Linear and exponential TAIL-PCR: a method for efficient and quick amplification of flanking sequences adjacent to Tn5 transposon insertion sites
Abstract Current genome walking methods are very time consuming, and many produce non-specific amplification products. To amplify the flanking sequences that are adjacent to Tn5 transposon insertion sites in Serratia marcescens FZSF02, we developed a genome walking method based on TAIL-PCR. This PCR...
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doaj-9f5be394c6a34314948a5084b761cc172020-11-25T00:41:46ZengSpringerOpenAMB Express2191-08552017-11-01711810.1186/s13568-017-0495-xLinear and exponential TAIL-PCR: a method for efficient and quick amplification of flanking sequences adjacent to Tn5 transposon insertion sitesXianbo Jia0Xinjian Lin1Jichen Chen2Institute of Soil and Fertilizer, Fujian Academy of Agricultural and SciencesInstitute of Soil and Fertilizer, Fujian Academy of Agricultural and SciencesInstitute of Soil and Fertilizer, Fujian Academy of Agricultural and SciencesAbstract Current genome walking methods are very time consuming, and many produce non-specific amplification products. To amplify the flanking sequences that are adjacent to Tn5 transposon insertion sites in Serratia marcescens FZSF02, we developed a genome walking method based on TAIL-PCR. This PCR method added a 20-cycle linear amplification step before the exponential amplification step to increase the concentration of the target sequences. Products of the linear amplification and the exponential amplification were diluted 100-fold to decrease the concentration of the templates that cause non-specific amplification. Fast DNA polymerase with a high extension speed was used in this method, and an amplification program was used to rapidly amplify long specific sequences. With this linear and exponential TAIL-PCR (LETAIL-PCR), we successfully obtained products larger than 2 kb from Tn5 transposon insertion mutant strains within 3 h. This method can be widely used in genome walking studies to amplify unknown sequences that are adjacent to known sequences.http://link.springer.com/article/10.1186/s13568-017-0495-xLETAIL-PCRGenome walkingLinear amplificationTn5 transposon |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Xianbo Jia Xinjian Lin Jichen Chen |
spellingShingle |
Xianbo Jia Xinjian Lin Jichen Chen Linear and exponential TAIL-PCR: a method for efficient and quick amplification of flanking sequences adjacent to Tn5 transposon insertion sites AMB Express LETAIL-PCR Genome walking Linear amplification Tn5 transposon |
author_facet |
Xianbo Jia Xinjian Lin Jichen Chen |
author_sort |
Xianbo Jia |
title |
Linear and exponential TAIL-PCR: a method for efficient and quick amplification of flanking sequences adjacent to Tn5 transposon insertion sites |
title_short |
Linear and exponential TAIL-PCR: a method for efficient and quick amplification of flanking sequences adjacent to Tn5 transposon insertion sites |
title_full |
Linear and exponential TAIL-PCR: a method for efficient and quick amplification of flanking sequences adjacent to Tn5 transposon insertion sites |
title_fullStr |
Linear and exponential TAIL-PCR: a method for efficient and quick amplification of flanking sequences adjacent to Tn5 transposon insertion sites |
title_full_unstemmed |
Linear and exponential TAIL-PCR: a method for efficient and quick amplification of flanking sequences adjacent to Tn5 transposon insertion sites |
title_sort |
linear and exponential tail-pcr: a method for efficient and quick amplification of flanking sequences adjacent to tn5 transposon insertion sites |
publisher |
SpringerOpen |
series |
AMB Express |
issn |
2191-0855 |
publishDate |
2017-11-01 |
description |
Abstract Current genome walking methods are very time consuming, and many produce non-specific amplification products. To amplify the flanking sequences that are adjacent to Tn5 transposon insertion sites in Serratia marcescens FZSF02, we developed a genome walking method based on TAIL-PCR. This PCR method added a 20-cycle linear amplification step before the exponential amplification step to increase the concentration of the target sequences. Products of the linear amplification and the exponential amplification were diluted 100-fold to decrease the concentration of the templates that cause non-specific amplification. Fast DNA polymerase with a high extension speed was used in this method, and an amplification program was used to rapidly amplify long specific sequences. With this linear and exponential TAIL-PCR (LETAIL-PCR), we successfully obtained products larger than 2 kb from Tn5 transposon insertion mutant strains within 3 h. This method can be widely used in genome walking studies to amplify unknown sequences that are adjacent to known sequences. |
topic |
LETAIL-PCR Genome walking Linear amplification Tn5 transposon |
url |
http://link.springer.com/article/10.1186/s13568-017-0495-x |
work_keys_str_mv |
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1725285729686257664 |