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genomic DNA

About: genomic DNA is a research topic. Over the lifetime, 15046 publications have been published within this topic receiving 663636 citations. The topic is also known as: genomic deoxyribonucleic acid & gDNA.


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TL;DR: This work describes a general experimental approach, using microscopic arrays of DNA fragments on glass substrates for differential hybridization analysis of fluorescently labeled DNA samples, and demonstrates the utility of DNA microarrays in the analysis of complex DNA samples.
Abstract: Detecting and determining the relative abundance of diverse individual sequences in complex DNA samples is a recurring experimental challenge in analyzing genomes. We describe a general experimental approach to this problem, using microscopic arrays of DNA fragments on glass substrates for differential hybridization analysis of fluorescently labeled DNA samples. To test the system, 864 physically mapped lambda clones of yeast genomic DNA, together representing >75% of the yeast genome, were arranged into 1.8-cm x 1.8-cm arrays, each containing a total of 1744 elements. The microarrays were characterized by simultaneous hybridization of two different sets of isolated yeast chromosomes labeled with two different fluorophores. A laser fluorescent scanner was used to detect the hybridization signals from the two fluorophores. The results demonstrate the utility of DNA microarrays in the analysis of complex DNA samples. This system should find numerous applications in genome-wide genetic mapping, physical mapping, and gene expression studies.

1,367 citations

Journal ArticleDOI
TL;DR: Sequence characterized amplified regions (SCARs) were derived from eight random amplified polymorphic DNA (RAPD) markers linked to disease resistance genes in lettuce, providing information on the molecular basis of RAPD markers.
Abstract: Sequence characterized amplified regions (SCARs) were derived from eight random amplified polymorphic DNA (RAPD) markers linked to disease resistance genes in lettuce. SCARs are PCR-based markers that represent single, genetically defined loci that are identified by PCR amplification of genomic DNA with pairs of specific oligonucleotide primers; they may contain high-copy, dispersed genomic sequences within the amplified region. Amplified RAPD products were cloned and sequenced. The sequence was used to design 24-mer oligonucleotide primers for each end. All pairs of SCAR primers resulted in the amplification of single major bands the same size as the RAPD fragment cloned. Polymorphism was either retained as the presence or absence of amplification of the band or appeared as length polymorphisms that converted dominant RAPD loci into codominant SCAR markers. This study provided information on the molecular basis of RAPD markers. The amplified fragment contained no obvious repeated sequences beyond the primer sequence. Five out of eight pairs of SCAR primers amplified an alternate allele from both parents of the mapping population; therefore, the original RAPD polymorphism was likely due to mismatch at the primer sites.

1,366 citations

Journal ArticleDOI
TL;DR: This work used the polymerase chain reaction to attach a 40-base-pair G + C-rich sequence, designated a GC-clamp, to one end of amplified DNA fragments that encompass regions of the mouse and human beta-globin genes, allowing the detection of mutations that were previously indistinguishable by DGGE.
Abstract: Denaturing gradient gel electrophoresis (DGGE) can be used to distinguish two DNA molecules that differ by as little as a single-base substitution. This method detects approximately 50% of all possible single-base changes in DNA fragments ranging from 50 to approximately 1000 base pairs. To increase the number of single-base changes that can be distinguished by DGGE, we used the polymerase chain reaction to attach a 40-base-pair G + C-rich sequence, designated a GC-clamp, to one end of amplified DNA fragments that encompass regions of the mouse and human beta-globin genes. We show that this GC-clamp allows the detection of mutations, including the hemoglobin sickle (HbS) and hemoglobin C (HbC) mutations within the human beta-globin gene, that were previously indistinguishable by DGGE. In addition to providing an easy way to attach a GC-clamp to genomic DNA fragments, the polymerase chain reaction technique greatly increases the sensitivity of DGGE. With this approach, DNA fragments derived from less than 5 ng of human genomic DNA can be detected by ethidium bromide staining of the gel, obviating the need for radioactive probes. These improvements extend the applicability of DGGE for the detection of polymorphisms and mutations in genomic and cloned DNA.

1,357 citations

Journal ArticleDOI
TL;DR: COBRA shows that methylation levels in the original DNA sample are represented by the relative amounts of digested and undigested PCR product in a linearly quantitative fashion across a wide spectrum of DNAmethylation levels.
Abstract: We report here on a quantitative technique called COBRA to determine DNA methylation levels at specific gene loci in small amounts of genomic DNA. Restriction enzyme digestion is used to reveal methylation-dependent sequence differences in PCR products of sodium bisulfite-treated DNA as described previously. We show that methylation levels in the original DNA sample are represented by the relative amounts of digested and undigested PCR product in a linearly quantitative fashion across a wide spectrum of DNA methylation levels. In addition, we show that this technique can be reliably applied to DNA obtained from microdissected paraffin-embedded tissue samples. COBRA thus combines the powerful features of ease of use, quantitative accuracy, and compatibility with paraffin sections.

1,340 citations

Journal ArticleDOI
TL;DR: This procedure utilizes simultaneous genomic DNA amplification of multiple widely separated sequences and should permit deletion scanning at any hemizygous locus and it is demonstrated the application of this multiplex reaction for prenatal and postnatal diagnosis of DMD.
Abstract: The application of recombinant DNA technology to prenatal diagnosis of many recessively inherited X-linked diseases is complicated by a high frequency of heterogeneous, new mutations (1). Partial gene deletions account for more than 50% of Duchenne muscular dystrophy (DMD) lesions, and approximately one-third of all cases result from a new mutation (2-5). We report the isolation and DNA sequence of several deletion prone exons from the human DMD gene. We also describe a rapid method capable of detecting the majority of deletions in the DMD gene. This procedure utilizes simultaneous genomic DNA amplification of multiple widely separated sequences and should permit deletion scanning at any hemizygous locus. We demonstrate the application of this multiplex reaction for prenatal and postnatal diagnosis of DMD.

1,323 citations


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Performance
Metrics
No. of papers in the topic in previous years
YearPapers
2023258
2022431
2021232
2020261
2019273
2018339