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.

RNA-programmed genome editing in human cells

Abstract: Type II CRISPR immune systems in bacteria use a dual RNA-guided DNA endonuclease, Cas9, to cleave foreign DNA at specific sites. We show here that Cas9 assembles with hybrid guide RNAs in human cells and can induce the formation of double-strand DNA breaks (DSBs) at a site complementary to the guide RNA sequence in genomic DNA. This cleavage activity requires both Cas9 and the complementary binding of the guide RNA. Experiments using extracts from transfected cells show that RNA expression and/or assembly into Cas9 is the limiting factor for Cas9-mediated DNA cleavage. In addition, we find that extension of the RNA sequence at the 3' end enhances DNA targeting activity in vivo. These results show that RNA-programmed genome editing is a facile strategy for introducing site-specific genetic changes in human cells.DOI:http://dx.doi.org/10.7554/eLife.00471.001.

Methods in yeast genetics : a Cold Spring Harbor Laboratory course manual

Abstract: Experiments: looking at yeast cells isolation and characterization of auxotrophic, temperature-sensitive and UV-sensitive mutants meiotic mapping mitotic recombination and random spore analysis transformation of yeast synthetic lethal mutants gene replacement isolation of ras2 suppressors manipulating cell types isolating mutants by insertional mutagenesis two-hybrid protein interaction method. Techniques and protocols: high-efficiency transformation of yeast "Lazy Bones" plasmid transformation of yeast colonies yeast DNA isolations yeast DNA miniprep (40 ml) yeast DNA miniprep (5 ml) a ten-minute DNA preparation from yeast yeast genomic DNA - glass bead preparation yeast protein extracts yeast RNA isolation hydroxylamine mutagenesis of plasmid DNA assay of beta-galactosidase in yeast plate assay for carboxypeptidase Y random spore analysis yeast vital stains yeast immunofluorescence actin staining in fixed cells PCR protocol for PCR-mediated gene disruption yeast colony PCR protocol measuring yeast cell density by spectrophotometry cell synchrony chromatin immunoprecipitation flow cytometry of yeast DNA logarithmic growth EMS mutagenesis tetrad dissection making a tetrad dissection needle picking zygotes determining plating efficiency. Appendices: media stock preservation yeast genetic and physical maps templates for making streak plates electrophoretic karyotypes of strains for southern blot mapping strains counting yeast cells with a standard hemocytometer chamber tetrad scoring sheet trademarks suppliers.

Analysis of enzymatically amplified beta-globin and HLA-DQ alpha DNA with allele-specific oligonucleotide probes.

Abstract: Allelic sequence variation has been analysed by synthetic oligonucleotide hybridization probes which can detect single base substitutions in human genomic DNA. An allele-specific oligonucleotide (ASO) will only anneal to sequences that match it perfectly, a single mismatch being sufficient to prevent hybridization under appropriate conditions. To improve the sensitivity, specificity and simplicity of this approach, we used the polymerase chain reaction (PCR) procedure to enzymatically amplify a specific segment of the beta-globin or HLA-DQ alpha gene in human genomic DNA before hybridization with ASOs. This in vitro amplification method, which produces a greater than 10(5)-fold increase in the amount of target sequence, permits the analysis of allelic variation with as little as 1 ng of genomic DNA and the use of a simple 'dot blot' for probe hybridization. As a further simplification, PCR amplification has been performed directly on crude cell lysates, eliminating the need for DNA purification.

Preparation of Genomic DNA from Bacteria

Abstract: Most protocols for the preparation of bacterial genomic DNA consist of lysis, followed by incubation with a nonspecific protease and a series of extractions prior to precipitation of the nucleic acids. Such procedures effectively remove contaminating proteins, but are not effective in removing exopolysaccharides which can interfere with the activity of enzymes such as restriction endonucleases and ligases. In this unit, however, the protease incubation is followed by a CTAB extraction whereby CTAB complexes both with polysaccharides and with residual protein, effectively removing both in the subsequent emulsification and extraction. This procedure is effective in producing digestible chromosomal DNA from a variety of gram-negative bacteria, all of which normally produce large amounts of polysaccharides. If large amounts of exceptionally clean DNA are required, the procedure can be scaled up and the DNA purified on a CsCl gradient, as described in the alternate protocol.

High sensitivity mapping of methylated cytosines.

Abstract: An understanding of DNA methylation and its potential role in gene control during development, aging and cancer has been hampered by a lack of sensitive methods which can resolve exact methylation patterns from only small quantities of DNA. We have now developed a genomic sequencing technique which is capable of detecting every methylated cytosine on both strands of any target sequence, using DNA isolated from fewer than 100 cells. In this method, sodium bisulphite is used to convert cytosine residues to uracil residues in single-stranded DNA, under conditions whereby 5-methylcytosine remains non-reactive. The converted DNA is amplified with specific primers and sequenced. All the cytosine residues remaining in the sequence represent previously methylated cytosines in the genome. The work described has defined procedures that maximise the efficiency of denaturation, bisulphite conversion and amplification, to permit methylation mapping of single genes from small amounts of genomic DNA, readily available from germ cells and early developmental stages.