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Biomedical subjects

M N Kronick

Publications and source records attributed to M N Kronick.

14 recordsLinked to original sources
PubMed1997

A generic sequencing based typing approach for the identification of HLA-A diversity.

Sequencing Based Typing (SBT) is a generic approach for the identification of HLA-A polymorphism. This approach includes the high resolution typing of the HLA-A broad reacting groups, HLA-A subtypes and will identify new alleles directly. The SBT approach described here uses a locus specific amplification of DNA from exon 1 to exon 5. The resulting 2,022 bp PCR product serves as a template for the subsequent sequencing reactions. Amplification is followed by direct sequencing of exons 2, 3 and 4 in both orientations with fluorescently labeled primers to define all polymorphic positions leading to a high resolution typing result. In this study the sequence of exons 2 and 3 of a panel of 49 cell lines was determined. In addition, the exon 4 region of 35 cell lines was also sequenced to evaluate the exon 4 polymorphism. The HLA-A type of most of the cells could be identified by sequencing only exons 2 and 3. However, the sequence of exon 4 was required to discriminate A*0201 from A*0209 and A*0207 from A*0215N. In this panel, an identical new "HLA-A*0103" was identified in two Caucasian samples.

Alleles↗
PubMed1996

Heterozygote and mutation detection by direct automated fluorescent DNA sequencing using a mutant Taq DNA polymerase.

We describe a method for direct cycle sequencing of PCR fragments amplified from genomic DNA or cDNA. DNA sequencing template is amplified using PCR and oligonucleotide primers flanking the region of interest. The amplified fragment is directly cycle sequenced using fluorescent sequencing primers, Sanger dideoxy sequencing chemistry and an enzyme mixture of a mutant Taq DNA polymerase and thermostable pyrophosphatase. The sequence ladders produced are analyzed on a real-time, automated four-color sequencing system. The method produces sequence ladders from unpurified PCR fragments of sufficiently high quality such that heterozygotes can be reproducibly detected and identified by software that recognizes signal-strength patterns indicative of mixed-base positions.

Base Sequence↗
PubMed1995

Efficient, automatic detection of heterozygous bases during large-scale DNA sequence screening.

A crucial factor in the success of positional cloning efforts is the ability to screen rapidly many different candidate genes for mutations. By modifying standard software, we have improved the detection of heterozygous base positions in PCR products sequenced by cycle sequencing. A key element of the method is the incorporation of a modified heterozygote detection algorithm that permits the use of DNA sequence data derived from PCR and sequencing reactions that have not been fully optimized. This allows sequencing runs of average quality to be used. We demonstrate that the sensitivity and specificity of the method are well suited to mutation detection applications such as positional cloning.

Algorithms↗
PubMed1993

Fluorescent approaches to diagnosis of Lesch-Nyhan syndrome and quantitative analysis of carrier status.

Lesch-Nyhan syndrome is an X-linked recessive disorder caused by molecular defects within the HPRT gene. Deletional forms of this syndrome, most of which are inherited, account for 15% of the cases. In addition, a large percentage of cases are due to de novo point mutations. We have used complementary fluorescence-based PCR assays to analyse disease-causing mutations in three unrelated families: (1) inheritance of dye-labelled PCR products of linked polymorphic loci mapping within and flanking the HPRT gene; (2) dye-labelled exon dosage analysis and (3) automated fluorescence-based DNA sequence analysis. Our results using fluorescent, dye-tagged PCR products show that inheritance of two polymorphic small tandem repeats, HPRTB [AGAT]n, mapping within intron 3 of the HPRT gene, and the CA-repeat at DXS294 can be used to establish linkage to the disease. In addition, we modified a previously described PCR protocol to use fluorescent dye-labelled oligoprimers and an ABI Gene Scanner in order to rapidly quantitate deletional forms of Lesch-Nyhan syndrome. Quantitative PCR analysis of individual exons followed by dosage analysis confirmed a deletion encompassing exon 9. A similar approach was used to confirm a previously described HPRT gene duplication involving exons 2 and 3. In this analysis, we co-amplified the HPRTB [AGAT]n and HUMARA [AGC]n repeats and confirmed increased exon dosage in carriers for the duplication. DNA sequence analysis remains the method of choice for delineating new disease-causing mutations, most of which are non-deletional forms of Lesch-Nyhan syndrome. We have also used a cycle-sequencing strategy employing dye-labelled dideoxy terminators and a laser-activated, fluorescence-emission DNA sequencer in order to define carrier status in 10 family members at risk for Lesch-Nyhan syndrome due to a splice donor mutation in intron 7. Our DNA sequence analyses corroborate small tandem repeat (STR) inheritance patterns in this family. Multiple fluorescence-based strategies should facilitate rapid diagnosis of the various Lesch-Nyhan disease-causing mutations.

Adolescent↗
PubMed1993

Alternative labeling techniques for automated fluorescence based analysis of PCR products.

We present here convenient and simple methods that utilize two new fluorescent tags, TOTO-1 and fluorescein-12-dUTP, in conjunction with analysis of PCR products on automated, fluorescence-based electrophoretic instruments. These methods should prove valuable in those applications wherein the effort or expense of covalently attaching a fluorescent tag onto one or both of the PCR primers is not justified. Advantages and disadvantages of the new labeling methods are then reviewed.

Deoxyuracil Nucleotides↗
PubMed1992

Application of automated DNA sizing technology for genotyping microsatellite loci.

Highly polymorphic microsatellite loci offer great promise for gene mapping studies, but fulfillment of this potential will require substantial improvements in methods for accurate and efficient genotyping. Here, we report a genotyping method based on fluorescently labeled PCR primers and size characterization of PCR products using an automated DNA fragment analyzer. We capitalize on the availability of three distinct fluorescent dyes to label uniquely loci that overlap in size, and this innovation increases by threefold the number of loci that can be analyzed simultaneously. We label size standards with a fourth dye and combine these with the microsatellite PCR products in each gel lane. Computer programs provide very rapid and accurate sizing of microsatellite alleles and efficient data management. In addition, fluorescence signals are linear over a much greater range of intensity than conventional autoradiography. This facilitates multiplexing of loci (since signal intensities often vary greatly) and helps distinguish major peaks from artifacts, thereby improving genotyping accuracy.

DNA, Satellite↗
PubMed1992

The use of fluorescence detection and internal lane standards to size PCR products automatically.

We have developed chemical procedures, optical and electrophoretic instrumentation and computer software automate the analysis of polymerase chain reaction (PCR) products. DNA molecules labeled with up to four different fluorescent dyes are analyzed within a single electrophoresis gel lane. A size calibration curve is created for each electrophoresis lane from the electrophoretogram of uniquely labeled DNA fragments belonging to an internal lane standard that co-electrophoreses with the PCR products. The unknown molecular lengths of PCR products are automatically calculated from the calibration curve. Data from control experiments with DNA segments of known molecular length demonstrate the accuracy and precision of such sizing. This system has been applied to the analysis of PCR products for research in the areas of human identification, genetic mapping and genetic disease.

Base Sequence↗
PubMed1991

Automated genetic analysis.

Automation of several new, non-traditional techniques for genetic analysis has now become possible. A new system is described that performs gel electrophoretic analysis of DNA including VNTRs, gene segments, and restriction enzyme digests. The instrument detects emitted fluorescence from labeled DNA segments in real-time as they electrophore through a gel matrix past a scanning laser beam. Molecular length determination and band quantification is accomplished by comparison to an in-lane standard. Since DNA segments can be labeled and detected with any of four different dyes, the simultaneous analysis of similar length segments from different reactions within a single lane is possible. PCR products are analyzed for research in the areas of human identification and genetic disease. These examples illustrate how automation will play key role in this new era of genetic analysis.

Automation↗
PubMed1989

Automated DNA sequencing methods involving polymerase chain reaction.

Polymerase chain reaction (PCR) as a method for preparing DNA templates has been used for several DNA sequencing applications. An in situ procedure for directly sequencing PCR products by the dideoxy-termination method has been developed by using fluorophore-labeled sequencing primers. Completed sequence reactions were combined and loaded into a single electrophoretic lane of a fluorescence-based DNA sequence analyzer. DNA targets devoid of a universal primer sequence could be sequenced with labeled universal primers by incorporating a universal primer sequence into the PCR product. With this method, the sequence of a 351-bp region in the bacteriophage lambda genome was fully analyzed in a single lane with automatic base identification accuracy of greater than 99%. An unknown sequence, 1.7 kb long, also was sequenced by this procedure, in combination with a "PCR gene walking" strategy. Comparison of the 1110 bases in overlapping sequence data from both strands yielded only two single-base ambiguities. Automated DNA sequence analysis of the highly polymorphic HLA-DQA-1 (alpha) region in the human genome can be performed with this simple methodology. Use of this PCR-sequencing method to analyze DNA extracted from a one-month-old blood sample from an individual who is heterozygous at this locus allowed unambiguous assignment of genotype.

Autoanalysis↗
PubMed1983

Immunoassay techniques with fluorescent phycobiliprotein conjugates.

We describe immunoassay techniques that take advantage of the novel properties of phycobiliprotein fluorescent dyes. These dyes, which can be isolated from a wide variety of algae, exhibit extremely high absorptivities, high quantum efficiencies, and excitation and emission bands across the visible spectrum. These stable, hydrophilic proteins can easily be linked to antibodies by conventional protein cross-linking reagents. One assay technique presented here demonstrates the use of phycobiliproteins in a solid-phase "sandwich" assay, which achieves picomolar sensitivity. In the other technique the unique spectral properties of phycobiliproteins are applied to fluorescence excitation transfer immunoassay.

Antibodies↗
PubMed1975

A new immunoassay based on fluorescence excitation by internal reflection spectroscopy.

A new immunoassay technique is described which uses totally internally reflected light to excite the fluorescence of fluorescein labeled antibody which has become bound to a hapten--protein conjugate absorbed on a quartz-plate in the antibody solution. The presence of any free hapten in solution reduces the amount of antibody free to bind to the surface and thus reduces the fluorescence signal. Measurement of the decrease of the fluorescent signal then gives a measure of the concentration of free hapten present. The technique is simple, fast and has high intrinsic sensitivity and specificity. It has been demonstrated for phenyl arsonic acid and morphine. Free morphine at a concentration of 2 X 10(-7) M is readily detected.

Binding Sites, Antibody↗