2.3. Identification of LDLR mutations

A re-sequencing microarray chip was used to identify candidate gene mutations from DNA samples provided by the five probands. The resequencing array was designed on the basis of photolithography and solid-phase DNA synthesis by Vita Genomics, Inc. and manufactured by Affymetrix (Santa Clara, CA).^[11] Each micro-array contained 12.6 kb of coding exon and flanking intron sequences (both sense and antisense) of the three most relevant FH causing genes, LDLR, ApoB, and PCSK9, in duplication.^[11] The FH arrays were scanned using the Affymetrix GeneChip scanner 3000 7G creating CEL files for subsequent analysis. According to the results of the microarray analysis, we amplified several mutated DNA fragments that would result in amino acid changes. Sanger sequencing was conducted to verify these mutations. Family verification was conducted by sequencing the DNA of both parents at the target segment. Novel mutations were tested in 50 unrelated healthy individuals. To confirm the heterozygous deletion mutation, clone sequencing analysis was conducted. T4 DNA ligase was used to ligate the target fragment to T vecto. Then the target fragment was transformed into Escherichia coli. Further the positive clonal were picked up and cultured. Plasmid extraction was conducted, and finally the desired fragment was amplified by PCR and sequenced.