Integrated portable genetic analysis microsystem for pathogen/infectious disease detection.

An integrated portable genetic analysis microsystem including PCR amplification and capillary electrophoretic (CE) analysis coupled with a compact instrument for electrical control and laser-excited fluorescence detection has been developed. The microdevice contains microfabricated heaters, temperature sensors, and membrane valves to provide controlled sample positioning and immobilization in 200-nL PCR chambers. The instrument incorporates a solid-state laser and confocal fluorescence detection optics, electronics for sensing and powering the PCR reactor, and high-voltage power supplies for conducting CE separations. The fluorescein-labeled PCR products are amplified and electrophoretically analyzed in a gel-filled microchannel in <10 min. We demonstrate the utility of this instrument by performing pathogen detection and genotyping directly from whole Escherichia coli and Staphylococcus aureus cells. The E. coli detection assay consists of a triplex PCR amplification targeting genes that encode 16S ribosomal RNA, the fliC flagellar antigen, and the sltI shigatoxin. Serial dilution demonstrates a limit of detection of 2-3 bacterial cells. The S. aureus assay uses a femA marker to identify cells as S. aureus and a mecA marker to probe for methicillin resistance. This integrated portable genomic analysis microsystem demonstrates the feasibility of performing rapid high-quality detection of pathogens and their antimicrobial drug resistance.

High-performance multiplex SNP analysis of three hemochromatosis-related mutations with capillary array electrophoresis microplates.

An assay is described for high-throughput single nucleotide polymorphism (SNP) genotyping on a microfabricated capillary array electrophoresis (CAE) microchip. The assay targets the three common variants at the HFE locus associated with the genetic disease hereditary hemochromatosis (HHC). The assay employs allele-specific PCR (ASPCR) for the C282Y (845g->a), H63D (187c->g), and S65C (193a->t) variants using fluorescently-labeled energy-transfer (ET) allele-specific primers. Using a 96-channel radial CAE microplate, the labeled ASPCR products generated from 96 samples in a reference Caucasian population are simultaneously separated with single-base-pair resolution and genotyped in under 10 min. Detection is accomplished with a laser-excited rotary four-color fluorescence scanner. The allele-specific amplicons are differentiated on the basis of both their size and the color of the label emission. This study is the first demonstration of the combined use of ASPCR with ET primers and microfabricated radial CAE microplates to perform multiplex SNP analyses in a clinically relevant population.

High speed single nucleotide polymorphism typing of a hereditary haemochromatosis mutation with capillary array electrophoresis microplates.

A single nucleotide polymorphism (SNP) typing assay is developed and evaluated on a microfabricated capillary array electrophoresis system. Using fluorescently labeled allele-specific primers, the S65C (193A-->T) substitution associated with hereditary haemochromatosis in the HFE gene is genotyped. The covalently labeled polymerase chain reaction (PCR) products are separated on a microfabricated radial capillary array electrophoresis microplate using nondenaturing gel media in under two minutes. Detection is accomplished with a laser-excited rotary confocal scanner. The Rox-labeled A-allele specific amplicon (211 bp) is differentiated from the R110-labeled T-allele specific amplicon (201 bp) by both size and color. This study demonstrates the feasibility of using allele-specific PCR with covalently labeled primers for high speed fluorescent SNP typing on microfabricated radial capillary array electrophoresis microplates.

Human 18 kDa phosphotyrosine protein phosphatase (ACP1) polymorphism: studies of rare variants provide evidence that substitutions within or near alternatively spliced exons affect splicing result.

The mammalian low molecular weight phosphotyrosine protein phosphatase is expressed as two distinct isoforms. The human 'fast' and 'slow' isoforms differ only in the sequence of an internal segment of 34 residues, and the ACP1 gene contains two adjacent exons (E3F and E3S) which encode these segments. We have previously suggested that the fast and slow isoforms are generated by mutually exclusive pre-mRNA splicing of E3F and E3S. The common alleles ACP1*A, *B and *C express the fast and slow isoforms in different ratios. The *A and *C alleles differ from *B by C --> T transitions in E3S and E3F respectively. To test the idea that the fast : slow ratio is determined by nucleotide substitutions in the E3F-I3F-E3S region, four groups of rare ACP1 variants with unusual fast : slow ratios and the rare *E and *R alleles, expressing fast∶slow ratios similar to *C and *B, respectively, were analysed. Gene segments of the I2-I3S region were amplified by PCR and analysed by SSCP and variant bands were excised and sequenced. For each of the rare isozymic variants one of six different nucleotide substitutions in E3F (nts+42, +85, +109, +110), I3F (nt+1) and I3S (nt+8) was observed. The *E and *R alleles showed C and B sequence, respectively, in accordance with the fast : slow ratio. The results support the hypothesis that the fast : slow ratio is constitutive.

A mitochondrial control region and cytochrome b phylogeny of sika deer (Cervus nippon) and report of tandem repeats in the control region.

Sika deer (Cervus nippon Temminck) are endemic to mainland and insular Asia. Numerous subspecies have been named, but they are not quantitatively well defined. Portions of the mitochondrial cytochrome b gene (450 bp) and control region (512 bp) were sequenced from 28 individuals belonging to five sika subspecies and two Cervus elaphus subspecies. Phylogenetic trees constructed using these sequences clearly demonstrated that sika are monophyletic with respect to C. elaphus. A survey of variation in the control region showed that approximately half the variation occurred in a 100-base segment between positions 150 and 250 in the left domain of the control region. Within this region there were three tandemly repeated copies of a 39-base motif. In addition, two of the samples (C. n. aplodontus and C. n. hortulorum) contained, respectively, two and four additional copies of the repeated motif.

High-throughput genetic analysis using microfabricated 96-sample capillary array electrophoresis microplates.

Capillary array electrophoresis (CAE) microplates that can analyze 96 samples in less than 8 min have been produced by bonding 10-cm-diameter micromachined glass wafers to form a glass sandwich structure. The microplate has 96 sample wells and 48 separation channels with an injection unit that permits the serial analysis of two different samples on each capillary. An elastomer sheet with an 8 by 12 array of holes is placed on top of the glass sandwich structure to define the sample wells. Samples are addressed with an electrode array that makes up the third layer of the assembly. Detection of all lanes with high temporal resolution was achieved by using a laser-excited confocal fluorescence scanner. To demonstrate the functionality of these microplates, electrophoretic separation and fluorescence detection of a restriction fragment marker for the diagnosis of hereditary hemochromatosis were performed. CAE microplates will facilitate all types of high-throughput genetic analysis because their high assay speed provides a throughput that is 50 to 100 times greater than that of conventional slab gels.

Extent of heterogeneity in mitochondrial DNA of sub-Saharan African populations.

Variation in the mitochondrial DNA (mtDNA) control region as detected by sequence-specific oligonucleotide (SSO) probes is described for 381 individuals from nine sub-Saharan African populations. Population diversity estimates for SSO types ranged from 0.23 to 0.97, while 102 SSO types were detected, none of these types was shared by more than four populations. Eighteen types occurred in > or = 10% of individuals in some populations; of these, 11 were population-specific. One type occurred in 15% of the total sample, but was shared among only three populations. African SSO types were characterized by high frequencies of blank variants, indicating that there was additional variation present at the nucleotide sequence level in regions where SSO probes hybridize. Analyses of molecular variance (AMOVA) incorporating genetic distances between SSO types showed that 30% of the total variation was due to differences among populations, indicating that there is statistically significant heterogeneity (p < 0.001). An AMOVA on mtDNA control region nucleotide sequence data from 12 populations showed that including all additional variation present at the sequence level increased the variance due to population subdivision to 34% (p < 0.001). Overall, when considering both the low diversity within some populations and high heterogeneity among populations, SSO typing of mtDNA may not be a desirable forensic DNA typing method for continental African populations. Further mtDNA sampling of African-derived populations of North America should be carried out to determine how much of the continental African mtDNA variation is of forensic significance. However, the existence of extensive mtDNA control region nucleotide sequence variation in African populations means that control region sequencing is still appropriate in forensic cases requiring mtDNA analysis.

High-speed DNA genotyping using microfabricated capillary array electrophoresis chips.

Capillary array electrophoresis (CAE) chips have been designed and fabricated with the capacity to rapidly (< 160 s) analyze 12 different samples in parallel. Detection of all lanes with 0.3 s temporal resolution was achieved using a laser-excited confocal-fluorescence scanner. The operation and capabilities of these CAE microdevices were first determined by performing electrophoretic separations of pBR322 MspI DNA samples. Genotyping of HLA-H, a candidate gene for the diagnosis of hereditary hemochromatosis, was then performed to demonstrate the rapid analysis of biologically relevant samples. Two-color multiplex fluorescence detection of HLA-H genotypes was accomplished by prelabeling the standard pBR322 MspI DNA ladder with a red emitting bis-intercalation dye (butyl TOTIN) and on-column labeling of the HLA-H DNA with thiazole orange. This work establishes the feasibility of using CAE chips for high speed, high-throughput genotyping.

Evaluation of new primers for CSF1PO.

We describe new primers for the detection of the STR polymorphism at the CSF1PO locus. These primers have been designed to produce shorter amplicons (150-182 bp) than the primers in standard use (295-327 bp). The reliability of the new primers for CSF1PO typing has been demonstrated by testing on known samples and by sequence analysis. These primers are superior to the original primers with regard to electrophoretic resolution and utility for typing of severely degraded DNA.

DNA sequencing using a four-color confocal fluorescence capillary array scanner.

The design, construction and operation of a four-color capillary array electrophoresis scanner are presented. The use of sensitive energy transfer primers facilitates four-color detection of the DNA sequencing fragments following excitation at a single laser wavelength (488 nm). This scanner collects fluorescence data from up to 25 capillaries in parallel. The resulting four-color image files are automatically reduced to four-color line plots, and a base-calling program (Sax) is used to call the sequence. The performance of this system for DNA sequencing is demonstrated by examining twelve different motifs of the hypervariable region I of human mitochondrial (mt) DNA obtained from a Sierra Leone population.

High-resolution capillary array electrophoretic sizing of multiplexed short tandem repeat loci using energy-transfer fluorescent primers.

Short tandem repeat regions (STRs) from the polymorphic loci VWFA, THO1, TPO and CSF were amplified by the multiplex polymerase chain reaction (PCR) and analyzed by capillary array electrophoresis with fluorescence detection of energy transfer (ET) labels. The fluorescent ET primers are labeled with one fluorescein at the 5' end and a second fluorescein at the position of the 7th or 9th (modified) base to produce fragments that fluoresce in the green (lambda max = 525 nm). M13 A-track sequencing fragments, used as an internal sizing standard, were generated with a universal primer that has a donor fluorescein at the 5' end and a rhodamine acceptor at the position of the 11th (modified) base to produce fragments fluorescing in the red (> 590 nm). The labeled DNA fragments were excited at 488 nm, and the fluorescence was detected with a two-color confocal fluorescence scanner. Separations were performed on arrays of hollow fused silica capillaries filled with denaturing and replaceable hydroxyethyl cellulose sieving matrices. Separations were complete in less than 50 min, and single base resolution as well as reproducible STR sizing was achieved. The relative standard deviation in sizing was below 0.6%. This work establishes the feasibility of high-resolution, high-speed and high-throughput STR typing of single-stranded DNA fragments using capillary array electrophoresis.

Characterization of a recombinant that locates the hereditary hemochromatosis gene telomeric to HLA-F.

The gene responsible for hereditary hemochromatosis has been shown to be closely linked to the HLA-A and D6S105 loci on the short arm of chromosome 6. Efforts at mapping the disease gene have been hindered, however, by a lack of informative recombinant in this region. We have identified two recombinant individuals in a single affected family and have confirmed recombination by analysis of 16 polymorphic markers located near HLA-A and D6S105. One of the recombinants provides evidence for the location of the hemochromatosis gene telomeric to HLA-F.

Rapid sizing of short tandem repeat alleles using capillary array electrophoresis and energy-transfer fluorescent primers.

Genetic typing of the short tandem repeat (STR) polymorphism HUMTHO1 has been performed using capillary array electrophoresis and energy-transfer fluorescent dye-labeled polymerase chain reaction primers. Target alleles were amplified by use of primers labeled with one fluorescein at the 5' end and another fluorescein at the position of the 15th (modified) base to produce fragments that fluoresce in the green (lambda max = 525 nm). Unknown alleles were electrophoretically separated together with a standard ladder made up of alleles having 6, 7, 8, and 9 four-base pair repeats, each of which was amplified with an energy-transfer primer having a donor fluorescein at the 5' end and a rhodamine acceptor at the position of the 7th (modified) base to produce standard fragments fluorescing in the red (> 590 nm). Separations were performed on arrays of hollow fused-silica capillaries filled with a replaceable sieving matrix consisting of 0.8% hydroxyethyl cellulose plus 1 microM 9-aminoacridine to enhance the resolution. The labeled DNA fragments were excited at 488 nm, and the fluorescence was detected with a two-color confocal fluorescence scanner. Separations are complete in less than 20 min and allow sizing with an average absolute error or accuracy of less than 0.4 base pair and an average standard deviation of approximately 0.5 base pair with no correction for mobility shift and cross-talk between the fluorescence channels. This work establishes the feasibility of high-speed, high-throughput STR typing of double-stranded DNA fragments using capillary array electrophoresis.

Exon structure at the human ACP1 locus supports alternative splicing model for f and s isozyme generation.

The human ACP1 locus encodes a genetically polymorphic cytoplasmic low-molecular-weight acid phosphatase. Each of the common alleles encodes two isoforms, f and s. Both isozymes are of equal length (157 residues) but differ in sequence over an internal 34 residue segment. Substantial portions of the ACP1*A, *B and *C alleles common to Europeans have been sequenced. Six linearly positioned exons containing codons 14 to 157 were identified. Two exons of equal length (114bp) interspaced by a short (41bp), probably nonfunctional, intron encode the specific f and s segments, respectively. These findings strongly support an alternative RNA splicing hypothesis. In addition, three allele-specific base substitutions were encountered.

Sexual abuse of children. The detection of semen on skin.

OBJECTIVE: The detection of semen on the skin of children who present within 72 hours of an episode of sexual assault is critical to medical, forensic, and legal personnel. The Wood's Lamp, a UV light that causes semen to fluoresce, and four forensic laboratory techniques were compared to determine their sensitivity and decline in sensitivity over time. DESIGN: A descriptive study. PARTICIPANTS: Eleven adult female volunteers. MEASUREMENTS/MAIN RESULTS: Semen was placed on the skin of the volunteers. Samples of the dried semen were assessed during a 28-hour period with the Wood's Lamp, microscopy, the acid phosphatase assay, and two assays for the prostatic protein p30 (counterimmunoelectrophoresis and enzyme-linked immunosorbent assay). The intensity of the Wood's Lamp fluorescence of semen diminished dramatically by 28 hours; in contrast, the fluorescence of urine persisted up to 80 hours. Over time, the p30-enzyme-linked immunosorbent assay technique was more sensitive than microscopy, the acid phosphatase assay, and p30-counterimmunoelectrophoresis in detecting semen on skin. CONCLUSIONS: The Wood's Lamp is not a sensitive screening tool and should be used with caution. To improve the detection of sexual abuse in children, we recommend that the p30-enzyme-linked immunosorbent assay be used because of its potential as a more sensitive assay than those in current clinical use.

Human red cell acid phosphatase (ACP1). The amino acid sequence of the two isozymes Bf and Bs encoded by the ACP1*B allele.

The pair of isozymes, Bf and Bs, encoded by the human red cell acid phosphatase ACP1*B allele has been sequenced. Similar but not identical primary structures were observed. Both isozymes consist of a single peptide chain of 157 amino acid residues, which is acetylated at the amino-terminal alanine residue. The Bf and Bs isozymes are not glycosylated, and the calculated molecular masses are 17,932 and 17,867 Da, respectively. They are identical except for the sequence segment 40-73, which is peculiar to the respective isozyme. This is consistent with our hypothesis that the two isozymes are generated as the result of alternative splicing of the primary RNA transcript. The finding of a signature sequence offers the basis for the characteristic differences in catalytic and molecular properties of the Bf and Bs isozymes. A high degree of homology was found between the Bs isozyme and the 18-kDa cytosolic acid phosphatase from bovine liver. No homology was observed with other sequenced proteins, and this establishes these low molecular weight acid phosphatases as products of a distinct gene family.

Analysis of genetic markers in forensic DNA samples using the polymerase chain reaction.

The ability to extract and type DNA from forensic evidentiary samples has revolutionized the field of forensic serology. Previously, genetic marker typing was limited to the analysis of blood group markers and soluble polymorphic protein markers. Because the number of suitable markers expressed in particular fluids and tissues is relatively small, and because mixtures of fluids cannot be separated for conventional genetic marker typing, a suspect frequently cannot be included or excluded as a fluid donor in a case. However, the development of methods to extract DNA from virtually all biological specimens has greatly expanded the potential for individual identification. Of particular importance was the ability to extract mixtures of sperm cells and epithelial cells found in sexual assault cases such that the DNA from the sperm cells could be typed independently of the DNA from the victim's epithelial cells. Restriction fragment length polymorphism (RFLP) analysis was the first DNA-based method applied to problems of individual identification. This method, while powerful in its ability to differentiate individuals, is limited by the quantity and quality of DNA required for an unambiguous result and by the amount of time it takes to obtain a result. Despite these limitations, several laboratories are using RFLP analysis successfully for the detection of polymorphisms in forensic DNA case samples. While the field of forensic serology was being revolutionized by the prospect of DNA analysis, the field of molecular biology was being revolutionized by the invention of the polymerase chain reaction (PCR), which ultimately has had an impact on every area of biological science. The PCR DNA amplification technology is ideally suited for the analysis of forensic DNA samples in that it is sensitive and rapid and not as limited by the quality of DNA as the RFLP method. The focus of this article is the use of the PCR for typing genetic markers, and we will address specifically the special considerations that arise from applying DNA amplification and typing technology to forensic materials.

Seminal plasma protein p30: simplified purification and evidence for identity with prostate specific antigen.

A rapid high yield purification scheme is described for the major 30,000 molecular weight glycoprotein (p30) in human seminal plasma. Immunological and protein sequence evidence demonstrates the identity of this protein with prostate specific antigen (PSA) and gamma-seminoprotein (gamma SM).