Frequent problems and their resolutions by using thermal asymmetric interlaced PCR (TAIL-PCR) to clone genes in Arabidopsis T-DNA tagged mutants

T-DNA insertional mutagenesis is a powerful tool in Arabidopsis functional genomics research. Previous studies have developed thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) as an efficient strategy in isolation of DNA sequences adjacent to known sequences in T-DNA tagged mutants....

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Published inBiotechnology, biotechnological equipment Vol. 29; no. 2; pp. 260 - 267
Main Authors Wu, Lei, Di, Dong-Wei, Zhang, Dan, Song, Bin, Luo, Pan, Guo, Guang-Qin
Format Journal Article
LanguageEnglish
Published England Taylor & Francis 04.03.2015
Taylor & Francis Ltd
Subjects
Agriculture and Environmental Biotechnology
Arabidopsis
Asymmetry
Cloning
complex T-DNA insertion
Deoxyribonucleic acid
DNA
Gene sequencing
high-throughput sequencing
Insertional mutagenesis
Mutagenesis
Mutants
Mutation
Next-generation sequencing
Nucleotide sequence
Polymerase chain reaction
T-DNA
T-DNA tagged mutants
TAIL-PCR
Tails
TAIL-PCR
high-throughput sequencing
T-DNA tagged mutants
complex T-DNA insertion
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Abstract T-DNA insertional mutagenesis is a powerful tool in Arabidopsis functional genomics research. Previous studies have developed thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) as an efficient strategy in isolation of DNA sequences adjacent to known sequences in T-DNA tagged mutants. However, a number of problems are encountered when attempts are made to clone flanking sequences in T-DNA tagged mutants. Therefore, it is necessary to improve the efficiency of cloning mutagenesis. Here, we present the most frequent problems and provide an improved method to increase TAIL-PCR efficiency. Even then, it is not always possible to successfully obtain flanking sequences; in such cases, we recommend using high-throughput sequencing to determine the mutations.
AbstractList T-DNA insertional mutagenesis is a powerful tool in Arabidopsis functional genomics research. Previous studies have developed thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) as an efficient strategy in isolation of DNA sequences adjacent to known sequences in T-DNA tagged mutants. However, a number of problems are encountered when attempts are made to clone flanking sequences in T-DNA tagged mutants. Therefore, it is necessary to improve the efficiency of cloning mutagenesis. Here, we present the most frequent problems and provide an improved method to increase TAIL-PCR efficiency. Even then, it is not always possible to successfully obtain flanking sequences; in such cases, we recommend using high-throughput sequencing to determine the mutations.
T-DNA insertional mutagenesis is a powerful tool in Arabidopsis functional genomics research. Previous studies have developed thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) as an efficient strategy in isolation of DNA sequences adjacent to known sequences in T-DNA tagged mutants. However, a number of problems are encountered when attempts are made to clone flanking sequences in T-DNA tagged mutants. Therefore, it is necessary to improve the efficiency of cloning mutagenesis. Here, we present the most frequent problems and provide an improved method to increase TAIL-PCR efficiency. Even then, it is not always possible to successfully obtain flanking sequences; in such cases, we recommend using high-throughput sequencing to determine the mutations.
T-DNA insertional mutagenesis is a powerful tool in functional genomics research. Previous studies have developed thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) as an efficient strategy in isolation of DNA sequences adjacent to known sequences in T-DNA tagged mutants. However, a number of problems are encountered when attempts are made to clone flanking sequences in T-DNA tagged mutants. Therefore, it is necessary to improve the efficiency of cloning mutagenesis. Here, we present the most frequent problems and provide an improved method to increase TAIL-PCR efficiency. Even then, it is not always possible to successfully obtain flanking sequences; in such cases, we recommend using high-throughput sequencing to determine the mutations.
Author Song, Bin
Luo, Pan
Zhang, Dan
Wu, Lei
Di, Dong-Wei
Guo, Guang-Qin
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Keywords TAIL-PCR
high-throughput sequencing
T-DNA tagged mutants
complex T-DNA insertion
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Snippet T-DNA insertional mutagenesis is a powerful tool in Arabidopsis functional genomics research. Previous studies have developed thermal asymmetric interlaced...
T-DNA insertional mutagenesis is a powerful tool in functional genomics research. Previous studies have developed thermal asymmetric interlaced polymerase...
T-DNA insertional mutagenesis is a powerful tool in Arabidopsis functional genomics research. Previous studies have developed thermal asymmetric interlaced...
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StartPage 260
SubjectTerms Agriculture and Environmental Biotechnology
Arabidopsis
Asymmetry
Cloning
complex T-DNA insertion
Deoxyribonucleic acid
DNA
Gene sequencing
high-throughput sequencing
Insertional mutagenesis
Mutagenesis
Mutants
Mutation
Next-generation sequencing
Nucleotide sequence
Polymerase chain reaction
T-DNA
T-DNA tagged mutants
TAIL-PCR
Tails
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Title Frequent problems and their resolutions by using thermal asymmetric interlaced PCR (TAIL-PCR) to clone genes in Arabidopsis T-DNA tagged mutants
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