Published online 8 November 2004
Nucleic Acids Research, Vol. 32 No. 19 © Oxford University Press 2004; all rights reserved
A method for cloning and sequencing long palindromic DNA junctions
GRCBL/NCI-FCRDC, PO Box B/ Building 539 Room 151, Frederick, MD 21702, USA
* Tel: +1 301 846 6715 (office), +1 301 846 7019 (lab); Fax: +1 301 846 6911; Email: rattray{at}ncifcrf.gov
Received July 13, 2004; Revised August 26, 2004; Accepted September 30, 2004
DNA sequences containing long adjacent inverted repeats (palindromes) are inherently unstable and are associated with many types of chromosomal rearrangements. The instability associated with palindromic sequences also creates difficulties in their molecular analysis: long palindromes (>250 bp/arm) are highly unstable in Escherichia coli, and cannot be directly PCR amplified or sequenced due to their propensity to form intra-strand hairpins. Here, we show that DNA molecules containing long palindromes (>900 bp/arm) can be transformed and stably maintained in Saccharomyces cerevisiae cells lacking a functional SAE2 gene. Treatment of the palindrome-containing DNA with sodium bisulfite at high temperature results in deamination of cytosine, converting it to uracil and thus reducing the propensity to form intra-strand hairpins. The bisulfite-treated DNA can then be PCR amplified, cloned and sequenced, allowing determination of the nucleotide sequence of the junctions. Our data demonstrates that long palindromes with either no spacer (perfect) or a 2 bp spacer can be stably maintained, recovered and sequenced from sae2
yeast cells. Since DNA sequences from mammalian cells can be gap repaired by their co-transformation into yeast cells with an appropriate vector, the methods described in this manuscript should provide some of the necessary tools to isolate and characterize palindromic junctions from mammalian cells.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  A. M. Casper, P. W. Greenwell, W. Tang, and T. D. Petes Chromosome Aberrations Resulting From Double-Strand DNA Breaks at a Naturally Occurring Yeast Fragile Site Composed of Inverted Ty Elements Are Independent of Mre11p and Sae2p Genetics, October 1, 2009; 183(2): 423 - 439. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Harada, M. Uchida, and N. Shimizu Episomal High Copy Number Maintenance of Hairpin-capped DNA Bearing a Replication Initiation Region in Human Cells J. Biol. Chem., September 4, 2009; 284(36): 24320 - 24327. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Tanaka, Y. Cao, D. A. Bergstrom, C. Kooperberg, S. J. Tapscott, and M.-C. Yao Intrastrand Annealing Leads to the Formation of a Large DNA Palindrome and Determines the Boundaries of Genomic Amplification in Human Cancer Mol. Cell. Biol., March 15, 2007; 27(6): 1993 - 2002. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. M. Lewis, S. Chen, J. N. Strathern, and A. J. Rattray New approaches to the analysis of palindromic sequences from the human genome: evolution and polymorphism of an intronic site at the NF1 locus Nucleic Acids Res., December 9, 2005; 33(22): e186 - e186. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. J. Rattray, B. K. Shafer, B. Neelam, and J. N. Strathern A mechanism of palindromic gene amplification in Saccharomyces cerevisiae Genes & Dev., June 1, 2005; 19(11): 1390 - 1399. [Abstract] [Full Text] [PDF]
|
|