title: Preparing long probes by an asymmetric polymerase chain reaction-based approach for multiplex ligation-dependent probe amplification.
abstract: To clearly discriminate the results of simultaneous screening and quantification of up to 40 different targets-DNA sequences, long probes from 100 to 500 nt, rather than smaller or similar-sized synthetic ones, were adopted for multiplex ligation-dependent probe amplification (MLPA). To prepare the long probes, asymmetric polymerase chain reaction (PCR) was employed to introduce non-complementary stuffers in between the two parts of the MLPA probe with specially designed primers, then restriction enzymes were selected to digest the double-stranded DNAs, and finally polyacrylamide gel electrophoresis was used to purify the single-stranded DNAs (i.e., the long probes). By using this approach, 12 long probes were prepared and used to identify genetically modified (GM) maize. Our experimental results show that the prepared long probes were in full accordance with the designed ones and could be assembled in 4-, 7-, and 10-plex MLPA analysis without losing result specificity and accuracy, showing they were as effective and reliable in MLPA analysis as those prepared with M13-derived vectors. This novel asymmetric PCR-based approach does not need expensive equipment, special reagents, or complicated operations when compared with previous methods. Therefore, our new approach could make MLPA analysis more independent, efficient, and economical.
authors.affiliation: Animal and Plant Inspection and Quarantine Technical Center, Shenzhen Entry-Exit Inspection and Quarantine Bureau, Shenzhen, Guangdong 518045, China. Electronic address: lingxingyuan@hotmail.com.
authors.forename: Xing-Yuan
authors.initials: XY
authors.lastname: Ling
authors.affiliation: Animal and Plant Inspection and Quarantine Technical Center, Shenzhen Entry-Exit Inspection and Quarantine Bureau, Shenzhen, Guangdong 518045, China.
authors.forename: Guiming
authors.initials: G
authors.lastname: Zhang
authors.affiliation: Animal and Plant Inspection and Quarantine Technical Center, Shenzhen Entry-Exit Inspection and Quarantine Bureau, Shenzhen, Guangdong 518045, China.
authors.forename: Guang
authors.initials: G
authors.lastname: Pan
authors.affiliation: Animal and Plant Inspection and Quarantine Technical Center, Shenzhen Entry-Exit Inspection and Quarantine Bureau, Shenzhen, Guangdong 518045, China.
authors.forename: Hai
authors.initials: H
authors.lastname: Long
authors.affiliation: Animal and Plant Inspection and Quarantine Technical Center, Shenzhen Entry-Exit Inspection and Quarantine Bureau, Shenzhen, Guangdong 518045, China.
authors.forename: Yinghui
authors.initials: Y
authors.lastname: Cheng
authors.affiliation: Animal and Plant Inspection and Quarantine Technical Center, Shenzhen Entry-Exit Inspection and Quarantine Bureau, Shenzhen, Guangdong 518045, China.
authors.forename: Caiyu
authors.initials: C
authors.lastname: Xiang
authors.affiliation: Animal and Plant Inspection and Quarantine Technical Center, Shenzhen Entry-Exit Inspection and Quarantine Bureau, Shenzhen, Guangdong 518045, China.
authors.forename: Lin
authors.initials: L
authors.lastname: Kang
authors.affiliation: Animal and Plant Inspection and Quarantine Technical Center, Shenzhen Entry-Exit Inspection and Quarantine Bureau, Shenzhen, Guangdong 518045, China.
authors.forename: Fei
authors.initials: F
authors.lastname: Chen
authors.affiliation: Shenzhen Academy of Inspection and Quarantine, Shenzhen, Guangdong 518001, China. Electronic address: 910774586@qq.com.
authors.forename: Zhinan
authors.initials: Z
authors.lastname: Chen
mesh_terms: D004277:DNA, Single-Stranded / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D060885:Multiplex Polymerase Chain Reaction
chemical_list: D004277:DNA, Single-Stranded
keywords: Asymmetric PCR; GM maize; Long MLPA probe; MLPA (multiplex ligation-dependent probe amplification)