Colposacropexy: an alternative technique

Colposacropexy procedures restore anatomically correct apical vaginal support on the levator plate at the ischial spine level. Venous hemorrhage resulting from laceration of presacral veins during suture fixation is the major hazard of this procedure. Titanium orthopedic bone anchor fixation minimizes this risk through precision placement of the bone anchor-suture unit.     

Membrane anchoring: an alternative technique

A technique for stabilizing membranes used in guided tissue regeneration is described. The technique does not require the purchase of new armamentaria or the acquisition of new skills.     

Transcriptional sequencing: A method for DNA sequencing using RNA polymerase

We have developed a sequencing method based on the RNA polymerase chain termination reaction with rhodamine dye attached to 3'-deoxynucleoside triphosphate (3'-dNTP). This method enables us to conduct a rapid isothermal sequencing reaction in <30 min, to reduce the amount of template required, and to do PCR direct sequencing without the elimination of primers and 2'-dNTP, which disturbs the Sanger sequencing reaction. An accurate and longer read length was made possible by newly designed four-color dye-3'-dNTPs and mutated RNA polymerase with an improved incorporation rate of 3'-dNTP. This method should be useful for large-scale sequencing in genome projects and clinical diagnosis.     

Technical report: an alternative mechanical technique of pseudoaneurysm compression therapy

Graded manual compression therapy under ultrasound guidance has become the standard first line treatment of post-catheterization femoral pseudoaneurysms. Although effective, this treatment is often poorly tolerated by both patient and operator. We describe a new mechanical technique which has proven successful in our department, and is well tolerated by both patients and staff.     

DNA sequencing on microfabricated electrophoretic devices

We present a model that quantitatively describes the performance of microfabricated electrophoretic devices filled with linear polyacrylamide as replaceable sieving material for single-stranded DNA analyses. The dependence of resolution on various separation parameters such as selectivity, diffusion, injector size, device length, and channel folding was investigated. A previously predicted dependence of longitudinal diffusion coefficient on electric field strength has been verified. We have used this model to develop and optimize microfabricated electrophoretic devices for DNA analyses. For single-color DNA sequencing mixtures, we routinely achieve separations of 400 bases in under 14 min at 200 V/cm, and separation of 350 bases in only 7 min at 400 V/cm, with a minimum resolution of R = 0.5. Our results also indicate reduced fragment biasing and efficient sample stacking for DNA sample loading on microfabricated devices.     

Preparation of PCR products for DNA sequencing

We demonstrate that routine PCR product analytical agarose gels can also serve as preparative gels for quick DNA template purification before sequencing. The band of interest is excised, placed into a Gel Nebulizer inside a Micropure separator and rapidly purified in a single centrifugation step. Gel-purified PCR product, suitable for manual and automated sequencing, is delivered within 10 min.     

Membrane-mediated sample loading for automated DNA sequencing

A new and improved sample loading method for DNA sequencing gels using oligo/polynucleotide binding membranes is described. The labeled DNA sequencing fragments were spotted onto a membrane sample loader, which was then placed in contact with the precast polyacrylamide separation gel. In this way, the time-consuming sample well loading by the tedious pipetting procedure was avoided. The spotted DNA fragments remain immobilized on the membrane until the separation process is initiated by the application of the electric field (an active DNA release mechanism). This novel technique enables sample loading outside of the separation platform, thereby allowing full utilization of various automated sample preparation and liquid handling (robotics) systems, resulting in "real" automated DNA sequencing. The loaded membranes can be stored for more than 24 hours for later use.     

New energy transfer dyes for DNA sequencing

We have synthesized a set of four energy transfer dyes and demonstrated their use in automated DNA sequencing. The donor dyes are the 5- or 6-carboxy isomers of 4'-aminomethylfluorescein and the acceptor dyes are a novel set of four 4,7-dichloro-substituted rhodamine dyes which have narrower emission spectra than the standard, unsubstituted rhodamines. A rigid amino acid linker, 4-aminomethylbenzoic acid, was used to separate the dyes. The brightness of each dye in an automated sequencing instrument equipped with a dual line argon ion laser (488 and 514 nm excitation) was 2-2.5 times greater than the standard dye-primers with a 2 times reduction in multicomponent noise. The overall improvement in signal-to-noise was 4- to 5-fold. The utility of the new dye set was demonstrated by sequencing of a BAC DNA with an 80 kb insert. Measurement of the extinction coefficients and the relative quantum yields of the dichlororhodamine components of the energy transfer dyes showed their values were reduced by 20-25% compared with the dichlororhodamine dyes alone.     

Base stacking: pH-mediated on-column sample concentration for capillary DNA sequencing

An on-column sample concentration method for capillary-based DNA sequencing was studied. This base-stacking method allows direct injection of unpurified products of dye-primer sequencing reactions onto capillaries without any pretreatment. On-column concentration of DNA fragments is achieved simply by electrokinetic injection of hydroxide ions. A neutralization reaction between these OH- ions and the cationic buffer component Tris+ results in a zone of lower conductivity, within which field focusing occurs. DNA fragments are concentrated at the front of this low-conductivity zone. With sample injection times as long as 360 s at 50 V/cm, resolution could still be restored by the stacking process. Using a 36/47-cm-long uncoated capillary, with poly(dimethylacrylamide) as the separation matrix, and electric field of 160 V/cm, a resolution of at least 0.5 could be generated for fragments up to 650 nucleotides long. Both resolution and signal strength are excellent relative to conventional injection of highly purified samples. No significant degradation of the capillary performance was observed over at least 20 sequencing runs using this new sample injection methods.     

Gd-DTPA: an alternative contrast medium for CT

OBJECTIVE: The purpose of this study was to demonstrate that gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA) could be used as a contrast medium in CT as an alternative to iodine-based compounds. MATERIALS AND METHODS: Solutions of different concentrations of Gd-DTPA and iopromide were scanned in a tissue equivalent phantom and it was shown that Gd-DTPA caused 2.5 times the attenuation of an equimolar solution of iopromide. From these in vitro studies an in vivo dose of 0.5 mmol/kg Gd-DTPA was calculated to be equivalent to 50 ml iopromide 300. RESULTS: Pre- and postenhancement CT was performed in a volunteer using Gd-DTPA intravenously, and adequate enhancement occurred in intracranial vessels. CONCLUSION: Gd-DTPA can be used to provide enhancement during CT and might be of value in iodine-sensitive patients.     

A quality control algorithm for DNA sequencing projects

Heterologous DNA sequences from rearrangements with the genomes of host cells, genomic fragments from hybrid cells, or impure tissue sources can threaten the purity of libraries that are derived from RNA or DNA. Hybridization methods can only detect contaminants from known or suspected heterologous sources, and whole library screening is technically very difficult. Detection of contaminating heterologous clones by sequence alignment is only possible when related sequences are present in a known database. We have developed a statistical test to identify heterologous sequences that is based on the differences in hexamer composition of DNA from different organisms. This test does not require that sequences similar to potential heterologous contaminants are present in the database, and can in principle detect contamination by previously unknown organisms. We have applied this test to the major public expressed sequence tag (EST) data sets to evaluate its utility as a quality control measure and a peer evaluation tool. There is detectable heterogeneity in most human and C.elegans EST data sets but it is not apparently associated with cross-species contamination. However, there is direct evidence for both yeast and bacterial sequence contamination in some public database sequences annotated as human. Results obtained with the hexamer test have been confirmed with similarity searches using sequences from the relevant data sets.     

An iterative and regenerative method for DNA sequencing

This paper presents, to our knowledge, the first iterative DNA sequencing method that regenerates the product of interest during each iterative cycle, allowing it to overcome the critical obstacles that impede alternative iterative approaches to DNA sequencing: loss of product and the accumulation of background signal due to incomplete reactions. It can sequence numerous double-stranded (ds) DNA segments in parallel without gel resolution of DNA fragments and can sequence DNA that is almost entirely double-stranded, preventing the secondary structures that impede sequencing by hybridization. This method uses ligation of an adaptor containing the recognition domain for a class-IIS restriction endonuclease and digestion with a class-IIS restriction endonuclease that recognizes the adaptor's recognition domain. This generates a set of DNA templates that are each composed of a short overhang positioned at a fixed interval with respect to one end of the original dsDNA fragment. Adaptor ligation also appends a unique sequence during each iterative cycle, so that the polymerase chain reaction can be used to regenerate the desired template-precursor before class-IIS restriction endonuclease digestion. Following class-IIS restriction endonuclease digestion, sequencing of a nucleotide in each overhang occurs by template-directed ligation during adaptor ligation or through a separate template-directed polymerization step with labeled ddNTPs. DNA sequencing occurs in strides determined by the number of nucleotides separating the recognition and cleavage domains for the class-IIS restriction endonuclease encoded in the ligated adaptor, maximizing the span of DNA sequenced for a given number of iterative cycles. This method allows the concurrent sequencing of numerous dsDNA segments in a microplate format, and in the future it can be adapted to biochip format.     

Pegaspargase: an alternative?

OBJECTIVE: To review the chemistry, pharmacology, pharmacokinetics, clinical activity, adverse effects, dosage, and administration guidelines for pegaspargase. DATA SOURCES: A MEDLINE search (1980-1996), a CANCERLIT search (1983-1996), and a CURRENT CONTENTS search (1980-1996) using the terms pegaspargase, PEG-asparaginase, PEG-L-asparaginase, polyethylene glycol L-asparaginase, polyethylene glycol conjugated L-asparaginase, and Oncaspar were conducted. STUDY SELECTION AND DATA EXTRACTION: All articles were considered for possible inclusion in this review. Abstracts were included only when they were judged to add critical information not otherwise available in the medical literature. DATA SYNTHESIS: L-Asparaginase has been a main component of treatment regimens for acute lymphocytic leukemia. A key limiting factor of L-asparaginase use has been the development of hypersensitivity to the drug. Recently, a polyethylene glycol (PEG) conjugated form of L-asparaginase, pegaspargase, has been made available. PEG modification of L-asparaginase has been shown to alter the tendency of the enzyme to induce an immune response and to extend the half-life of the drug. The majority of patients with hypersensitivity to the native enzyme preparations tolerate pegaspargase without further clinical hypersensitivity. The adverse effect profile of pegaspargase is similar to that of the native forms of L-asparaginase. The recommended dosage of pegaspargase is 2500 IU/m2 administered by intramuscular or intravenous injection every 2 weeks in combination with other chemotherapeutic agents. CONCLUSIONS: Pegaspargase is a safe, effective alternative to L-asparaginase in patients who have had clinical hypersensitivity reactions to both Escherichia coli- and Erwinia carotovora-derived L-asparaginase. However, pegaspargase should not be routinely substituted for L-asparaginase.     

Thermo Sequenase DNA polymerase and T. acidophilum pyrophosphatase: new thermostable enzymes for DNA sequencing

A combination of thermostable enzymes has been developed that produces higher quality cycle sequences. Thermo Sequenase DNA polymerase is a thermostable enzyme engineered to catalyze the incorporation of ddNTPs with an efficiency several thousandfold better than other thermostable DNA polymerases. Since the enzyme also catalyzes pyrophosphorolysis at dideoxy termini, a thermostable inorganic pyrophosphatase is needed to remove the pyrophosphate produced during sequencing reactions. Thermoplasma acidophilum inorganic pyrophosphatase (TAP) is thermostable and effective for converting pyrophosphate to orthophosphate. The use of the combination of Thermo Sequenase polymerase and TAP for cycle sequencing yields sequence data with uniform band intensities, allowing the determination of longer, more accurate sequence reads. Uniform band intensities also facilitate interpretation of sequence anomalies and the presence of mixed templates. Sequencing PCR products of DNA amplified from heterozygous diploid individuals results in signals of equal intensity from each allele.     

Semiskeletonization of internal thoracic artery: alternative harvest technique

A simple technique of harvesting the internal thoracic artery is described. Harvesting the internal thoracic artery as a thin pedicle without the endothoracic fascia is advantageous in terms of obtaining its maximum length, dissecting a narrow space, avoiding pleural opening, and facilitating handling of the internal thoracic artery during the coronary anastomoses. This alternative-technique requires almost the same skill and time as the conventional wide pedicle technique.     

Elaboration of an alternative, segmental, cartilage-sparing tip graft technique: experience in 405 cases

Despite the value of tip grafting in many rhinoplasty patients, adequate donor cartilage may be unavailable in secondary and even primary patients whose donor sites have been harvested previously or whose septal cartilage is calcified. Furthermore, by enlarging the lobule, tip grafts can create undesirable postoperative disproportions in some patients. These two observations have stimulated the elaboration of a tip graft method (which evolved from the Sheen technique) that uses small amounts of autogenous donor material to augment only those lobular segments that require increased contour or support, without necessarily increasing overall lobular volume. This article reports experience with; the technique in a 405-patient study group. Segmental tip grafting is performed endonasally through access incisions along the caudal edge of one alar cartilage. Grafts augment each third of the tip lobule and anterior columella (corresponding to each of the alar cartilage crura) depending on the aesthetic objective; multiple grafts are always placed. Selective augmentation limits the overall increase in lobular size. The method is not suitable for those patients needing substantial augmentation (58 of 463 tip-grafted patients in the 6-year study period), in which case the author still prefers the Sheen technique. The records of the 405-patient study group (40 percent primary rhinoplasty, 60 percent secondary rhinoplasty) indicate a total nasal revision rate of 14 percent; 6 percent were tip revisions. Tip revisions were more frequent in secondary patients but not in patients with thin skin. Reoperation percentages decreased during the study term, so that the tip revision rate was 12 percent in the first 12 months of study but only 4 percent in the last 12 months (p < 0.0008). The primary indication for tip grafting has evolved since the author's earlier practice experience: in the past 3 years of the study, 77 percent of primary patients and 80 percent of secondary patients underwent grafting principally to improve lobular contour, not tip projection (p < 0.0005). A segmental, cartilage-sparing tip graft technique can provide both projection and contour for primary and secondary rhinoplasty patients. Nevertheless, tip imperfections remain the most common reason for revision in the author's practice.