Topic
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 has developed a highly sensitive quantitative RT-PCR for β-actin, using the TaqManTM chemistry, and was able to quantitatively detect 10 β-Actin molecules per 100 ng of cDNA without coamplification of pseudogenes or genomic DNA.
Abstract: The use of common reverse transcription (RT)-PCR reference target sequences can produce false-positive results by amplification of either contaminating DNA or processed pseudogenes. Furthermore, qualitative RT-PCR alone cannot distinguish between high- and poor-quality cDNA preparations, which again may be crucial for the interpretation of low-abundance transcripts. We have developed a highly sensitive quantitative RT-PCR for β-actin, using the TaqManTM chemistry. Through this technique, we were able to quantitatively detect 10 β-actin molecules per 100 ng of cDNA without coamplification of pseudogenes or genomic DNA. Thus, the presented method may be advantageous for the interpretation of quantitative RT-PCR results.
PCR analysis may be difficult to interpret unless a reference target sequence is amplified in parallel. This control reaction is necessary to evaluate whether a sufficient amount of amplifiable material is present in the sample investigated. Thus, negative PCR can be defined as such only when amplification of a reference gene reveals a positive result. Amplifying a reference is especially important in RT-PCR because RNA can be degraded rapidly before or during cDNA synthesis.
As reference sequences for RT-PCR, so called “housekeeping” genes are preferred because they are constitutionally expressed by all cell types. β-Actin is an attractive candidate for reference coamplification because it exhibits only minor intraindividual kinetic changes and is not primarily affected by any human disease (1). However, a major concern with β-actin and other commonly used references such as glyceraldehyde-3-phosphate dehydrogenase is that processed pseudogenes can be coamplified by primers that are originally restricted to cDNA. This problem is difficult to circumvent because sequence homologies of >90% exist between mRNA and the respective pseudogenes (2); thus, the pseudogenes usually cannot be distinguished in gel electrophoresis. Furthermore, as long as genomic DNA contamination cannot safely be excluded, DNA may be coamplified together with cDNA. Thus, positive analysis of …
205 citations
TL;DR: Mouse and human DNA used as in vitro-labeled "high-complexity" probes revealed hybridization between specific herpesvirus DNA fragments on Southern transfers and repetitive sequences present at 10(3) to 10(5) copies per mammalian cell genome.
205 citations
TL;DR: Screening a bank of mouse genomic DNA, cloned in Charon 4A, indicates that the number of actin-related sequences in the mouse genome is much higher than 20, and five phages have been isolated representing part of a sub-family of 20 to 50 similar but non-identical sequences, only weakly homologous to actin cDNA probes.
205 citations
TL;DR: The development of a method that allows direct sequence analysis of the dystrophin gene in a rapid, accurate, and economical fashion is reported, and is applicable to other genes and should allow the development of widely available assays for any number of large, multiexon genes.
Abstract: Mutations in the dystrophin gene result in both Duchenne and Becker muscular dystrophy (DMD and BMD), as well as X-linked dilated cardiomyopathy. Mutational analysis is complicated by the large size of the gene, which consists of 79 exons and 8 promoters spread over 2.2 million base pairs of genomic DNA. Deletions of one or more exons account for 55%–65% of cases of DMD and BMD, and a multiplex polymerase chain reaction method—currently the most widely available method of mutational analysis—detects ∼98% of deletions. Detection of point mutations and small subexonic rearrangements has remained challenging. We report the development of a method that allows direct sequence analysis of the dystrophin gene in a rapid, accurate, and economical fashion. This same method, termed “SCAIP” (single condition amplification/internal primer) sequencing, is applicable to other genes and should allow the development of widely available assays for any number of large, multiexon genes.
204 citations
TL;DR: It is shown here that 5‐methylcytosine occurs in the DNA of D.melanogaster at a level of ∼1 in 1000–2000 cytosine residues in adult flies.
Abstract: It is commonly accepted that the DNA of Drosophila melanogaster does not contain 5-methylcytosine, which is essential in the development of most eukaryotes. We have developed a new, highly specific and sensitive assay to detect the presence of 5-methylcytosine in genomic DNA. The DNA is degraded to nucleosides, 5-methylcytosine purified by HPLC and, for detection by 1D- and 2D-TLC, radiolabeled using deoxynucleoside kinase and [γ-32P]ATP. Using this assay, we show here that 5-methylcytosine occurs in the DNA of D.melanogaster at a level of ∼1 in 1000–2000 cytosine residues in adult flies. DNA methylation is detectable in all stages of D.melanogaster development.
204 citations