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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Main Authors: Xianbo Jia, Xinjian Lin, Jichen Chen
Format: Article
Language:English
Published: SpringerOpen 2017-11-01
Series:AMB Express
Subjects:
Online Access:http://link.springer.com/article/10.1186/s13568-017-0495-x
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spelling 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
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AT xinjianlin linearandexponentialtailpcramethodforefficientandquickamplificationofflankingsequencesadjacenttotn5transposoninsertionsites
AT jichenchen linearandexponentialtailpcramethodforefficientandquickamplificationofflankingsequencesadjacenttotn5transposoninsertionsites
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