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Detection of ligation products of DNA linkers with 5'-OH ends by denaturing PAGE silver stain.

ABSTRACT: To explore if DNA linkers with 5'-hydroxyl (OH) ends could be joined by commercial T4 and E. coli DNA ligase, these linkers were synthesized by using the solid-phase phosphoramidite method and joined by using commercial T4 and E. coli DNA ligases. The ligation products were detected by using denaturing PAGE silver stain and PCR method. About 0.5-1% of linkers A-B and E-F, and 0.13-0.5% of linkers C-D could be joined by T4 DNA ligases. About 0.25-0.77% of linkers A-B and E-F, and 0.06-0.39% of linkers C-D could be joined by E. coli DNA ligases. A 1-base deletion (-G) and a 5-base deletion (-GGAGC) could be found at the ligation junctions of the linkers. But about 80% of the ligation products purified with a PCR product purification kit did not contain these base deletions, meaning that some linkers had been correctly joined by T4 and E. coli DNA ligases. In addition, about 0.025-0.1% of oligo 11 could be phosphorylated by commercial T4 DNA ligase. The phosphorylation products could be increased when the phosphorylation reaction was extended from 1 hr to 2 hrs. We speculated that perhaps the linkers with 5'-OH ends could be joined by T4 or E. coli DNA ligase in 2 different manners: (i) about 0.025-0.1% of linkers could be phosphorylated by commercial T4 DNA ligase, and then these phosphorylated linkers could be joined to the 3'-OH ends of other linkers; and (ii) the linkers could delete one or more nucleotide(s) at their 5'-ends and thereby generated some 5'-phosphate ends, and then these 5'-phosphate ends could be joined to the 3'-OH ends of other linkers at a low efficiency. Our findings may probably indicate that some DNA nicks with 5'-OH ends can be joined by commercial T4 or E. coli DNA ligase even in the absence of PNK.

SUBMITTER: Gao F

PROVIDER: S-EPMC3384673 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Detection of ligation products of DNA linkers with 5'-OH ends by denaturing PAGE silver stain.

Gao Feng F Zhou Huafu H Li Wei W Zhang Xuerong X

PloS one 20120627 6

To explore if DNA linkers with 5'-hydroxyl (OH) ends could be joined by commercial T4 and E. coli DNA ligase, these linkers were synthesized by using the solid-phase phosphoramidite method and joined by using commercial T4 and E. coli DNA ligases. The ligation products were detected by using denaturing PAGE silver stain and PCR method. About 0.5-1% of linkers A-B and E-F, and 0.13-0.5% of linkers C-D could be joined by T4 DNA ligases. About 0.25-0.77% of linkers A-B and E-F, and 0.06-0.39% of li ...[more]

PMID: 22761747

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Project description:IntroductionThe aim of this study was to determine whether Electronic Nicotine Delivery Systems (ENDS) device type (disposable devices, replaceable cartridges, and refillables) at initial or first ENDS use predicts subsequent initiation of combustible tobacco products (cigarettes, hookah, cigars) among adolescents and/or differentiates between those who initiate use of both ENDS and combustible tobacco products at the same time.MethodsThe study examined data from the Texas Adolescent Tobacco and Marketing Surveillance System (TATAMS), a longitudinal population-based cohort of students in major metropolitan areas of Texas (n = 3907; N = 461 069). Data were collected every 6 months, from 2014 to 2018; 33.9% (n = 1324; N = 151 784) of the sample initiated ENDS use across this period. Unadjusted and adjusted logistic regression models were used to assess the odds of initiating combustible tobacco products at a subsequent or similar wave as ENDS initiation, given initial ENDS device type.ResultsAfter adjusting for sociodemographic factors, the odds of initiating combustible tobacco use subsequent to ENDS initiation were significantly lower among those who reported using Cartridges as their initial device type compared to those who reported Refillables as their initial device type (adjusted odds ratio = 0.42 [0.18-0.98], p = .05). In addition, after controlling for sociodemographic variables, the odds of initiating combustible tobacco use in the same wave as ENDS initiation were significantly higher among those who reported using Cartridges as their initial device type, compared with those who reported Refillables as their initial device type (adjusted odds ratio = 2.31 [1.05-5.10], p = .04). No significant differences were found in adjusted models when Disposables were compared to Refillables and Cartridges to Disposables.ConclusionENDS device type differentiates between adolescents who start using combustible tobacco products at the same time as initial ENDS use, or subsequently.ImplicationsPrevious research has shown ENDS use predicts subsequent combustible use among adolescents, but there is lack of research on the role of specific ENDS device types and the timing of initiation. Findings from this longitudinal study show that initiation of combustible tobacco product use varies by initial ENDS device type among adolescents. These findings can become a focal point for developing interventions for adolescents and could have regulatory implications for ENDS products.

Dataset Information

Detection of ligation products of DNA linkers with 5'-OH ends by denaturing PAGE silver stain.

Publications

Detection of ligation products of DNA linkers with 5'-OH ends by denaturing PAGE silver stain.

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