Detection of differential gene copy number using denaturing high performance liquid chromatography.
Genomic rearrangements leading to deletion or duplication of gene(s) resulting in alterations in gene copy number underlie the molecular lesion in several genetic disorders. Methods currently used to determine gene copy number including real time PCR, southern hybridization, fluorescence in situ hybridization, densitometric scanning of PCR product etc. have certain disadvantages and are also expensive and time consuming. Herein, we describe a simple and rapid method to assess gene copy number using denaturing high performance liquid chromatography (dHPLC). We used X chromosome genes as model to compare the gene copy numbers present on this chromosome in males and females. DNA from these samples were amplified by biplex PCR using primer pairs specific for X chromosome genes only (target gene) and for genes present on both X and Y chromosomes (internal control). Amplified products were analyzed using HPLC under non-denaturing conditions. The ratio of peak areas (target gene/internal control) of the amplified products was approximately twice in female samples than male samples (p < 0.001) demonstrating that the differential gene copy number can be easily detected using this method. This method can potentially be used for diagnostic purpose where the need is to distinguish samples based on the differential gene copy numbers.
Kumar
Jitender
J
Institute of Genomics and Integrative Biology, Mall Road, Delhi-110007, India.
Kumar
Arun
A
Das
Swapan Kumar
SK
Shukla
Garima
G
Sengupta
Shantanu
S
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004247:DNA / Q000032:analysis / Q000235:genetics; D005260:Female; D018628:Gene Dosage / Q000235:genetics*; D006801:Humans; D008297:Male; D009691:Nucleic Acid Denaturation
D004247:DNA