A Novel Universal Primer-Multiplex-PCR Method with Sequencing Gel Electrophoresis Analysis

Abstract

In this study, a novel universal primer-multiplex-PCR (UP-M-PCR) method adding a universal primer (UP) in the multiplex PCR reaction system was described. A universal adapter was designed in the 5′-end of each specific primer pairs which matched with the specific DNA sequences for each template and also used as the universal primer (UP). PCR products were analyzed on sequencing gel electrophoresis (SGE) which had the advantage of exhibiting extraordinary resolution. This method overcame the disadvantages rooted deeply in conventional multiplex PCR such as complex manipulation, lower sensitivity, self-inhibition and amplification disparity resulting from different primers, and it got a high specificity and had a low detection limit of 0.1 ng for single kind of crops when screening the presence of genetically modified (GM) crops in mixture samples. The novel developed multiplex PCR assay with sequencing gel electrophoresis analysis will be useful in many fields, such as verifying the GM status of a sample irrespective of the crop and GM trait and so on.

Development of SNAP markers based on nucleotide variability of WRKY genes in coconut and their validation using multiplex PCR

“…It was also important to adjust primer concentration according to the sizes of amplicons (Liu and Wu 2012) since there was competition among primers in multiplex PCR (Sint et al 2012). In multiplex PCR, some primers and their concentrations affect the efficiency of PCR reactions (Xu et al 2012).…”

“…Agarose gel electrophoresis can only differentiate 30 bp differences in fragments of less than 300 bp while larger DNA fragment requires larger size differences to be able to score the amplicons unambiguously (Le et al 2012;Liu and Wu 2012;Culley et al 2013). Although multiplex PCR has advantages, the potential disadvantages should also be considered before applying the approach such as selection among different sets of primers and low amplification efficiency because of primer competition (Xu et al 2012).…”

Randomly broken fragment PCR with 5′ end-directed adaptor for genome walking

“…A amount of 120 mg sample was measured for genomic DNA extraction using the CTAB method12. The DNA concentrations were determined spectrophotometrically at 260 nm using a UV/VIS spectrometer (Kontron, Neufahrn, Germany).…”

Development of multiplex PCR system for identification of glyphosate-tolerant sugar beet

“…ϳäáèðàþ÷è îïòèìàëüí³ óìîâè ïðîâåäåííÿ ÏËÐ, âèçíà÷àëè òàê³ ïàðàìåòðè ðåàêö³¿: ñêëàä ðåàêö³éíî¿ ñóì³ø³ -êîíöåíòðàö³ÿ ïðàéìåð³â, Taq-ïîë³ìåðàçè, Mg 2+ ; ïðîãðàìà àìïë³ô³êàö³¿ -òåìïåðàòóðà ã³áðèäèçàö³¿ ïðàéìåð³â, òðèâàë³ñòü êîaeíîãî êðîêó öèêëó àìïë³ô³êàö³¿, ê³ëüê³ñòü öèêë³â, òðèâàë³ñòü ïîïåðåäíüî¿ äåíàòóðàö³¿. Äàë³, âèêîðèñòîâó-þ÷è äàí³, îòðèìàí³ ó ïðîöåñ³ ïðîâåäåííÿ ìîíîïëåêñíèõ ðåàêö³é [14], áóâ ï³ä³áðàíèé ñêëàä ðåàêö³éíî¿ ñóì³ø³ äëÿ ìóëüòèïëåêñ-íî¿ ÏËÐ ñèñòåìè, ùî çàáåçïå÷óº ñòàá³ëüíå íàïðàöþâàííÿ àìïë³êîí³â [15][16][17][18][19][20].…”