Improved methods for the formation and stabilization of R-loops

David B. Kaback; Lynne M. Angerer; Norman Davidson

doi:10.1093/nar/6.7.2499

Improved methods for the formation and stabilization of R-loops

David B. Kaback, Lynne M. Angerer, Norman Davidson

New Jersey Medical School (NJMS), Microbiology

Research output: Contribution to journal › Article › peer-review

103 Scopus citations

Abstract

Improved methods for the formation and stabilization of R-loops for visualization in the electron microscope are presented. The two complementary strands of a duplex DNA are photochemically crosslinked once every 1 to 3 kb using 4, 5′, 8 trimethylpsoralen. R-loops are then formed by incubation with RNA in 70% formamide at a temperature above the DNA melting temperature. Finally, the R-loops are stabilized by modifying the free single strand of DNA with glyoxal, thus minimizing the displacement of the hybridized RNA by branch migration. In this manner R-loops can be formed and visualized at a high frequency irrespective of the base composition of the nucleic acid of interest.

Original language	English (US)
Pages (from-to)	2499-2517
Number of pages	19
Journal	Nucleic acids research
Volume	6
Issue number	7
DOIs	https://doi.org/10.1093/nar/6.7.2499
State	Published - Jun 11 1979

All Science Journal Classification (ASJC) codes

Genetics

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10.1093/nar/6.7.2499

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title = "Improved methods for the formation and stabilization of R-loops",

abstract = "Improved methods for the formation and stabilization of R-loops for visualization in the electron microscope are presented. The two complementary strands of a duplex DNA are photochemically crosslinked once every 1 to 3 kb using 4, 5′, 8 trimethylpsoralen. R-loops are then formed by incubation with RNA in 70% formamide at a temperature above the DNA melting temperature. Finally, the R-loops are stabilized by modifying the free single strand of DNA with glyoxal, thus minimizing the displacement of the hybridized RNA by branch migration. In this manner R-loops can be formed and visualized at a high frequency irrespective of the base composition of the nucleic acid of interest.",

author = "Kaback, {David B.} and Angerer, {Lynne M.} and Norman Davidson",

note = "Funding Information: We are indebted to Fiona Gibson, Lynna Hereford and Michael Rosbash for Poly(A) RNA; the Paul D. Elder Co. for Trioxsalen; and Peter Chandler, James Casey and Pauline Yen for valuable discussions. DBK was supported by a Damon Runyon-Walter Winchell Cancer Research Foundation Fellowship and an U.S.P.H.S. National Research Service Award. This research was supported by grants GM 10991 and GM 20927 from the U.S.P.H.S. and RR 07003, Biomedical Research Grant.",

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doi = "10.1093/nar/6.7.2499",

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T1 - Improved methods for the formation and stabilization of R-loops

AU - Kaback, David B.

AU - Angerer, Lynne M.

AU - Davidson, Norman

N1 - Funding Information: We are indebted to Fiona Gibson, Lynna Hereford and Michael Rosbash for Poly(A) RNA; the Paul D. Elder Co. for Trioxsalen; and Peter Chandler, James Casey and Pauline Yen for valuable discussions. DBK was supported by a Damon Runyon-Walter Winchell Cancer Research Foundation Fellowship and an U.S.P.H.S. National Research Service Award. This research was supported by grants GM 10991 and GM 20927 from the U.S.P.H.S. and RR 07003, Biomedical Research Grant.

PY - 1979/6/11

Y1 - 1979/6/11

N2 - Improved methods for the formation and stabilization of R-loops for visualization in the electron microscope are presented. The two complementary strands of a duplex DNA are photochemically crosslinked once every 1 to 3 kb using 4, 5′, 8 trimethylpsoralen. R-loops are then formed by incubation with RNA in 70% formamide at a temperature above the DNA melting temperature. Finally, the R-loops are stabilized by modifying the free single strand of DNA with glyoxal, thus minimizing the displacement of the hybridized RNA by branch migration. In this manner R-loops can be formed and visualized at a high frequency irrespective of the base composition of the nucleic acid of interest.

AB - Improved methods for the formation and stabilization of R-loops for visualization in the electron microscope are presented. The two complementary strands of a duplex DNA are photochemically crosslinked once every 1 to 3 kb using 4, 5′, 8 trimethylpsoralen. R-loops are then formed by incubation with RNA in 70% formamide at a temperature above the DNA melting temperature. Finally, the R-loops are stabilized by modifying the free single strand of DNA with glyoxal, thus minimizing the displacement of the hybridized RNA by branch migration. In this manner R-loops can be formed and visualized at a high frequency irrespective of the base composition of the nucleic acid of interest.

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JO - Nucleic acids research

JF - Nucleic acids research

IS - 7

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Improved methods for the formation and stabilization of R-loops

Abstract

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