum or the supplemented serum-free medium without either amelogenin peptide. The tooth germs were cultured for 6 days, then fixed and paraffin embedded by standard procedures. The tissue blocks were serially sectioned and stained with hematoxylin-eosin (H&E), or antibodies to collagen 1 (Col1), phosphophoryn (DMP2), or cementum attachment protein (CAP). CAP, DMP2, and Col1 expression was enhanced by the addition of the amelogenin peptides, as compared to the 0% fetal bovine serum (FBS) controls, but the peptides showed different effects. Expression of DMP2, characteristic of dentin matrix, was upregulated by [A + 4], whereas CAP, characteristic of cementum, was upregulated by [A - 4]. Since the recombinant peptides are active, their corresponding tissue forms may be important in the stimulation of mesenchymal tissue differentiation. Thus, these specific amelogenin proteins may be involved in tooth morphogenesis.

The human tuftelin gene: cloning and characterization.

Tuftelin has been suggested to play an important role during the development and mineralization of enamel. We isolated the full-length human tuftelin cDNA using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (5' RACE and 3' RACE) methods. Sequence analysis of the tuftelin cDNA revealed an open reading frame of 1170 bp encoding a 390 amino acid protein with a molecular mass of 44.3 kDa and an isoelectric point of 5.7. The human tuftelin protein shares 89 and 88% amino acid sequence identity with the bovine and mouse tuftelin, respectively. It contains a coiled-coil region, recently reported to be involved with tuftelin self-assembly and with the interaction of tuftelin with TIP39 (a novel tuftelin interacting protein). Detailed DNA analysis of the cloned genomic DNA revealed that the human tuftelin gene contains 13 exons and is larger than 26 kb. Two alternatively spliced tuftelin mRNA transcripts have now been identified in the human tooth bud, one lacking exon 2, and the other lacking exon 2 and exon 3. Primer extension analysis, corroborated by RT-PCR and DNA sequencing, revealed multiple transcription initiation sites. The cloned 1.6 kb promoter region contained several GC boxes and several transcription factor binding sites such as those for activator protein 1 and stimulatory protein 1. Our blast search of the human and mouse expressed sequence tag data bases, as well as our RT-PCR and DNA sequencing results, and a previous study using Northern blot analysis revealed that tuftelin cDNA sequences are also expressed in normal and cancerous non-mineralizing soft tissues, suggesting that tuftelin has a universal function. We have now identified and characterized different alternatively spliced mouse tuftelin mRNAs in several non-mineralizing tissues. These results provide an important baseline for future understanding of the biological role of tuftelin.

Reconstruction of kinship by fecal DNA analysis of orangutans.

Genetic analysis is a useful tool for assigning biological relationships. Thus, it will improve genetic management of wild animal populations and breeding colonies. Kinship analysis will give new insights into the behavior, sociobiology and genetic management of orangutans. In this study, chromosomal DNA from orangutan (Pongo pygmaeus ssp.) was extracted from excrements. Feces samples were screened for up to nine microsatellite markers from related zoo populations of orangutans (Pongo pygmaeus ssp.) kept at the Zoological Garden Berlin and the Zoological Garden Heidelberg, Germany. Family structures are documented in the "International Studybook of the Orangutan" (Perkins 1995) and the "Europäisches Erhaltungszucht Programm 1998" (Becker 1998). To examine whether human short tandem repeat loci (STR) are suitable for the reconstruction of kinship in orangutans, nine STRs, commonly used in forensic studies and the amelogenin system, were amplified in a multiplex-PCR approach (AmpFlSTR Profiler Plus). We were able to show that five of the nine human autosomal STRs in question amplified successfully in orangutans. Thus, we could reconstruct kinship structures of the Berlin and Heidelberg populations.

Histological analysis and ancient DNA amplification of human bone remains found in caius iulius polybius house in pompeii.

Thirteen skeletons found in the Caius Iulius Polybius house, which has been the object of intensive study since its discovery in Pompeii 250 years ago, have provided an opportunity to study either bone diagenesis by histological investigation or ancient DNA by polymerase chain reaction analysis. DNA analysis was done by amplifying both X- and Y-chromosomes amelogenin loci and Y-specific alphoid repeat locus. The von Willebrand factor (vWF) microsatellite locus on chromosome 12 was also analyzed for personal identification in two individuals showing alleles with 10/11 and 12/12 TCTA repeats, respectively. Technical problems were the scarcity of DNA content from osteocytes, DNA molecule fragmentation, microbial contamination which change bone structure, contaminating human DNA which results from mishandling, and frequent presence of Taq DNA polymerase inhibiting molecules like polyphenols and heavy metals. The results suggest that the remains contain endogenous human DNA that can be amplified and analyzed. The amplifiability of DNA corresponds to the bone preservation and dynamics of the burial conditions subsequent to the 79 A.D. eruption.

Evolutionary analysis of "hagfish amelogenin".

Hagfishes lack mineralized tissues and teeth. Part of a cDNA strand, allegedly from amelogenin, the major gene involved in enamel formation in mammals, has recently been cloned in a hagfish (Slavkin and Diekwish, Anat. Rec., 1996;245:131-150). This cloning is of great interest because it could change the current view about the evolution of mineralized tissues, but no phylogenetic analysis of this piece of DNA has been made by the authors. Phylogenetic analysis of this part of cDNA has been conducted using both phenetic and cladistic methods. The cDNA amplified in hagfish does not fit with a nonmammalian origin but fits well with a degraded rodent sequence. The gene cloned in hagfish is probably of mammalian origin due to contamination during PCR.

Derived protein and cDNA sequences of hamster amelogenin.

Hamster enamel protein extracts were analyzed by RP-HPLC and the isolated fractions by SDS-and Western blotting using polyclonal antibodies against recombinant mouse amelogenin and anti-peptide antibodies against the mouse exon 4-encoded sequence. Total RNA was extracted from enamel organ epithelia and, using a 3' rapid amplification of cDNA ends (3' RACE) technique, the coding regions for three different amelogenin isoforms were cloned along with the 3' non-coding region. DNA sequencing revealed that the hamster amelogenin isoforms are 180, 73 and 59 amino acids in length, respectively. The 59-residue amelogenin corresponds to the leucine-rich amelogenin protein (LRAP), the 73-residue amelogenin corresponds to LRAP with the inclusion of the exon 4-encoded sequence, while the 180-residue amelogenin is the most abundant amelogenin isoform. Edman degradation was performed on purified hamster amelogenin, which provided the amino acid sequence in the region encoded by the 5' PCR amplification primer used in cloning. Therefore, the entire derived amino acid sequence of hamster amelogenin was revealed. The hamster amelogenin amino acid sequence was aligned with all its known homologues. Hamster differs from rat and mouse amelogenin at only three amino acid positions. Southern blot analysis using a panel of restriction enzymes gave the same pattern for hamster DNA obtained from males and females, suggesting that in hamster, as in mouse, amelogenin is expressed from a single gene located on the X chromosome.

Tuftelin--aspects of protein and gene structure.

The acidic enamel protein tuftelin has now been cDNA cloned, sequenced and characterized in a number of vertebrate species. Recently, the bovine tuftelin gene structure was elucidated. Cloning of the human tuftelin gene and partial sequencing of a number of exons have also been achieved. Immunologically, the protein has been shown to be conserved throughout 550 million years of vertebrate evolution. The gene has been localized to the long arm of the autosomal chromosome 1. The mapping of the human tuftelin gene to a well-defined cytogenetic region could be important in understanding the etiology of autosomally inherited amelogenesis imperfecta, the most common hereditary disease of enamel. The present paper reviews the primary structure, mRNA/cDNA structure, and gene structure of tuftelin. It describes its immunolocalization at the light microscope level and at the ultrastructural level in both the ameloblast cells and in the extracellular enamel matrix. The timing of tuftelin expression and its possible roles in enamel formation are discussed.

Infrared fluorescent detection of PCR amplified gender identifying alleles.

An automated DNA sequencer utilizing high sensitivity infrared (IR) fluorescence technology together with Polymerase Chain Reaction (PCR) methodology was used to detect several sex differentiating loci on the X and Y chromosomes from various samples often encountered in forensic case work. Amplifications of the X-Y homologous amelogenin gene, the alpha-satellite (alphoid) repeat sequences and the X and Y chromosome zinc finger protein genes ZFX and ZFY (ZFX/ZFY) were performed. DNA extracted from various forensic specimens was amplified using either Taq, Tth or ThermoSequenase. Multiplexing using primers for all three loci in one reaction tube was achieved using Tth and ThermoSequenase. Two IR labeling strategies for detection of PCR products were utilized. In the first strategy, one of the PCR primers contained a 19-base extension at its 5' end identical to an IR-labeled universal M13 Forward (-29) primer which was included in the amplification reactions. During PCR the tailed primer generates sequence complementary to the M13 primer which subsequently primes the initial amplification products, thereby generating IR-labeled PCR products. In the second strategy, dATP labeled with an IR dye (IR-dATP) was included in the amplification reaction. During amplification IR-dATP was utilized by the polymerase and incorporated into the synthesized DNA, thus resulting in IR-labeled PCR products. X and Y specific bands were readily detected using both labeling methodologies. Amplified products were electrophoretically resolved using denaturing Long-Ranger gels and detected with an automated detection system using IR laser irradiation. A separation distance of 15 cm allowed run times of less than 2 h from sample loading to detection. Because the gels could be run more than once, at least 120 samples (2 loads x 60 samples/load) can be typed using a single gel.