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H Kambara

Publications and source records attributed to H Kambara.

At least 19 recordsLinked to original sources
PubMed1999

A capillary-array electrophoresis system using side-entry on-column laser irradiation combined with glass rod lenses.

We have developed a simple and high-throughput capillary-array electrophoresis system that uses side-entry on-column laser irradiation. The number of capillaries in an array is generally limited by laser-power attenuation along the array due to reflection and divergence. We overcame these problems by placing the capillaries in water and adding glass rod lenses between the capillaries. As a result, up to 45 capillaries could be simultaneously irradiated with a single laser beam and the fluorescence from all the capillaries could be detected with high sensitivity. We demonstrated the high throughput of 12 kbp/h with a 45 capillary array using this system.

Electrophoresis, Capillary↗
PubMed1998

Characteristics of selective polymerase chain reaction (PCR) using two-base anchored primers and improvement of its specificity.

We have developed a reliable method for eliminating base-mispair amplification in selective polymerase chain reaction (PCR), which is utilized for amplifying unknown sequence fragments produced by restriction enzyme reaction. The proposed procedure applies amplified fragment length polymorphism (AFLP) with high fidelity. Selective PCR utilizes the known polymerase reaction characteristic that the complementary strand extension is strongly affected by matching a template with the 3'-terminus of the primers. However, false positive amplification is frequently observed because the specificity of terminal bases for discrimination of fragments (usually, 1-3 anchor sequences) is not enough to separate each fragment. A protocol for the selective PCR separation of every fragment was therefore investigated. A single-base mismatch was artificially introduced on the 4th base position from the 3' end of the primers to improve the hybridization specificity of anchored 2-bases at the 3' termini of primers. PCR reaction was carried out at 66 degrees C to prevent false positive amplification. The concentration of the primers having anchored-base sequences of AA, AT, TA, and TT must be three times larger than that of other primers because the Tm values for these sequences are lower than the others. As all the fragments can be separated into groups with high fidelity, the improved selective PCR will be applied to gene finding and analyzing differences on genome sequences based on AFLP.

DNA Primers↗
PubMed1997

Analysis of single-strand conformation polymorphisms by capillary electrophoresis with laser induced fluorescence detection.

Detection of point mutations in genomic DNA is important for diagnosis of inherited characteristics and genetic diseases. A point mutation in a specific region of DNA amplified by polymerase chain reaction (PCR) can be detected with single-strand conformation polymorphism (SSCP) analysis. Analysis of SSCP by laser-induced fluorescence capillary electrophoresis in entangled polymer solution (CE-LIF) has been developed in the present paper. K-ras genes including seven mutations were amplified with primer labeled with Texas Red at its 5' end. The labeled PCR products were dissociated to single strands by heating and separated with capillary gel electrophoresis and He-Ne laser-excited fluorescence detection. Our results suggest that all fragments having normal (Gly) and mutated (Ala, Arg, Cys, Ser, Val, Asp) sequences at codon 12 can be distinguished. Analysis of SSCPs with CE-LIF is well suited for clinical analysis of SSCPs because of its high sensitivity, resolution, reproducibility and speed.

Codon↗
PubMed1996

Paternity testing by the detection of D1S80 VNTR using fluorescence image analyzer (Dualcolour system).

An improved method for DNA polymorphism typing of D1S80 VNTR locus and its application to paternity testing are described. For accurate estimation of the length of polymorphic DNA fragments, the size marker was labeled with fluorescence different from that of PCR primers, and co-electrophoresed as an internal standard. The dualcolour system of fluorescence image analyzer was used to detect the fragments and determine their size. This internal marker method could successfully overcome the problems of band pattern distortion and tailing, besides it allows easy and accurate interpretation of the DNA types. Our results indicate that the internal marker method is much more accurate than the method of using size marker in gel, even with the presence of distortion or tailing of the band patterns. Family studies applying this method showed complete agreement between the observed and predicted types.

Electrophoresis, Polyacrylamide Gel↗
PubMed1996

Fragment walking for long DNA sequencing by using a library as small as 16 primers.

A DNA sequence can be rapidly and efficiently determined by digesting it into segments small enough to be sequenced at one time, then assembling them into contigs by searching for overlaps. Fragment walking achieves this without subcloning or preparing many kinds of primers; fragments obtained by digesting a template DNA are sequenced in parallel directly from the fragment mixture by using a set of 16 primers. Since the sequence adjacent to each cutting site is determined together with the fragment sequence, the contiguous fragment can be easily determined. The complete template DNA sequence can thus efficiently determined. The sequencing of pUC19 (2.7 kb) from both sides was done in one step by using only 15 fragments. The redundancy was only about 1.3, greatly reducing base-reading redundancy. Automating this strategy would increase the speed and efficiency of large-scale DNA sequencing.

Base Sequence↗
PubMed1996

Temperature profile of buffer-filled electrophoresis capillaries using air convection cooling.

Temperature profiles of buffer-filled capillaries (capillary temperature), using both natural and forced air convection for cooling, were obtained both theoretically and experimentally in order to achieve stable capillary electrophoresis for accurate DNA sequencing analysis. The capillary temperature was calculated using the temperature dependency of the buffer resistivity, and the threshold values of the electric field and the capillary temperature for temperature instability were characterized. The capillary temperature became unstable at about 420 V/cm and at about 62 degrees C regardless of the outer diameter of the capillary; these values approximately coincide with the measured values obtained using a very thin thermocouple. The sets of values of electric field, air temperature, and air flow velocity in forced air cooling were obtained to keep the capillary temperature at the 40-60 degrees C needed for accurate DNA sequencing in a Tris-boric acid-EDTA buffer.

Buffers↗
PubMed1996

Characteristics of single-stranded DNA separation by capillary gel electrophoresis.

The optimization of electrophoretic conditions for fast separation of single stranded DNA by capillary gel electrophoresis was investigated. Mobilities and band broadening of the DNA fragments were measured for a 4% T, 5% C gel at 100-300 V/cm. The resolution of DNA peaks was evaluated for band spacing and band width. Band spacing decreased with increasing electric field strengths as well as DNA fragment size; resolution for large DNA fragments also decreased at higher electric field strengths. The measured mobilities in a 4% T, 5% C gel were analyzed by the reptation theory and compared with those in a 9% T, 0% C matrix. The reptation plots indicate that the onset of reptation in a 4% T, 5% C gel occurs at a DNA size of about 200 bases and about 150 bases in a 9% T, 0% C matrix. The 4% T, 5% C gel proved superior to the 9% T, 0% C matrix with respect to resolution and analysis time. The optimum electric field strength and migration distance for specific DNA fragment length (200, 400 and 700 bases) in the shortest time were calculated from band spacing and the measured band width for the 4% T, 5% C gel at Rs = 0.5. Under optimized conditions, analysis of DNA fragments up to 200, 400 and 700 bases could be carried out in about 5, 50, and 230 min, respectively.

Acrylic Resins↗
PubMed1996

Selecting and amplifying one fragment from a DNA fragment mixture by polymerase chain reaction with a pair of selective primers.

A new method for selecting and amplifying a single DNA fragment from a mixture is proposed. This method is applicable for the rapid classification of DNA fragments from a mixture and for preparation of sequencing templates. DNAs of several to tens of kilobases (kb) are digested with a four-base recognition restriction enzyme to produce smaller fragments. The complementary strand extension reactions are then carried out to produce fluorophore-labeled DNA fragments from the digestion products. These fragments can be rapidly classified according to their terminal-base sequences and their sizes are analyzed by capillary-array gel electrophoresis (CAGE). Electropherograms are used to characterize the fragments and to select polymerase chain reaction (PCR) primers. Any fragment in a digestion mixture can be amplified by PCR with a pair of primers selected from a primer pool by referring to the electropherograms of the fragments. This method was successfully used to compare the electropherograms of two different DNA strands and to sequence a several-kb DNA fragment without subcloning. Combined with CAGE, this method could be used to dramatically simplify DNA fragment analysis.

DNA↗
PubMed1995

DNA probe assay based on exonuclease III digestion of probes hybridized on target DNA.

A quantitative DNA probe assay process has been developed that uses exonuclease III. The fluorophore-labeled DNA probe is hybridized with specific sequences of the target DNA and then enzymatically digested. As these probe hybridization and digestion cycle reactions are repeated at a fixed temperature, digested probes (shortened probes) accumulate in the reaction mixture in a manner similar to a DNA polymerase chain reaction. Investigation of the digestion characteristics of the DNA probe showed that a slight digestion of a free single-stranded probe produces a large background signal, which results in low detection sensitivity. The digestion of single-stranded DNA probes is caused by double-stranded formations in the molecules. This digestion decreases and the double-stranded-specific digestion increases with increasing reaction temperature. When the reaction occurs at 45 degrees C, the association rate of the enzyme on the double-stranded DNA is 700 times faster than that on single-stranded DNA. This enables selective digestion of double-stranded DNA. The detection limit is 9 x 10(-19) mol for a M13-phage DNA.

Animals↗
PubMed1995

Effects of nisoldipine in silent myocardial ischemia after healing of acute myocardial infarction.

Silent myocardial ischemia occurring after acute myocardial infarction is classified as Cohn type II and has a frequency of 20-30% in all patients with acute myocardial infarction. Follow-up data of patients with either silent or anginal ischemia show a poor prognosis. Thus, all ischemic episodes occurring after myocardial infarction should be treated aggressively. Many multicenter studies have evaluated whether drug treatment can improve prognosis or protect from a nonfatal second attack. Calcium antagonists, especially those that increase heart rate, have not been considered as drugs of choice for this purpose, despite the many beneficial effects shown on myocardial tissue in experimental studies. In the study reported here, the effect of nisoldipine on postinfarction silent myocardial ischemia was evaluated by ambulatory left ventricular function monitoring. Ten patients were selected for study who showed silent myocardial ischemia after their first acute infarction. Blood pressure fell significantly (p < 0.05) after 4 weeks of treatment with nisoldipine (5-10 mg/day), but heart rate showed no change at rest. Exercise time improved (p < 0.05), with increased peak double products. During exercise, there was no significant change in end-diastolic volume but there was a marked improvement in end-systolic volume, and at the submaximal point the ejection fraction was significantly (p < 0.05) increased. Ejection fraction at rest also improved. The deterioration in ejection fraction due to dipyridamole was ameliorated by nisoldipine. Ejection fraction and blood pressure improved during the calculation test, and work performance also improved.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1995

Gender difference in long-term prognosis after myocardial infarction--clinical characteristics in 1000 patients. The Kyoto and Shiga Myocardial Infarction (KYSMI) Study Group.

To examine gender differences in the long-term prognosis of patients with myocardial infarction, 1000 patients with myocardial infarction were studied after coronary arteriography. Over a follow-up period of 3.3 +/- 2.0 years, 65 patients died from cardiac causes and 301 experienced cardiac events (death, reinfarction and revascularization). Overall, the 5-year cardiac mortality was 8%: that in females (12.4%) was significantly higher than that in males (6.6%) (p = 0.0073). The overall 5-year cardiac event-rate was 35%, with no significant difference between females and males (41.1% vs 33.3%). Univariate analysis revealed that differences in age (57.8 +/- 9.8 years in males vs 64.8 +/- 8.9 years in females, p < 0.0001), presence of smoking habit, obesity, hypercholesterolemia, hypertension, heart failure, right coronary artery disease, nicorandil administration, hypolipidemic, diuretic and anti-hypertensive treatment, and warfarin administration were present between men and women. The mortality rate in elderly females tended to be higher than that in their male counterparts. Multivariate analysis demonstrated that number of diseased vessels, post-infarction angina and left main trunk disease were significant predictors for cardiac death in both sexes, while gender was not. Therefore, gender did not appear to affect the long-term prognosis after age-adjustment among patients with myocardial infarction in the Western part of Japan.

Adult↗
PubMed1995

[Sudden death among 1,000 patients with myocardial infarction: incidence and contributory factors. KYSMI Study Group].

Clinical indicators and the incidence of sudden death among 1,000 consecutive patients (816 males and 184 females) with myocardial infarction were investigated between 1983 and 1987 after coronary arteriography. Twenty-four patients died suddenly during a study period of 3.3 +/- 2.0 yrs. The yearly incidence of sudden death calculated by the Kaplan-Meier method was 0.7%, nearly half of the incidence of cardiac death (1.5%/yr). Univariate analysis demonstrated statistically significant differences in the number of cases with coronary thrombolysis in the acute phase, degree of coronary artery stenosis, the frequency and degree of congestive heart failure and angina in the acute and chronic phase, the presence of serious post-infarction arrhythmia, ejection fraction, recurrent myocardial infarction, and administration of digitalis and diuretics. between the sudden-death group and the cardiac and other patient groups. There was also a tendency to higher frequency of history of diabetes mellitus, presence of acute mitral regurgitation, nitrates and nicorandil treatment, and coronary artery bypass graft in the sudden-death group. Multivariate analysis revealed that digitalis and nitrates treatment and left anterior descending artery involvement were major contributing factors. Re-infarction, left ventricular ejection fraction, and nicorandil therapy were not significant and minor contributing factors. The incidence of sudden death increased with digitalis therapy (Odds' ratio of 9.59), and left anterior descending artery disease, and decreased with nitrates (0.34).

Adult↗
PubMed1994

Separation of long DNA fragments by capillary gel electrophoresis with laser-induced fluorescence detection.

Long DNA fragments were separated by capillary gel electrophoresis with laser-induced fluorescence detection, and two approaches to obtaining high-resolution separation of DNA fragments were examined. One method achieved high resolution at the expense of detection time, while the other achieved both high resolution and fast detection. The former approach used a 300 cm long capillary with a gel concentration of 4% T and an electric field strength of 70 V/cm. The resolution limit in this electrophoresis was 800 bases with a resolution value of 0.5 for adjacent peaks. Under these conditions, the measurement time was 44 h. The latter approach used a 200 cm long capillary with a gel concentration of 3% T and an electric field strength of 170 V/cm. The resolution limit in this electrophoresis was 680 bases with a resolution value of 0.5 for adjacent peaks, and the measurement time was only 10 h. With both approaches, fragments with less than 600 bases were efficiently separated. The resolution values for adjacent peaks with less than 500 bases are greater than 1.0, and those for peaks with less than 100 bases are greater than 2.7. These approaches thus improve the accuracy of the base sequence determination.

DNA↗
PubMed1994

DNA sequencing directly from a mixture using terminal-base-selective primers.

DNA cloning is often used to select and amplify one DNA species from a mixture. However, the cloning process is complex and labor-intensive. We have developed a new two-step method for DNA sequencing directly from a mixture. The first is the introduction of a known oligonucleotide (common part) into the terminus of unknown DNA by ligation. The second is selective DNA sequencing using primers with two additional nucleotides at the 3' terminus in addition to the common part (terminal-base-selective primers). The primers work only for templates on which the primers perfectly hybridized. This method was found to be effective for the HindIII digestion products of lambda phage.

Bacteriophage lambda↗