12/25/2023 0 Comments T4 dna polymerase klenow fragment![]() By providing near base pair (bp) resolution of protein-DNA interactions, structural insights into protein complex organization are gained. It uses lambda exonuclease to digest sonicated chromatin to the formaldehyde-induced protein-DNA cross-linking point 6. ChIP-exo was developed as a variation of ChIP-seq to improve sensitivity and increase positional resolution by up to two orders of magnitude. Next, a protein of interest is immunoprecipitated and its attached DNA identified by either PCR, microarrays 3, or deep sequencing (ChIP-seq) 4, 5 listed in order of increased genome coverage and resolution. After quenching, chromatin is isolated and fragmented. Formaldehyde is used to covalently trap proteins at their in vivo binding locations. The simplicity of ChIP-exo now makes it a highly appropriate substitute for ChIP-seq, and for broader adoption.Ĭhromatin immunoprecipitation (ChIP) is a long-standing method for detecting protein-DNA interactions in vivo 1, 2. It is suitable for high-throughput parallelization. Importantly, the new ChIP-exo assays allow high-resolution detection of some protein-DNA interactions in organs and in as few as 27,000 cells. In comparing assays, we reveal substantial limitations in other ChIP-based assays. Greater library yields, lower processing time, and lower costs are achieved. This is achieved through assay optimization and use of Tn5 tagmentation and/or single-stranded DNA ligation. Here we describe greatly simplified ChIP-exo methods, each with use-specific advantages. Construction of ChIP-exo libraries is technically difficult. Consequently, and unlike other genomic assays, ChIP-exo provides structural information on genome-wide binding proteins. In contrast, ChIP-exo has relatively low noise and achieves near-base pair resolution. Although ChIP-seq is widely adopted in academic research, it has inherently high noise. Klenow fragment induced both possible transitions and deletions of 2 and 4 base pairs.ChIP-seq and ChIP-exo identify where proteins bind along any genome in vivo. ![]() The predominant mutations were sequenced and found to be transitions of G.C to A.T for T4 and modified T7 DNA polymerases, and A.T to G.C for Taq polymerase. The error rate for T4 DNA polymerase was not more than 3 x 10(-6) error per base duplication. The error rate induced in the 104-base-pair low-temperature melting domain of exon 3 of the human hypoxanthine/guanine phosphoribosyltransferase (HPRT) gene was approximately 3.4 x 10(-5) for modified T7, 1.3 x 10(-4) for Klenow fragment, and 2.1 x 10(-4) for Taq polymerases after a 10(6)-fold amplification. Incorrectly synthesized sequences were separated from the wild type by DGGE as mutant/wild-type heteroduplexes and the heteroduplex fraction was used to calculate the average error rate (mutations per base duplication). The strategy permitted direct enumeration and identification of point mutations created by T4, modified T7, Klenow fragment of polymerase I, and Thermus aquaticus (Taq) DNA polymerases. Denaturing gradient gel electrophoresis (DGGE) was used to separate and isolate the products of DNA amplification by polymerase chain reaction (PCR). ![]()
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