TY - JOUR
T1 - PCR-free mutation detection of BRCA1 on a zip-code microarray using ligase chain reaction.
AU - Girigoswami, Agnishwar
AU - Jung, Cheulhee
AU - Mun, Hyo Young
AU - Park, Hyun Gyu
N1 - Funding Information:
This work was supported by the Brain Korea 21 (BK21) program, the Korea Research Foundation and the Centre for Ultramicrochemical Process Systems.
PY - 2008/4/24
Y1 - 2008/4/24
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=48649089429&partnerID=8YFLogxK
U2 - 10.1016/j.jprot.2008.01.005
DO - 10.1016/j.jprot.2008.01.005
M3 - Article
C2 - 18276013
AN - SCOPUS:48649089429
SN - 1874-3919
VL - 70
SP - 897
EP - 902
JO - Journal of Proteomics
JF - Journal of Proteomics
IS - 6
ER -