Chemical linkage of nucleic acids to neutral and phosphorylated cellulose powders and isolation of specific sequences by affinity chromatography

Shih, Thomas Y.; Martin, Malcolm A.

doi:10.1021/bi00713a036

Cited by 45 publications

(22 citation statements)

References 27 publications

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“…DNA, either in liquid or bound to immobilized supports, has been used to hybridize and thereby sequester specific mRNAs from the plethora of cellular RNA species. These methods have included isolation of RNA-DNA hybrids by: selective binding to hydroxylapatite (1); selective exclusion through agarose (2) or Sepharose 4B (3); the use of DNA covalently bound to cellulose (4,5) or Sepharose (6); DNA bound directly to nitrocellulose (7)(8)(9); DNA enzymatically synthesized and covalently bound to oligo(dT)-cellulose (10).…”

mentioning

confidence: 99%

Purification and mapping of specific mRNAs by hybridization-selection and cell-free translation.

Ricciardi¹,

Miller²,

Roberts³

1979

Proc. Natl. Acad. Sci. U.S.A.

444

139

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We describe a simple procedure for isolating specific mRNAs and for mapping them to the regions of the DNA from which they originate. The method involves hybridization of total cytoplasmic RNA to restriction fragments of DNA that have been fractionated in agarose gels and immobilized on nitrocellulose filters. The hybridization-selected RNAs are eluted and translated in a cell-free system in order to identify their encoded polypeptides. Optimal hybridization and filter washing conditions are given for selection of mRNAs from adenovirus 2-infected cells and transformed cells.Synthesis of individual polypeptides directed by purified mRNAs in a cell-free translation system provides compelling evidence for the expression of specific genes. The ability to purify functional mRNAs is a prerequisite for understanding some of the detailed mechanisms involved in gene regulation. Characterization of purified mRNAs by cell-free translation, in combination with other techniques, can yield information about the spatial and topological arrangement of transcripts along the genome, the location of protein coding regions within the mRNA, mRNA structure, and mRNA abundance.Several methods for isolating specific mRNAs have been developed. DNA, either in liquid or bound to immobilized supports, has been used to hybridize and thereby sequester specific mRNAs from the plethora of cellular RNA species. These methods have included isolation of RNA-DNA hybrids by: selective binding to hydroxylapatite (1); selective exclusion through agarose (2) or Sepharose 4B (3); the use of DNA covalently bound to cellulose (4, 5) or Sepharose (6); DNA bound directly to nitrocellulose (7-9); DNA enzymatically synthesized and covalently bound to oligo(dT)-cellulose (10).Here we describe an efficient method by which specific mRNAs can be purified and used to determine the location of these mRNAs with respect to their DNA coding regions. This method relies on hybridization of total cytoplasmic RNA to restriction fragments of DNA which have been immobilized on nitrocellulose filters. The hybridization-selected mRNAs are eluted from the DNA and identified by the polypeptide products that are synthesized in a reticulocyte cell-free system. The map positions of the mRNA transcripts on the DNA can be determined directly because the genomic coordinates of the DNA restriction fragments are known.This procedure has several advantages. Most importantly, purification of the DNA restriction fragments is not required. Rather, DNA restriction fragments are fractionated by electrophoresis in agarose gels and then directly transferred to the nitrocellulose filter membrane by the method of Southern (11). In this way, many different DNA fragments may be easily handled in a single experiment. In addition, the nitrocellulose filters that contain the bound DNA fragments can be used several times. By this procedure, it is possible to identify the translation product of a specific mRNA which is otherwise obscured by the large number of polypeptides that are synthesized...

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mentioning

confidence: 99%

Purification and mapping of specific mRNAs by hybridization-selection and cell-free translation.

Ricciardi¹,

Miller²,

Roberts³

1979

Proc. Natl. Acad. Sci. U.S.A.

444

139

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“…Adsorption of nucleic acids to CM-cellulose leads to materials with high stability and good performance but the mechanism of the immobilization reaction is not well understood [45,46]. Affinity adsorbents with covalently immobilized polynucleotide ligands suffer from a low ligand concentration [40] or the introduction of additional charges on the matrix as a result of CNBr activation [29,55]. Small polynucleotides are best immobilized after introduction of a spacer-arm [56] which can on the other hand give rise to undesirable hydrophobic interactions with proteins.…”

Section: The Choice Of the Methods Of Immobilizationmentioning

confidence: 99%

“…It is, however, essential that the molecular weight of the polynucleotide does not exceed a limit above which non-specific interactions of the ligand with the polysaccharide matrix cannot be avoided. Because of the exceptional stability of the resulting adsorbents to temperature and formamide they are particularly useful materials for the chromatography of nucleic acids which hybridize to the ligand [5,38,40] and furthermore they have been used as insoluble templates for DNA-and RNA polymerase and initiators of the terminal deoxynucleotidyltransferase [41].…”

Section: Activation Of Phosphate Terminimentioning

confidence: 99%

“…Materials consisting ofpolynucleotides linked to phosphocellulose [6,40], nitrocellulose [42,43], aminoethylcellulose [8], and m-diazobenzoyloxymethylcellulose [44] have been developed and applied mainly to nucleic acid hybridization.…”

Section: Immobilization Onto Different Cellulose Deriva-mentioning

confidence: 99%

“…Besides hybridization on nitrocellulose filters, affinity chromatography of nucleic acids on polynucleotides covalently immobilized onto different matrices has been successfully applied to their base-specific separation [7,40,44,55].…”

Section: Application Of Immobilized Nucleic Acids To Affmity Chromatomentioning

confidence: 99%

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Affinity chromatography on columns containing nucleic acids

Potuzak

Dean

1978

FEBS Letters

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Immobilization of DNA onto a polymer support and its potentiality as immunoadsorbent

Kato

Ikada

2000

Biotechnol. Bioeng.

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Immobilization of DNA to the surface of poly(ethylene terephthalate) (PET) microfibers with a high specific surface area of 0.83 m2/g was carried out to give the fiber surface an affinity for anti‐DNA antibody. Following ozone oxidation, the microfibers were subjected to graft polymerization of monomers including acrylic acid, methacryloyloxyethyl phosphate, N,N‐dimethylaminoethyl methacrylate, N‐vinylformamide, and glycidyl methacrylate. Calf thymus DNA was immobilized to the grafted fiber surface through either covalent binding or polyion complexation with the grafted polymer chains. The highest surface density of DNA immobilized (0.6 μg/cm2) was obtained when DNA was immobilized through formation of phosphodiester linkage between the hydroxyl group of DNA and the phosphate group in grafted poly(methacryloyloxyethyl phosphate) using 1,1‐carbonyldiimidazole, or through polyion complexation between the anionic DNA and the cationic grafted poly(N,N‐dimethylaminoethyl methacrylate) chains. Batch adsorption of anti‐DNA antibody to the grafted PET fibers with and without DNA immobilized on their surface was conducted with serum obtained from systemic lupus erythematosus model mice. The DNA‐immobilized PET fibers exhibited a higher adsorption capacity and specificity than the others. In addition, the DNA‐immobilized fibers effectively adsorbed human anti‐DNA antibody.

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Chemical linkage of nucleic acids to neutral and phosphorylated cellulose powders and isolation of specific sequences by affinity chromatography

Abstract: Abbreviations used are: SV40, simian virus 40; Mes, sodium 2-(A-morpholino)ethanesulfonate; CMC, l-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate.

Cited by 45 publications

References 27 publications

Purification and mapping of specific mRNAs by hybridization-selection and cell-free translation.

Purification and mapping of specific mRNAs by hybridization-selection and cell-free translation.

Affinity chromatography on columns containing nucleic acids

Immobilization of DNA onto a polymer support and its potentiality as immunoadsorbent

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