Genomic sequencing by ligation mediated polymerase chain reaction using direct blotting and non-radioactive detection

Abstract

Genomic sequencing has become an important tool for analyzing uncloned cellular DNA with regard to the methylation status of cytidines as well as to ONA-protein interactions within cells. The hybridization step of the genomic sequencing procedure requires a very high sensitivity, rendering the method fairly difficult. Using a modified ligation mediated polymerase chain reaction procedure (LMPCR) and a sensitive non-radioactive detection method, we have developed a procedure avoiding the high amounts of radioactivity formerly needed for detection of chemically cleaved genomic DNA. The detection limit of our method of genomic sequencing is less than 1 μg mammalian DNA, which is much better than the detection limit of the original genomic sequencing method and comparable with the detection limit of radioactive detection after the LMPCR procedure. In addition to the advantages of the non-radioactive detection technique we simplified the blotting step of the genomic sequencing procedure by using the direct blotting electrophoresis method. The method was applied to a region 5′ to the human c-myc promoter of HeLa cells and was able to verify the sequence obtained by other authors and to specify the methylation status of five CpG-pairs within this sequence.

Key Words:

• direct blotting electrophoresis
• DNA methylation
• genomic sequencing
• ligation mediated PCR