Comparative analysis of T4 DNA ligases and DNA polymerases used in chromosome conformation capture assays

Michal Schwartz, Avital Sarusi, Rachel T. Deitch, Moran Tal, Dana Raz, Myong Hee Sung, Tommy Kaplan, Ofir Hakim*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

6 Scopus citations

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.

Original languageAmerican English
Pages (from-to)195-199
Number of pages5
JournalBioTechniques
Volume58
Issue number4
DOIs
StatePublished - 2015

Bibliographical note

Publisher Copyright:
© 1983-2010 BioTechniques.

Keywords

  • 3C
  • 4C
  • Chromosome conformation capture
  • Nuclear architecture

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