TY - JOUR
T1 - Trapping and breaking of in vivo nicked DNA during pulsed field gel electrophoresis
AU - Khan, Sharik R.
AU - Kuzminov, Andrei
N1 - Funding Information:
This work was supported by grant GM 073115 from the National Institutes of Health.
PY - 2013
Y1 - 2013
N2 - Pulsed field gel electrophoresis (PFGE) offers a high-resolution approach to quantify chromosomal fragmentation in bacteria, measured as percentage of chromosomal DNA entering the gel. The degree of separation in pulsed field gel (PFG) depends on the size of DNA as well as various conditions of electrophoresis such as electric field strength, time of electrophoresis, switch time, and buffer composition. Here we describe a new parameter, the structural integrity of the sample DNA itself, that influences its migration through PFGs. We show that subchromosomal fragments containing both spontaneous and DNA damage-induced nicks are prone to breakage during PFGE. Such breakage at single-strand interruptions results in artifactual decrease in molecular weight of linear DNA making accurate determination of the number of double-strand breaks difficult. Although breakage of nicked subchromosomal fragments is field strength independent, some high-molecular-weight subchromosomal fragments are also trapped within wells under the standard PFGE conditions. This trapping can be minimized by lowering the field strength and increasing the time of electrophoresis. We discuss how breakage of nicked DNA may be mechanistically linked to trapping. Our results suggest how to optimize conditions for PFGE when quantifying chromosomal fragmentation induced by DNA damage.
AB - Pulsed field gel electrophoresis (PFGE) offers a high-resolution approach to quantify chromosomal fragmentation in bacteria, measured as percentage of chromosomal DNA entering the gel. The degree of separation in pulsed field gel (PFG) depends on the size of DNA as well as various conditions of electrophoresis such as electric field strength, time of electrophoresis, switch time, and buffer composition. Here we describe a new parameter, the structural integrity of the sample DNA itself, that influences its migration through PFGs. We show that subchromosomal fragments containing both spontaneous and DNA damage-induced nicks are prone to breakage during PFGE. Such breakage at single-strand interruptions results in artifactual decrease in molecular weight of linear DNA making accurate determination of the number of double-strand breaks difficult. Although breakage of nicked subchromosomal fragments is field strength independent, some high-molecular-weight subchromosomal fragments are also trapped within wells under the standard PFGE conditions. This trapping can be minimized by lowering the field strength and increasing the time of electrophoresis. We discuss how breakage of nicked DNA may be mechanistically linked to trapping. Our results suggest how to optimize conditions for PFGE when quantifying chromosomal fragmentation induced by DNA damage.
KW - DNA trapping
KW - Double-strand breaks
KW - Field strength
KW - Single-strand breaks
KW - Time of electrophoresis
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U2 - 10.1016/j.ab.2013.06.001
DO - 10.1016/j.ab.2013.06.001
M3 - Article
C2 - 23770235
AN - SCOPUS:84888299036
SN - 0003-2697
VL - 443
SP - 269
EP - 281
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 2
ER -