A method for cloning and sequencing long palindromic DNA junctions.
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 sae2Delta 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.
Rattray
Alison J
AJ
GRCBL/NCI-FCRDC, PO Box B/ Building 539 Room 151, Frederick, MD 21702, USA. rattray@ncifcrf.gov
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000737:chemistry*; D004720:Endonucleases; D008969:Molecular Sequence Data; D010957:Plasmids / Q000737:chemistry; D016133:Polymerase Chain Reaction; D012091:Repetitive Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA / Q000379:methods*; D013447:Sulfites / Q000737:chemistry
C107849:SAE2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D013447:Sulfites; D004247:DNA; D004720:Endonucleases; C009279:sodium bisulfite