Comparative analysis of T4 DNA ligases and DNA polymerases used in chromosome conformation capture assays.
Biotechniques
; 58(4): 195-9, 2015 Apr.
Article
in En
| MEDLINE
| ID: mdl-25861932
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.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
DNA Ligases
/
Chromosomes
/
DNA-Directed DNA Polymerase
Language:
En
Journal:
Biotechniques
Year:
2015
Document type:
Article
Affiliation country:
Israel