Abi 3730xl pop7 dna sequencing analysis 5

Genomic DNA was isolated from peripheral blood of participants using the Wizard Genomic DNA Purification Kit(Promega, Madison, WI, USA). The complete coding sequence of human BMPR-IA gene (GenBank Accession No: NM004329.2) was amplified by polymerase chain reaction (PCR) using a standard protocol [23 (link)]. The DNA fragments containing the exon sequences of BMPR-IA gene was then respectively amplified using ten pairs of specific primers (Table 1). The PCR products were analyzed by direct sequencing using BigDye Terminator cycle sequencing on an ABI 3730XL POP7 DNA sequencing analysis 5.2 (Applied Biosystems, Carlsbad, CA, USA).Table 1

Ten pairs of primers were used to amplify the complete exon sequences of human BMPR-IA gene by PCR using a standard protocol

Primer	Forward	Reverse	Annealing temperature (°C)
Primer1	GCGTTGGATGGGAGCGATAA	GGAAGCTGCGCACAGTGTTG	58
Primer2	CTCACGTCGGTCCTGTCC	CCCTGCTCCATGCCTCAC	61
Primer3	CGGAGGAGTTTATCACCTCAGCAGAGC	TTCCATCATGGCCAAAAGTTACTAGCA	60
Primer4	AGAGATTGGAATCCGCCTGCCGGGCTT	ACCCGCGAGTGGGAGACAAAAGAGG	55
Primer5	GGACTATTGAGATTGTTTAATATAC	GAAAGAACAGAAGCAAGAAATAGTG	55
Primer6	GGACTATTGAGATTGTTTAATATAC	GAAAGAACAGAAGCAAGAAATAGTG	55
Primer7	CCACAATGCATCTGGCCCCAAGGAG	TGTGATTATTACACATGGCATGCCTGTATC	55
Primer8	ACATCAGATTACTGGGAGCC	TATAGCAAAGCAGCTGGAG	52
Primer9	AAGCCTTAAGAAGATAAATGA	ATTACCCTAATGAAGTTTTTG	62
Primer10	TGATTAGTGTCTCCAGTCAAGC	TCTCAGGTAAAAGGCAAAGTC	50

Genomic DNA was isolated using Wizard Genomic DNA Purification Kits (Promega, USA) from each individual included in the study. The entire BMP2 coding sequence was amplified in overlapping fragments of 300–600 base pairs by polymerase chain reaction (PCR). In terms of the BMP2 gene, several SNPs were already reported [21] (link), [22] (link). We analyzed rs2273073 (T/G) and rs235768 (A/T) among 11 SNPs within BMP2 gene that have been previously described. Primers were designed in close proximity to the selected SNPs which are summarized in Table 1 and Table 2; the sequences of the BMP2 gene were obtained from http://www.ncbi.nim.nih.gov. The PCR products including the SNPs were genotyped by direct sequencing using BigDye Terminator cycle sequencing on an ABI 3730XL POP7 DNA sequencing analysis 5.2 (Applied Biosystems, USA).