PCR-free mutation detection of BRCA1 on a zip-code microarray using ligase chain reaction.
We describe here ligation-based strategy to detect mutations in BRCA1 utilizing zip-code microarray technology. In our first approach, PCR was performed to amplify the genomic regions containing the mutation sites. The PCR products were then used as templates in a subsequent ligation reaction using two ligation primers that flanked the mutation site. The primary allele-specific primer is designed to contain a base of mutation site at its 3' end with 5' complementarity to the respective zip-code sequence while the secondary common primer is modified by biotin at its 3' end. Depending on the genotype of samples at the mutation site, the nick between the two ligation primers can be sealed in the presence of DNA ligase. The ligation products were then hybridized on the zip-code microarray followed by staining with streptavidine-cy3 to generate a fluorescent signal. Using this strategy we successfully genotyped selected Korean-specific mutation sites in exon 11 of BRCA1 with a wild type and two heterozygote mutant samples. Furthermore, we also demonstrated that ligase chain reaction using unamplified genomic DNA as direct templates is enough to generate sufficient signals for correct genotypings in a multiplexed manner, verifying first that PCR is not essential for this microarray-based strategy.
Girigoswami
Agnishwar
A
Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology (KAIST), 373-1 Guseong-dong, Yuseong-gu, Daejeon, 305-701, Republic of Korea.
Jung
Cheulhee
C
Mun
Hyo Young
HY
Park
Hyun Gyu
HG
D044466:Asian People / Q000235:genetics; D019313:BRCA1 Protein / Q000032:analysis* / Q000235:genetics* / Q000378:metabolism; D011088:DNA Ligases / Q000378:metabolism*; D004252:DNA Mutational Analysis; D005091:Exons / Q000235:genetics; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction
D019313:BRCA1 Protein; D011088:DNA Ligases