Segregation of partly melted molecules: isolation of CpG islands by polyacrylamide gel electrophoresis

Abstract The technique of segregation of partly melted molecules (SPM) is a convenient and efficient method to isolate DNA fragments associated with CpG islands. The approach is conceptually simple and uses denaturant gradient gel electrophoresis to separate DNA molecules digested with restriction endonucleases. The SPM methodology has successfully been applied to the identification of genes from anonymous, unsequenced DNA fragments and CpG islands methylated in human cancer. In this article the theoretical background and practical application of the SPM method is reviewed.

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Isolation of DNA fragments associated with methylated CpG islands in human adenocarcinomas of the lung using a methylated DNA binding column and denaturing gradient gel electrophoresis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.96.6.2913 ◽

1999 ◽

Vol 96 (6) ◽

pp. 2913-2918 ◽

Cited By ~ 29

Author(s):

M. Shiraishi ◽

Y. H. Chuu ◽

T. Sekiya

Keyword(s):

Dna Binding ◽

Gel Electrophoresis ◽

Denaturing Gradient Gel Electrophoresis ◽

Cpg Islands ◽

Dna Fragments ◽

Methylated Dna ◽

Gradient Gel Electrophoresis

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Affinity purification and subunit structures of β-N-acetylhexosaminidases A and B from boar epididymis

Biochemical Journal ◽

10.1042/bj2191009 ◽

1984 ◽

Vol 219 (3) ◽

pp. 1009-1015 ◽

Cited By ~ 7

Author(s):

H C Parkes ◽

J L Stirling ◽

P Calvo

Keyword(s):

Sodium Dodecyl Sulphate ◽

Gel Electrophoresis ◽

Affinity Purification ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Deae Cellulose ◽

Gradient Gel Electrophoresis ◽

Sepharose 4B ◽

Electrophoretic Transfer ◽

Peptide Maps

beta-N-Acetylhexosaminidase from boar epididymis was separated into two forms, A and B, on DEAE-cellulose. Both these forms were excluded from Sepharose S-200 and had apparent Mr values of 510 000 on gradient gel electrophoresis under non-denaturing conditions. Affinity chromatography on 2-acetamido-N-(6-aminohexanoyl)-2-deoxy-beta-D-glucopyranosylam ine coupled to CNBr-activated Sepharose 4B was used to separate and purify beta-N-acetylhexosaminidases A and B that had specific activities of 115 and 380 mumol/min per mg of protein respectively. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of denatured beta-N-acetylhexosaminidase A gave a single major component of Mr 67 000. beta-N-Acetylhexosaminidase B also had this component, and in addition had polypeptides of Mr 29 000 and 26 000. All these polypeptides were glycosylated. Antiserum to the B form precipitated form A from solution and reacted with the 67 000-Mr component or form A after electrophoretic transfer from sodium dodecyl sulphate/polyacrylamide gels to nitrocellulose sheets. The 67 000-Mr components of forms A and B yielded identical peptide maps when digested with Staphylococcus aureus V8 proteinase, and the 29 000-Mr and 26 000-Mr components in form B may be related to the 67 000-Mr polypeptide.

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Nearly all single base substitutions in DNA fragments joined to a GC-clamp can be detected by denaturing gradient gel electrophoresis

Nucleic Acids Research ◽

10.1093/nar/13.9.3131 ◽

1985 ◽

Vol 13 (9) ◽

pp. 3131-3145 ◽

Cited By ~ 364

Author(s):

Richard M. Myers ◽

Stuart G. Fischer ◽

Leonard S. Lerman ◽

Tom Maniatis

Keyword(s):

Gel Electrophoresis ◽

Denaturing Gradient Gel Electrophoresis ◽

Dna Fragments ◽

Single Base ◽

Base Substitutions ◽

Gradient Gel Electrophoresis

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The Electrophoretic Separation of Curved Cisplatin-Modifled DNA Fragments on Polyacrylamide Gels Is Dependent on the Voltage Gradient

Zeitschrift für Naturforschung C ◽

10.1515/znc-1998-9-1023 ◽

1998 ◽

Vol 53 (9-10) ◽

pp. 921-923

Author(s):

Julia Yaneva ◽

Jordanka Zlatanova

Keyword(s):

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Threshold Level ◽

Voltage Gradient ◽

Dna Fragments ◽

Electrophoretic Separation ◽

Polyacrylamide Gels ◽

Curved Dna ◽

Electrophoretic Behavior ◽

Param Eters

Polyacrylamide gel electrophoresis has been widely used to study DNA fragments containing sequence-dependent curvature. The anomalous electrophoretic behavior of curved DNA fragments on such gels allows their separation from straight fragments of the same length. Here we demonstrate that polyacrylamide gels can be successfully used to resolve DNA fragments modified at a single site by the antitumor drug cis-diamminedichloroplatinum(II) (cis-DDP, cisplatin) from their unmodified counterparts. However, the resolution strongly depends on the voltage gradient, being completely lost when it drops below a certain threshold level. The param eters of the electric field do not affect separation of ‘normal’ DNA fragments of comparable length.

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