Genomiphi dna amplification kit

Genomic DNA was extracted from peripheral blood leukocytes, by GoldMag extraction method (GoldMag Co. Ltd, Xi’an, China)^{23 (link),24 (link)} and stored at −80 °C until the beginning of the project. DNA concentration was measured using spectrometry (DU530UV/VIS spectrophotometer, Beckman Instruments, Fullerton, CA, USA). The following primers were used for CRYAB C-802G (rs14133): 5′-TTGACCATCACTGCTCTCTT-3′ and 5′-TTGGCAATGTGACA CATACC-3′; for CRYAB A-1215G (rs2228387): 59-ACCTGTTGGAGTCTGATCTT-39 and 59-ATGCACCTCAATCACATCTC-39; for CRYAB intron 2 (rs2070894): 59-GTCTAGAAGACTAAGTTAGG-39 and 59-AGAGAAGTCACAACTCAAGT-39. DNA amplification was performed with the GenomiPhi DNA amplification kit (Amersham Biosciences, Piscataway, NJ). According to the manufacturer’s protocols, SEQUENOM’s MassARRAY iPLEX assay was used to perform genotyping. To confirm the genotyping method, we also analyzed 5% of randomly selected samples using direct sequencing. These results for the two independent technicians were 100% concordant. Moreover, approximately 5% of the total samples were randomly selected for genotyping in duplicate using two methods to yielding 100% congruent results.

During surgery to control the patient’s disease, two 1 mm³ papilloma biopsies were collected and preserved in TRIzol. At the coordinating center, DNA was extracted from the laryngeal biopsies according to the manufacturer’s guidelines (Invitrogen, Carlsbad, CA, USA). To assess the quantity and quality of the isolated DNA, samples were tested by spectrophotometry using a ND-1000 spectrometer (NanoDrop) and by PCR using the GH20 (59-GAAGAGCCAAGGACAGGTAC-39) and PC04 (59-CAACTTCATCCACGTTCACC-39) primers, which span a 268-bp segment of the β-globin gene [7 (link)]. During the early phase of establishing the specimen collection, several samples failed to yield β-globin amplicons. Thus, the specimens that failed PCR were subjected to whole genome amplification (WGA) using the GenomiPhi DNA Amplification Kit from Amersham Biosciences (Piscataway, NJ, USA) following the manufacturer’s instructions. After the extraction of the first 45 laryngeal specimens, we decided to systematically carry out WGA on all extracted DNA. The products of the WGA reactions were used as template to PCR amplify the β-globin gene using GH20 and PC04 primers.