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Paper Information

Journal:   SHAHID CHAMRAN UNIVERSITY JOURNAL OF SCIENCE   SUMMER 2006 , Volume NEW SERIES , Number 15 (SECTION B); Page(s) 35 To 44.
 
Paper: 

IMPROVED WHOLE GENOME AMPLIFICATION PROTOCOL AT SINGLE CELL LEVEL

 
 
Author(s):  GALEHDARI HAMID*, FOROUGHMAND A.M., ANDASHTI B.
 
* DEPT. OF BIOLOGY, SHAHID CHAMRAN UNIVERSITY, AHVAZ, IRAN
 
Abstract: 

Genetic analysis of limited amounts of genomic DNA plays an important role in many molecular biology investigations. A prerequisite for molecular analysis of single or few cells is a reliable and reproducible method for DNA preparation for subsequent polymerase chain reaction (PCR) analyses. We investigated the use of degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) to generate a template for LOH (loss of heterozygosity)- methodology and mutation detection. DOP-PCR employs a degenerate primer to produce non-specific uniform amplification of DNA. This approach has been successfully applied to microsatellite analyzing. We compared amplified whole genome performed by DOP-PCR to the genomic DNA as a template. Results were analyzed with respect to feasibility, amplification efficiency and reproducibility. DOP-PCR yielded overall satisfactory results. Accuracy and quality of genotypes generated from the DOP-PCR template also depends on several conditions such as the choice of DNA polymerase and cell treatment. The conclusion is that we have successfully used DOP-PCR to amplify our genomic DNA from single cell for subsequent genotyping and mutation analysis as a standard process.

 
Keyword(s): WGA, DOP-PCR, PEP-PCR
 
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