Preparative and analytical purification of DNA from agarose.

Vogelstein, Bert; Gillespie, D. A. F.

doi:10.1073/pnas.76.2.615

Cited by 1,361 publications

(621 citation statements)

References 18 publications

Supporting

Mentioning

603

Contrasting

Unclassified

Order By: Relevance

“…Capture of long DNA fragments (genomic DNA) on flat glass surfaces is surprisingly rapid and efficient in the presence of chaotropic salts. The DNA binding properties of glass slides are similar to glass tubes [4] but DNA recovery in small volumes is facilitated by the flat slides compared to the curved glass surfaces. The capacity of DNA binding on the interior walls of 10 × 75 mm glass tubes was reported to be ~1 μg [4] but the capacity of the glass slide surfaces was not studied here.…”

Section: Discussionmentioning

confidence: 99%

“…The DNA binding properties of glass slides are similar to glass tubes [4] but DNA recovery in small volumes is facilitated by the flat slides compared to the curved glass surfaces. The capacity of DNA binding on the interior walls of 10 × 75 mm glass tubes was reported to be ~1 μg [4] but the capacity of the glass slide surfaces was not studied here. DNA recovery using the glass slide method is somewhat variable, but may improve when the glass slides are incorporated in microfluidic devices.…”

Section: Discussionmentioning

confidence: 99%

“…After Boom et al [1] published their "1 step" method for DNA isolation using silica particles, the use of monolithic glass surfaces [4] was not further examined by the scientific community. The glass slides are not convenient for routine purification of nucleic acids from complex biological samples compared to the easy-to-use commercial spin columns.…”

Section: Discussionmentioning

confidence: 99%

“…Therefore, DNA purification devices must be designed to use the least expensive materials possible, and without unnecessary engineering. Early researchers showed that the curved inner surfaces of glass test tubes could be used to purify DNA from agarose gels, but their binding capacity was deemed too small for most preparative experiments [4]. Currently, it is standard practice to maximize DNA binding surface area, for example by using silica glass bead matrices.…”

mentioning

confidence: 99%

See 3 more Smart Citations

Capture of genomic DNA on glass microscope slides

Nanassy

Haydock

Reed

2007

Analytical Biochemistry

View full text Add to dashboard Cite

It is well known that DNA strands bind to silica surfaces in the presence of high concentrations of chaotropic salts. We developed simple methods to evaluate binding and recovery of DNA on flat glass microscope slides and compared their properties to commercially available silica "spin columns". Surprisingly, genomic DNA was recovered efficiently from untreated glass slides. Binding and elution times from glass slides were optimized in experiments with DNA samples of various sizes and defined buffers. Average DNA recovery from 500 ng of input genomic DNA varied from 25-53 % for the glass slide protocol. Yields were comparable to spin columns. Human serum albumin and plasma components decreased DNA binding to glass several-fold in a concentration dependent manner. These results support the concept of using flat glass slides as DNA purification surfaces in microfluidics devices for PCR sample preparation. Keywords DNA extraction; silica surfaces; PCR; microfluidicsThe use of powdered glass or other high surface area silica materials has been widely adopted as a basic technique for purifying DNA from biological samples [1]. DNA strands bind efficiently to silica particles in the presence of high concentrations of chaotropic salts such as guanidinium thiocyanate or sodium perchlorate. After washing away contaminating substances with high salt buffers, the bound DNA is released from the silica surface in the presence of water or low salt buffer. The recovered DNA is suitable for use in PCR or other amplification methods. These methods are used extensively in medical diagnostics and forensics, in recombinant technology and for molecular genetics.Silica-based DNA purification is compatible with microfluidic diagnostic devices. Such "lab on a chip" devices may help bring the benefits of PCR-based molecular diagnosis to users and point-of-care settings that lack the capacity for standard laboratory-based DNA purification [2] [3]. In such applications there are significant pressures to keep costs low. Therefore, DNA purification devices must be designed to use the least expensive materials possible, and without unnecessary engineering. Early researchers showed that the curved inner surfaces of glass test tubes could be used to purify DNA from agarose gels, but their binding capacity was deemed too small for most preparative experiments [4]. Currently, it is standard practice to maximize DNA binding surface area, for example by using silica glass bead matrices. However, such engineering measures may not be necessary to achieve satisfactory DNA purification yields.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Capture of genomic DNA on glass microscope slides

Nanassy

Haydock

Reed

2007

Analytical Biochemistry

View full text Add to dashboard Cite

show abstract

“…12 Microdissection to optimize tumor cell content was performed, if necessary. RNA concentration was measured using a nanodrop spectrophotometer.…”

Section: Detection Of Fus-chop and Ewsr1-chop Fusion Genementioning

confidence: 99%

Primary retroperitoneal myxoid/round cell liposarcoma is a nonexisting disease: an immunohistochemical and molecular biological analysis

et al. 2009

View full text Add to dashboard Cite

Almost all primary retroperitoneal liposarcomas can be classified as well-/dedifferentiated liposarcoma. Rarely, however, primary retroperitoneal liposarcoma is classified as myxoid/round cell liposarcoma, based on the presence of myxoid areas and vascular crow's feet pattern, which has resulted in a debate on the classification of liposarcoma in the retroperitoneum. Genetically, myxoid/round cell liposarcoma and well-/dedifferentiated liposarcoma are different diseases. Myxoid/round cell liposarcoma is characterized by a translocation causing FUS-CHOP or EWSR1-CHOP fusion, whereas well-/dedifferentiated liposarcoma is characterized by an amplification of the 12q13-15 region, including MDM2 and CDK4 genes. As myxoid/round cell liposarcoma is highly radio-and chemosensitive, differentiation between subtypes is important to optimize treatment. We studied whether primary retroperitoneal liposarcomas diagnosed as myxoid/round cell liposarcoma represent molecularly true myxoid/round cell liposarcoma or are histopathological mimics and represent well-/dedifferentiated liposarcoma. Primary retroperitoneal myxoid/round cell liposarcoma (n ¼ 16) were compared to primary extremity myxoid/round cell liposarcoma (n ¼ 20). Histopathological and immunohistochemical features were studied. Amplification status of the 12q13-15 region was studied using a multiplex ligation-dependent probe amplification analysis, and FUS-CHOP or EWS-CHOP translocations were studied using RT-PCR. In primary retroperitoneal myxoid/round cell liposarcoma, MDM2 and CDK4 staining was both positive in 12 of 15 cases. In primary extremity myxoid/round cell liposarcoma, MDM2 was negative in 18/20 and CDK4 was negative in all cases. Multiplex ligation-dependent probe amplification showed the amplification of 12q13-15 region in 16/16 primary retroperitoneal myxoid/round cell liposarcomas and in 1/20 primary extremity myxoid/round cell liposarcomas. Translocation was present in all (18/18) primary extremity myxoid/round cell liposarcomas, but absent in all primary retroperitoneal myxoid/round cell liposarcomas. On the basis of immunohistochemical and molecular characteristics, apparent primary retroperitoneal myxoid/round cell liposarcoma can be recognized as well-/dedifferentiated liposarcoma with morphological features mimicking myxoid/round cell liposarcoma. In these cases, treatment should probably be specifically designed as for well-/dedifferentiated liposarcoma. Moreover, finding of myxoid/round cell liposarcoma translocations in a retroperitoneal localization is highly suggestive of metastasis and should prompt search for a primary localization outside the retroperitoneum. Keywords: liposarcoma; retroperitoneal; myxoid/round cell; well-/dedifferentiated; RT-PCR; multiplex ligation-dependant probe amplification Liposarcomas are a rare type of malignancy with a very heterogeneous morphological appearance and clinical course. The therapeutic approach is based on histopathological classification and is dependent on primary localization.Liposarcoma occurs...

show abstract

Structure, chromosomal localization, and brain expression of human Cx36 gene

et al. 1999

View full text Add to dashboard Cite

Rat connexin-36 (Cx36) is the first gap junction protein shown to be expressed predominantly in neuronal cells of the mammalian central nervous system. As a prerequisite for studies devoted to the investigation of the possible role of this connexin in human neurological diseases, we report the cloning and sequencing of the human Cx36 gene, its chromosomal localization, and its pattern of expression in the human brain analyzed by radioactive in situ hybridization. The determination of the human gene sequence revealed that the coding sequence of Cx36 is highly conserved (98% identity at the protein level with the mouse and rat Cx36 and 80% with the ortholog perch and skate Cx35), and that the gene structure is that typical of the Cx35/36 subgroup observed in the other species (presence of a single intron located within the coding region, 71 bp after the translation initiation site). The distribution of Cx36 in several regions of the human central nervous system is similar to that previously observed in rat brain. The most intense signal among the cerebral areas examined by in situ hybridization was observed in the inferior olivary complex, both in principal and accessory nuclei. A moderate labeling was also observed in several myelencephalic nuclei, in specific cells of the the cerebellar cortex, in a relatively large subpopulation of cells in the cerebral cortex, in the hilus of the dentate gyrus, and in the strata radiatum and oriens of hippocampal subfields. Moreover, labeled cells were revealed in all the lamina of the spinal cord gray matter. The chromosomal localization of the human Cx36 gene was determined by fluorescence in situ hybridization. The results allowed assignment of the gene to band 15q14, thus making it a possible candidate gene for a form of familial epilepsy previously linked to the same chromosomal band. The knowledge of the human Cx36 gene sequence, of its chromosomal localization, and of its pattern of expression opens new avenues for the analysis of its possible involvement in human genetic and acquired neuropathology.

show abstract

Preparative and analytical purification of DNA from agarose.

Cited by 1,361 publications

References 18 publications

Capture of genomic DNA on glass microscope slides

Capture of genomic DNA on glass microscope slides

Primary retroperitoneal myxoid/round cell liposarcoma is a nonexisting disease: an immunohistochemical and molecular biological analysis

Structure, chromosomal localization, and brain expression of human Cx36 gene

Contact Info

Product

Resources

About