Amplification and sequence analysis of DNA flanking integrated proviruses by a simple two-step polymerase chain reaction method

We describe a two-step polymerase chain reaction method that can be used for the amplification of cellular DNA sequences adjacent to an integrated retroviral provirus. The technique involves a partly degenerate, arbitrary primer that will hybridize in the provirus-flanking cellular DNA. By using this primer in combination with a biotinylated provirus-specific primer, a provirus-cellular DNA junction fragment can be isolated from the nonspecific amplification products by using streptavidin-coated magnetic beads. A second amplification employing a nested provirus-specific primer and a biotinylated nondegenerate primer derived from the partly degenerate primer followed by purification with streptavidin-coated beads enhances the specificity and the efficiency of recovery of a fragment(s) containing the unknown flanking sequences. In addition to being relevant in studies of viral integration sites, the method should be generally useful to analyze DNA sequences either upstream or downstream from a known sequence.

[1]  J. Horowitz,et al.  Germ line integration of a murine leukemia provirus into a retroviruslike sequence , 1987, Journal of virology.

[2]  J. Horowitz,et al.  Molecular and biological characterization of the endogenous ecotropic provirus of BALB/c mice , 1985, Journal of virology.

[3]  J. Riley,et al.  A novel, rapid method for the isolation of terminal sequences from yeast artificial chromosome (YAC) clones. , 1990, Nucleic acids research.

[4]  T. Grundström,et al.  SL3-3 enhancer factor 1 transcriptional activators are required for tumor formation by SL3-3 murine leukemia virus , 1991, Journal of virology.

[5]  T. Grundström,et al.  Binding of SL3-3 enhancer factor 1 transcriptional activators to viral and chromosomal enhancer sequences , 1991, Journal of virology.

[6]  L. Lanier,et al.  Polymerase chain reaction with single-sided specificity: analysis of T cell receptor delta chain. , 1989, Science.

[7]  W. Herr,et al.  Nucleotide sequence of AKV murine leukemia virus , 1984, Journal of virology.

[8]  F. Pedersen,et al.  Mammalian expression-and-transmission vector derived from Akv murine leukemia virus. , 1986, Gene.

[9]  J. Lenz,et al.  Determination of the leukaemogenicity of a murine retrovirus by sequences within the long terminal repeat , 1984, Nature.

[10]  Henry A. Erlich,et al.  Characterization of β-thalassaemia mutations using direct genomic sequencing of amplified single copy DNA , 1987, Nature.

[11]  F. Pedersen,et al.  Different relative expression from two murine leukemia virus long terminal repeats in unintegrated transfected DNA and in integrated retroviral vector proviruses , 1989, Journal of virology.

[12]  J. Silver,et al.  Novel use of polymerase chain reaction to amplify cellular DNA adjacent to an integrated provirus , 1989, Journal of virology.

[13]  P. Rabinovitch,et al.  Targeted gene walking polymerase chain reaction. , 1991, Nucleic acids research.

[14]  D. Kemp,et al.  A procedure for in vitro amplification of DNA segments that lie outside the boundaries of known sequences. , 1988, Nucleic acids research.

[15]  S. Winistorfer,et al.  Sequence specific generation of a DNA panhandle permits PCR amplification of unknown flanking DNA. , 1992, Nucleic acids research.

[16]  D. Hartl,et al.  27 – AMPLIFICATION OF FLANKING SEQUENCES BY INVERSE PCR , 1989 .

[17]  A. Rosenthal,et al.  Genomic walking and sequencing by oligo-cassette mediated polymerase chain reaction. , 1990, Nucleic acids research.

[18]  W. Gilbert,et al.  One-sided polymerase chain reaction: the amplification of cDNA. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[19]  F. Pedersen,et al.  The nucleotide sequence of the Akv murine leukemia virus genome. , 1984, Virology.

[20]  M. Uhlén,et al.  Direct solid phase sequencing of genomic and plasmid DNA using magnetic beads as solid support. , 1989, Nucleic acids research.