PALINDROMIC SEQUENCE WITH PALLENDROME DRIVERS
Long inverted repeats that may reflect de novo palindromes have been found in tumor cells and cancer cell lines, and are likely drivers of gene amplification. Natural AT-rich palindromes (PATRRs) exist at sites of some recurrent chromosomal rearrangements in humans and cause genetic disorders. The propensity of palindromes to adopt secondary structure interferes with DNA replication, transcription and repair, and leads to genome instability. Palindromes are also underrepresented in high-throughput sequencing results generated from libraries constructed by PCR amplification or sequencing steps that involve emulsion PCR amplification (Yang H. Palindromes longer than 200 bp cannot be amplified by traditional PCR using DNA polymerases with low strand displacement activity, nor can they be stably maintained in Escherichia coli. This is because perfect and near perfect palindromes, where a sequence is immediately followed by its exact inverse complement with very little or no spacer, are able to intrastrand anneal to form hairpin structures. Long DNA palindromes are difficult to directly analyze using standard molecular genetics methods. It will be useful for studying human cancers and other diseases associated with palindromes. The GAP-Seq approach underscores the importance of developing new tools for identifying and characterizing palindromes, and provides a new strategy to systematically assess palindromes in genomes. Because this protocol eliminates many of the false positives that plague earlier techniques, we have improved palindrome identification. Using MCF-7 breast cancer cell line as the proof-of-principle analysis, we have identified 35 palindrome candidates and physically characterized the top 5 candidates and their junctions. It also indicates the location of the novel junction, which can then be recovered. Unlike earlier protocols, it does not involve restriction enzymatic digestion prior to DNA snap-back thereby preserving longer DNA sequences. We developed a new protocol to identify palindromes that couples the S1 nuclease treated Cot0 DNA (GAPF) with high-throughput sequencing (GAP-Seq).
Because of their role in genomic instability and gene amplification in some human cancers, it is important to develop systematic approaches to detect and characterize DNA palindromes. The ability to form these hairpins results in genome instability, difficulties in maintaining clones in Escherichia coli and major problems for most DNA sequencing approaches. 2012.Closely spaced long inverted repeats, also known as DNA palindromes, can undergo intrastrand annealing to form DNA hairpins. When the complementary strand is read backwards, the sequence is 5’-GGATCC-3’ which is identical to the first one, making it a palindromic sequence. This is the sequence where the restriction endonuclease, BamHI, binds to and cleaves at a specific cleavage site. An example of a palindromic sequence is 5’-GGATCC-3’, which has a complementary strand, 3’-CCTAGG-5’. So if a sequence is palindromic, the nucleotide sequence of one strand would be the same as its reverse complementary strand. The pairing of nucleotides within the DNA double-helix is complementary which consist of Adenine (A) pairing with either Thymine (T) in DNA or Uracil (U) in RNA, while Cytosine (C) pairs with Guanine (G). Recognition sites of many restriction enzymes are palindromic. The sequence is the same when one strand is read left to right and the other strand is read right to left. A DNA sequence whose 5'-to-3' sequence is identical on each DNA strand.
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