title: Comparative analysis of T4 DNA ligases and DNA polymerases used in chromosome conformation capture assays.
abstract: Three-dimensional (3-D) genome organization in the nuclear space affects various genomic functions. Circular chromosome conformation capture (4C-seq) is a powerful technique that allows researchers to measure long-range chromosomal interactions with a locus of interest across the entire genome. This method relies on enzymatic cleavage of cross-linked chromatin and consecutive ligation to create ligation junctions between physically adjacent loci, followed by PCR amplification of locus-specific associating loci. The enzymes used must meet 4C standards because variations in their efficiency and performance may affect the quality of the obtained data. Here we systematically compare the efficiency and reliability of different T4 DNA ligases and PCR DNA polymerases, assessing the most critical and technically challenging steps in 4C. The results of this analysis enable the use of cost-effective enzymes with superior specificity and efficiency for 4C and save time in screening for appropriate primers. This information provides users with flexibility in their experimental design and guidelines for adapting and testing any enzyme of choice for obtaining standardized results.
authors: The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel.
authors: Michal
authors: M
authors: Schwartz
authors: The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel.
authors: Avital
authors: A
authors: Sarusi
authors: The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel.
authors: Rachel T
authors: RT
authors: Deitch
authors: The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel.
authors: Moran
authors: M
authors: Tal
authors: The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel.
authors: Dana
authors: D
authors: Raz
authors: Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, National Institutes of Health, Bethesda, MD.
authors: Myong-Hee
authors: MH
authors: Sung
authors: School of Computer Science and Engineering, The Hebrew University of Jerusalem, Jerusalem, Israel.
authors: Tommy
authors: T
authors: Kaplan
authors: The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel.
authors: Ofir
authors: O
authors: Hakim
mesh_terms: D002874:Chromosome Mapping / Q000379:methods; D002875:Chromosomes / Q000378:metabolism* / Q000648:ultrastructure; D003432:Cross-Linking Reagents; D011088:DNA Ligases / Q000737:chemistry*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D008968:Molecular Conformation; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction / Q000379:methods
chemical_list: D003432:Cross-Linking Reagents; D004259:DNA-Directed DNA Polymerase; D011088:DNA Ligases
keywords: 3C; 4C; chromosome conformation capture; nuclear architecture