Amino acid pattern in the liver and blood of fattening turkeys suffering from hepatic lipidosis.

Hepatic lipidosis (HL) is a well-known disease in fattening and in parent turkey flocks. Among others, dietary effects like (a lack of) essential amino acids (AA) as lipotrophic factors (e.g., methionine) have been considered as potentially predispositing for HL. Several studies have reported abnormal AA profiles in hepatic diseases of humans and other livestock. The ratio of branched-chain amino acids (BCAA) to aromatic amino acids (AAA) in plasma is used to predict hepatic cirrhosis. In this study, the state of supply of AA was investigated by comparing non-affected (NA) animals and those affected by HL. The AA pattern in the liver and blood can provide potential indications of pathogenesis of HL. In cooperation with German poultry veterinarians, 3 cases of HL on 3 different fattening turkey farms were visited (13/14 wk old, "B.U.T. Big 6" and "TP7"). Overall, 73 birds were examined, of which 42 birds suffered from HL and 31 were not affected. Feeding samples of the respective actual feed were taken and analyzed. The selection of animals was carried out (NA randomly) by clinical signs such as apathy and dyspnea and the diagnosis was made at necropsy, which could be confirmed by crude fat content in liver tissue (HL: 309, NA: 155). In liver tissue, the CP and AA contents were lower among animals with HL than among NA (P < 0.05). In blood samples, the sum of AA, ammonia, and urea was more than 3 times higher among animals with HL (431 mg/dL serum) than among NA (114 mg/dL serum; P < 0.01). The ratio of BCAA to AAA was also significantly different between the groups (HL: 0.85, NA: 1.42; P < 0.05). In the case of HL, entire herds were not affected and the "non-affected" ones were comparable with healthy slaughtered animals. There seems to be a clear change in protein and AA metabolism of HL animals, which could lead to an optimization in feeding practice in repeated cases of HL.

Time-resolved fluorescence imaging of slab gels for lifetime base-calling in DNA sequencing applications.

A compact time-resolved near-IR fluorescence imager was constructed to obtain lifetime and intensity images of DNA sequencing slab gels. The scanner consisted of a microscope body with f/1.2 relay optics onto which was mounted a pulsed diode laser (repetition rate 80 MHz, lasing wavelength 680 nm, average power 5 mW), filtering optics, and a large photoactive area (diameter 500 microns) single-photon avalanche diode that was actively quenched to provide a large dynamic operating range. The time-resolved data were processed using electronics configured in a conventional time-correlated single-photon-counting format with all of the counting hardware situated on a PC card resident on the computer bus. The microscope head produced a timing response of 450 ps (fwhm) in a scanning mode, allowing the measurement of subnano-second lifetimes. The time-resolved microscope head was placed in an automated DNA sequencer and translated across a 21-cm-wide gel plate in approximately 6 s (scan rate 3.5 cm/s) with an accumulation time per pixel of 10 ms. The sampling frequency was 0.17 Hz (duty cycle 0.0017), sufficient to prevent signal aliasing during the electrophoresis separation. Software (written in Visual Basic) allowed acquisition of both the intensity image and lifetime analysis of DNA bands migrating through the gel in real time. Using a dual-labeling (IRD700 and Cy5.5 labeling dyes)/two-lane sequencing strategy, we successfully read 670 bases of a control M13mp18 ssDNA template using lifetime identification. Comparison of the reconstructed sequence with the known sequence of the phage indicated the number of miscalls was only 2, producing an error rate of approximately 0.3% (identification accuracy 99.7%). The lifetimes were calculated using maximum likelihood estimators and allowed on-line determinations with high precision, even when short integration times were used to construct the decay profiles. Comparison of the lifetime base calling to a single-dye/four-lane sequencing strategy indicated similar results in terms of miscalls, but reduced insertion and deletion errors using lifetime identification methods, improving the overall read accuracy.

Infrared fluorescent detection of D1S80 alleles from blood and body fluid collected on IsoCode devices.

A genetic locus D1S80 (pMCT 118) containing variable number of tandem repeats (VNTR) has been used extensively in forensic analysis and paternity testing. In the current research, DNA was isolated from blood saliva and nasal secretions collected on two types of IsoCode paper-based devices. The D1S80 locus was amplified using PCR technology, and the alleles were separated by gel electrophoresis and then detected using an infrared (IR) fluorescence automated DNA sequencer. IR-labeled amplification products were generated from human genomic DNA using oligonucleotide primers, which were covalently linked to an infrared fluorescent dye (IRD41) at the 5' end. This system combines IR fluorescence chemistry and laser technology, thus eliminating the need for post-electrophoretic gel handling for the detection of the alleles. Real-time detection after separation of the alleles is valuable for visualization of the data. The VNTR alleles are displayed as familiar autoradiogram-like images, which can also be analyzed by computer. Since DNA is eluted from the IsoCode devices only with sterile distilled water and without time-consuming methods of extraction, amplification can be performed from numerous samples within a short period of time.

An infrared fluorescent dATP for labeling DNA.

Near-infrared fluorescence provides a nonradioactive method of detection with high sensitivity and low background. An infrared fluorophore has been attached covalently to the nucleotide deoxyadenosine triphosphate (dATP) to provide a reagent for enzymatic labeling of various types of DNA molecules and for facilitating their detection with an automated DNA sequencing and analysis system. DNA sequencing reaction products can be labeled internally by performing limited polymerization utilizing infrared-labeled dATP (IR-dATP) as the sole source of adenine deoxynucleotide prior to a dideoxy-specific termination reaction. PCR products can be labeled fluorescently by the addition of limited quantities of IR-dATP to the amplification reaction. This latter strategy has been utilized for detection of short tandem repeat polymorphisms (STRPs) which are useful for gene mapping, genetic diagnostics, forensic analysis, and paternity testing. Restriction fragments can be labeled also by fill-in reactions of appropriate 5' overhangs. Diminutive amounts of such fluorescently labeled DNA molecules can be visualized rapidly and conveniently using infrared detection technology.

Analysis of tyrosinase gene mutations using direct automated infrared fluorescence DNA sequencing of amplified exons.

The ability to correctly diagnose the molecular cause of genetic diseases is becoming increasingly important in medicine. This requires an efficient method for the analysis of the DNA sequence of specific genes and the detection of mutations in affected individuals. We report a method to determine the mutations responsible for tyrosinase related albinism (OCA1) using a combination of polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) analysis and direct DNA cycle sequencing using fluorescently labeled oligonucleotides and an automated DNA sequencer based on infrared fluorescence technology. This method allows DNA from several individuals to be sequenced quickly and simultaneously so that the specific location of each mutation and the carrier status of family members can be determined.

Continuous, on-line DNA sequencing using a versatile infrared laser scanner/electrophoresis apparatus.

A new apparatus for continuously detecting fluorescently labeled DNA fragments is based on infrared fluorescence technology. This technology combines state-of-the-art developments in chemistry, laser technology, and detection, while achieving improved reliability, sensitivity, and flexibility for applications including DNA sequencing. DNA molecules labeled with a novel infrared fluorophore are detected during electrophoresis using a scanning infrared fluorescence microscope. The microscope consists of a laser diode for exciting the fluorophore and a silicon avalanche photodiode for detecting the infrared emission. Optimum conditions for detection and throughput are obtained by adjusting electrophoresis, scanning and imaging parameters. Typical DNA sequencing runs (test templates) allow identification of over 500 bases per sample with greater than 99% accuracy.

Continuous, on-line DNA sequencing using oligodeoxynucleotide primers with multiple fluorophores.

A method for sequencing DNA by using a difluoresceinated primer and laser excitation is described. Dideoxy protocols have been determined that provide sequences for 600 bases starting with base 1 with less than 1% error in a single load. Electrophoresis is at 20 W and the bands are detected 24 cm from the bottom of the loading well with a scanning fluorescence detector. Bands are imaged on a TV screen in two dimensions. The sequences can be read from the TV screen manually or semiautomatically by using a simple software program. The system allows more bases to be read with a lower error rate than any other reported automated sequencing method.