Search PubMed⌕ Search

Biomedical subjects

M A Marino

Publications and source records attributed to M A Marino.

At least 19 recordsLinked to original sources
PubMed2000

DNA microsatellite analysis using ion-pair reversed-phase high-performance liquid chromatography.

Genotyping based on short tandem repeat (STR) regions is used in human identification and parentage testing, gene mapping studies, cancer diagnostics, and diagnosis of hereditary diseases. Analysis of STR systems using slab gel electrophoresis requires lengthy and labor-intensive procedures. Therefore, alternative methods such as capillary electrophoresis or ion-pair reversed-phase high-performance liquid chromatography (IPRP HPLC) have been used to analyze DNA. IPRP HPLC offers an attractive substitute to gel electrophoresis for STR analysis because of the reduced analysis time, and there is no need for the waste disposal associated with radioisotopic, enzyme-linked, or fluorescence detection systems. We evaluated the use of IPRP HPLC for the sizing and typing of STR alleles from the HUMTHO1 locus. The IPRP HPLC conditions (column temperature, flow rate, percent organic modifier per minute) were optimized for the separation of PCR products. Using the optimized separation conditions, the alleles of the HUMTHO1 system were sized in their native state (double standard) with the use of internal markers. The typing results correlated 100% to accepted methods of DNA typing. The analysis time for the HUMTHO1 locus was less than 14 min, and the alleles could be peak captured for further examination following such as sequencing.

Alleles↗
PubMed1999

Optimization of intercalation dye concentration for short tandem repeat allele genotyping using capillary electrophoresis with laser-induced fluorescence detection.

DNA analysis using capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection requires that polymerase chain reaction products either be prepared using primers with fluorescent molecules covalently bonded to them, or stained with a fluorescent intercalation dye following amplification. The intercalation technique has the advantage of allowing fluorescence detection of any double-stranded DNA (dsDNA) product regardless of the amplification primers used. The increased sensitivity of LIF detection is sometimes compromised by the intercalation dye changing the mass to charge ratio of the DNA. The purpose of this study was to evaluate the changes of migration rate, resolution and fluorescent intensity of dye-DNA complexes during electrophoretic separations, and to establish the optimal parameters for short tandem repeats alleles profiling. The alleles of three STR loci THO1, F13A01 and vWFA31 were intercalated with the monomeric dyes TOPRO-1 and YOPRO-1, and their corresponding dimers, TOTO-1 and YOYO-1 (Molecular Probes, Eugene, OR, USA). Alleles intercalated before injection onto the CE column resulted in loss of resolution and sensitivity when compared to the on-column labeling technique. The results of this experimentation were then applied to a STR typing assay using a commercially available polymer and buffer matrix. This assay included development of a unique internal standard used for migration time normalization assignment of alleles. Consequently, the 9 allele and the 9.3 microvariant of the THOI locus were separated and typed correctly with a resolution of 0.49 in less than 20 min, and the only sample preparation necessary after amplification was a dilution step.

Alleles↗
PubMed1998

Spectral measurements of intercalated PCR-amplified short tandem repeat alleles.

Short tandem repeat (STR) alleles are popular for use as forensic markers due to their highly polymorphic nature. Commonly they are separated by gel electrophoresis and visualized using intercalation dyes. The purpose of this study was to determine the changes in absorbance and fluorescence of DNA-intercalation dye complexes as a function of base pair (bp)-to-dye ratio. The DNA samples consisted of STR alleles from loci THO1, F13A01, and vWFA31. The alleles were PCR amplified and HPLC purified to ensure that only the desired DNA fragment was present in each sample. Alleles ranged in size from 151 bp for locus vWFA (allele 17) to 199 bp for the locus F13A01 (allele 8). The adenine and thymine (AT) content varied from 48% for the THO1 locus to 69% for F13A01 and vWFA31 loci. The homozygous alleles of each locus were mixed individually with the bis-intercalators TOTO-1 and YOYO-1 and their corresponding monomeric dyes TOPRO-1 and YOPRO-1. The absorbance of the DNA-dye complex at 260 nm increased with addition of each intercalation dye. Subtraction of the dye absorbance rendered the DNA absorbance constant at 260 nm. Fluorescence emission increased dramatically upon intercalation of both the monomeric and dimeric dyes into the DNA helix. A plateau of fluorescence intensity was observed at base pair-to-dye ratios of 10/1 for the bis-intercalator TOTO-1 and 5/1 for YOYO-1 for all three loci. The greatest fluorescence intensity response was obtained with the intercalator YOYO-1 using allele 8 of the F13A01 locus, which had the greatest AT concentration.

Adenine↗
PubMed1998

Sequencing using capillary electrophoresis of short tandem repeat alleles separated and purified by high performance liquid chromatography.

Polymerase chain reaction (PCR) amplified alleles need to be isolated and purified before carrying out additional analysis to confirm sequence, number of repeats and microvariants within a short tandem repeat (STR) locus. Also, PCR amplification of tetranucleotide repeat loci, used in DNA typing assays, often result in heteroduplex formation, adding to the complexity of analysis. Sequencing reactions require single specific target DNA for reliable sequencing analysis. Alkylated poly(styrene-divinylbenzene) columns at elevated temperature and gradient elution conditions increase the efficiency of separation to allow for the purification of PCR products. Using the separation technique of ion-pairing reverse-phase (IPRP) high performance liquid chromatography (HPLC), molecular biologists can separate and purify DNA fragments without alteration to the double-stranded DNA sequencing properties. In this study, the IP-RP chromatography technique has been demonstrated by separation of alleles of the short tandem repeat loci of TH01, vWA31, F13A01 and FES/ FPS. Alleles differing in size range of 12 to 4 base pairs were separated by IPRP/HPLC and individual alleles were peak-captured, then cycle-sequenced. These HPLC fractions required no additional steps prior to cycle sequencing. Capillary electrophoresis (CE) was used to sequence the alleles. Furthermore, CE offers advantages over traditional slab methods via automation and higher applied voltages. Interestingly, unlike traditional gel electrophoresis, samples were introduced into the sieving matrix by electrokinetic injection, which allows for multiple injections from a single sample, a key feature for method development. Applied voltage was 320 V per centimeter using a nonderivatized fused silica capillary with an interior diameter of 50 microm and a total length of 47 centimeters. The total analysis time including capillary filling and pre-electrophoresis was less than 30 min for a 220-bp fragment. A sequencing rate of 530 bp/h was achieved using these conditions. By combining the techniques of HPLC separation and CE sequencing, the results confirmed the sequence and number of nucleotide repeats for each STR loci. An average sequencing efficiency of 97% was achieved. Additionally, this method defined the absence of a 9.3 microvariant for a TH01 heterozygous individual previously typed as a 9, 9.3/10 using slab gel electrophoresis. The techniques described can be applied to other DNA purification and isolation problems.

Alleles↗
PubMed1996

Automated polymerase chain reaction product sample preparation for capillary electrophoresis analysis.

The analysis of crude polymerase chain reaction (PCR) products by capillary electrophoresis (CE) is often compromised due to the presence of a high concentration of salt. Salt interferes with the electrokinetic injection and induces localized heating within the column; hence, PCR products must be desalted or cleaned-up prior to CE analysis. A variety of commercial clean-up systems are available that have been traditionally used to prepare PCR products for cloning, sequencing and digestion with restriction enzymes. These systems were tested for their effectiveness in preparing PCR products for CE analysis and were evaluated based on CE resolution, salt removal, DNA recovery, processing time and cost. One particularly effective clean-up system, membrane dialysis, was automated using a robotic workstation.

Automation↗
PubMed1996

Characterization of mitochondrial DNA using low-stringency single specific primer amplification analyzed by laser induced fluorescence--capillary electrophoresis.

Polymerase chain reaction (PCR)-based DNA typing is routinely used in forensics for identity testing. Those assays that distinguish single nucleotide polymorphisms (SNPs) require other biochemical reactions in addition to PCR to identify the sequence polymorphisms. Low-stringency sequence-specific PCR (LSSP-PCR) is an example of a recent method that does not require additional biochemical treatments. The analysis of LSSP-PCR by capillary electrophoresis (CE) to discriminate the highly polymorphic mitochondrial DNA (mtDNA) D-loop region is described. The DNA from five individuals were amplified (first step) using sequence-specific primers to produce 1021 bp fragments containing the D-loop region. Each fragment was isolated by electroelution using CE and UV detection, and subjected to a second amplification (second step) using a single primer annealed under low stringency conditions. This generated a range or profile of PCR products for each sample, which were resolved and analyzed by CE with the intercalator TOTO-1 and laser-induced fluorescence (LIF) detection. The LSSP-PCR profiles were unique for each individual, indicating that this technique may be applicable for forensic identity testing.

DNA Primers↗
PubMed1996

PCR-based human leukocyte antigen (HLA) DQ alpha typing of blood stained light and dark blue denim fabric.

Obtaining typable PCR products from DNA purified from blood stained blue denim has been difficult since inhibitors of PCR in blue denim apparently co-purify with the DNA. Organic and chelex extraction methods were tested for their ability to purify typable DNA from either light or dark blue denim fabric both stained with blood. DNA purified from the light blue denim using either method was successfully used in obtaining correct HLA-DQ alpha typing results. The chelex, but not the organic, procedure was able to yield typable DNA when the dark blue denim was the substrate. Therefore, the chelex method may be more effective than the organic method in preventing compounds that inhibit PCR from co-purifying with the DNA.

Blood Stains↗
PubMed1996

The evaluation of fast purification methods for preparing polymerase chain reaction (PCR) products for capillary electrophoresis analysis.

The analysis of crude polymerase chain reaction (PCR) products by capillary electrophoresis (CE) is often compromised by the presence of a high concentration of salt. Salt interferes with the electrokinetic injection and induces localized heating within the column, hence, PCR products must be desalted or cleaned-up prior to CE analysis. A variety of commercial clean-up systems are available that have been traditionally used to prepare PCR products for cloning, sequencing, and digestion with restriction enzymes. These systems were tested for their effectiveness in preparing PCR products for CE analysis and were evaluated based on CE resolution, salt removal, DNA recovery, processing time, and cost.

DNA↗
PubMed1995

Surgical correction of aortic disease using intraluminal, crimped bovine pericardial graft.

We propose the use of a crimped bovine pericardial graft joined to a rigid and grooved ring for the treatment of aortic dissections and aneurysms. This device was developed to support common tubular vascular prostheses of any material, length, diameter, or form, transforming them into intraluminal ones during the surgical procedure. During March 1989 to May 1994, 74 patients underwent an operation on the aorta in which this intraluminal ring, prepared from a tubular prosthesis of bovine pericardium, was used. Twenty-seven patients had an acute type A aortic dissection, 6 had an acute type B aortic dissection, 10 had annuloaortic ectasia, 6 had a descending aortic aneurysm, 3 had a thoracoabdominal aneurysm, and 22 had an abdominal aneurysm. The global mortality rate of 10.8% in the immediate postoperative period cannot be related to the surgical technique or the type of prosthesis used. We conclude from our findings that this modification of the sutureless intraluminal graft, which allows it to be used to replace diseased segments of the aorta, affords the quickest and simplest means of repair and is attended by the least likelihood of anastomotic complications.

Adult↗
PubMed1995

Automated DNA purification and amplification from blood-stained cards using a robotic workstation.

A new method for the purification of DNA on blood-stained cards was developed. This method was implemented into a high-throughput automated system using a Biomek 1000 robotic workstation. In addition, the processes of DNA purification and amplification were coupled into a completely automated and uninterrupted prototype system, and the resultant PCR products generated by this system were subjected to automated desalting for capillary electrophoresis analysis.

Autoanalysis↗
PubMed1995

The analysis of simple sequence repeat DNA in soybean by Capillary Gel Electrophoresis.

The objective of this work is to examine the presence of simple sequence repeat (SSR) DNA in soybean plant genotypes by Capillary Gel Electrophoresis (CGE). The SSR DNA length polymorphism in soybean determines the variation in polymerase chain reaction (PCR) product lengths. Loci were chosen where amplification produced one PCR product per genotype (M.S. Akkaya et al., 1992). The F1 hybrids of parents carrying different alleles produced two PCR products identical to the two parents. The CGE system used a 3%T,3%C polyacrylamide gel capillary with an effective length of 40 cm. The PCR products with lengths of 150 to 200 base pairs were monitored at 260 nm. The analysis time was under 50 minutes. CGE is capable of separating these PCR products by base pair number the same as conventional sequencing gel techniques. CGE offers an automated, high speed, high resolution analytical method for determining soybean SSR allele sizes as compared with the traditional methodologies.

Autoanalysis↗
PubMed1994

Analysis of DNA restriction fragments and polymerase chain reaction products by capillary electrophoresis.

Capillary electrophoresis (CE) is a new, high-resolution tool for the analysis of DNA restriction fragments and DNA amplified by the polymerase chain reaction (PCR). By combining many of the principles of traditional slab gel methods in a capillary format, it is possible to perform molecular size determinations of human and plant PCR amplification products and DNA restriction fragments. DNA restriction fragments and PCR products were analyzed by dynamic sieving electrophoresis (DSE) and capillary gel electrophoresis (CGE). As part of this study, sample preparation procedures, injection modes, and the use of molecular mass markers were evaluated. Optimum separations were performed using the uPage-3 (3% T, 3% C) CGE columns with UV detection at 260 nm. Membrane dialysis and ultrafiltration/centrifugation proved to be nearly equivalent methods of sample preparation. Reproducibility studies demonstrated that blunt-ended, non-phosphorylated markers (specifically allele generated markers) provide the most accurate calibration for PCR product analysis. This study demonstrates that CE offers a high-speed, high-resolution analytical method for accurately determining molecular size and/or allelic type as compared with traditional methodologies.

Bacteriophage phi X 174↗
PubMed1994

Molecular size determinations of DNA restriction fragments and polymerase chain reaction products using capillary gel electrophoresis.

Commercially available capillary electrophoresis (CE) systems offer advantages over traditional slab gel methodologies. The capillary format allows the use of higher voltages (225 V/cm), which results in faster migration, higher resolution and greater efficiency without excessive heating. The ability to automate the system increases the unattended sample analysis throughput. For this study, the CE system was configured with a muPAGE 3% T, 3% C polyacrylamide gel capillary with an effective length of 40 cm and microPAGE Tris-borate urea buffer system. The analysis of DNA restriction fragments and polymerase chain reaction products, with an internal standard of Boehringer Mannheim DNA marker XI were completed in less than 70 min. All samples were analyzed at 260 nm. The data establishes automated capillary gel electrophoresis as a high-resolution, reproducible method for analysis of samples under 1000 base pairs.

Base Composition↗
PubMed1994

High-dose cyclophosphamide for mobilization of circulating stem cells in chronic myeloid leukemia.

Experimental and clinical data suggest that Ph-negative myeloid progenitor cells are present, albeit suppressed, in the bone marrow of chronic myeloid leukemia (CML) patients. These residual Ph-negative cells might, in certain circumstances, regain their proliferative advantage over the leukemic Ph-positive clone. Treating CML patients with intensive chemotherapy might allow the harvest, in the early phase of recovery, of Ph-negative stem cells to be used as graft after myeloablative regimen. In our study, 6 CML patients were admitted to a program of autograft with circulating stem cells (CSC) collected after high-dose (5 or 7 g/m2) cyclophosphamide (HD-CY) mobilization. All were autografted, using busulphan 16 mg/kg and melphalan 60 mg/m2. As graft, 4 patients received CSC only, while 2 patients were also given bone marrow, as their peripheral blood CFU-GM yield was unsatisfactory. Two previously alpha-IFN-responding patients showed a slow hematologic recovery, but achieved a marked and further reduction of their Ph-positive metaphases post-graft. Moreover, in one of them, cytogenetic analyses performed on apheresis product showed a more pronounced reduction of his Ph-positive metaphases, as compared to bone marrow samples, suggesting a potential purging effect of the mobilization procedure.

Adolescent↗