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Find video protocols related to scientific articles indexed in Pubmed.
Glycosylases and AP-cleaving enzymes as a general tool for probe-directed cleavage of ssDNA targets.
Nucleic Acids Res.
PUBLISHED: 01-15-2010
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The current arsenal of molecular tools for site-directed cleavage of single-stranded DNA (ssDNA) is limited. Here, we describe a method for targeted DNA cleavage that requires only the presence of an A nucleotide at the target position. The procedure involves hybridization of a complementary oligonucleotide probe to the target sequence. The probe is designed to create a deliberate G:A mismatch at the desired position of cleavage. The DNA repair enzyme MutY glycosylase recognizes the mismatch structure and selectively removes the mispaired A from the duplex to create an abasic site in the target strand. Addition of an AP-endonuclease, such as Endonuclease IV, subsequently cleaves the backbone dividing the DNA strand into two fragments. With an appropriate choice of an AP-cleaving enzyme, the 3- and 5-ends of the cleaved DNA are suitable to take part in subsequent enzymatic reactions such as priming for polymerization or joining by DNA ligation. We define suitable standard reaction conditions for glycosylase/AP-cleaving enzyme (G/AP) cleavage, and demonstrate the use of the method in an improved scheme for in situ detection using target-primed rolling-circle amplification of padlock probes.
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Genome-wide methylation screen in low-grade breast cancer identifies novel epigenetically altered genes as potential biomarkers for tumor diagnosis.
FASEB J.
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Aberrant DNA methylation constitutes a well-established epigenetic marker for breast cancer. Changes in methylation early in cancer development may be clinically relevant for cancer detection and prognosis-based therapeutic decisions. In the present study, a combination of methyl-CpG immunoprecipitation (MCIp) and human CpG island (CGI) arrays was applied to compare genome-wide DNA methylation profiles in 10 low-grade in situ and invasive breast cancers against 10 normal breast samples. In total, 214 CGIs were found to be hypermethylated in ?6 of 10 tumors. Functional term enrichment analyses revealed an overrepresentation of homeobox genes and genes involved in transcription and regulation of transcription. Significant hypermethylation of 11 selected genes in tumor vs. normal tissue was validated in two independent sample sets (45 tumors and 11 controls, 43 tumors and 8 controls) using quantitative EpiTyper technology. In tumors, median methylation levels of BCAN, HOXD1, KCTD8, KLF11, NXPH1, POU4F1, SIM1, and TCF7L1 were ?30% higher than in normal samples, representing potential biomarkers for tumor diagnosis. Using the 90th percentile of methylation levels in normal tissue as cutoff value, 62-92% of in situ samples (n=13), 72-97% of invasive samples from the first validation set (n=32), and 86-100% of invasive samples from the second validation set (n=43) were classified as hypermethylated. Hypermethylation of KLF11 and SIM1 might also be associated with increased risk of developing metastases. In summary, early methylation changes are frequent in the low-grade pathway of breast cancer and may be useful in the development of differential diagnostic and possibly also prognostic markers.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.