T-linker-specific ligation PCR (T-linker PCR): an advanced PCR technique for chromosome walking or for isolation of tagged DNA ends.
Dozens of PCR-based methods are available for chromosome walking from a known sequence to an unknown region. These methods are of three types: inverse PCR, ligation-mediated PCR and randomly primed PCR. However, none of them has been generally applied for this purpose, because they are either difficult or inefficient. Here we describe a simple and efficient PCR strategy--T-linker-specific ligation PCR (T-linker PCR) for gene or chromosome walking. The strategy amplifies the template molecules in three steps. First, genomic DNA is digested with 3' overhang enzymes. Secondly, primed by a specific primer, a strand of the target molecule is replicated by Taq DNA polymerase and a single A tail is generated on the 3' unknown end of the target molecule, and then a 3' overhang-T linker (named T-linker) is specifically ligated onto the target. Thirdly, the target is amplified by two rounds of nested PCR with specific primers and T-linker primers. T-linker PCR significantly improves the existing PCR methods for walking because it uses specific T/A ligation instead of arbitrary ligation or random annealing. To show the feasibility and efficiency of T-linker PCR, we have exploited this method to identify vector DNA or T-DNA insertions in transgenic plants.
Yuanxin
Yan
Y
The National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871, China.
Chengcai
An
A
Li
Li
L
Jiayu
Gu
G
Guihong
Tan
T
Zhangliang
Chen
C
D017360:Arabidopsis / Q000235:genetics; D036022:Capsid Proteins / Q000235:genetics; D016386:Chromosome Walking / Q000379:methods*; D004247:DNA / Q000302:isolation & purification*; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial / Q000032:analysis; D003627:Data Interpretation, Statistical; D005814:Genes, Viral; D018745:Genome, Plant; D023281:Genomics / Q000379:methods; D016254:Mutagenesis, Insertional; D009154:Mutation; D012275:Oryza / Q000235:genetics; D030821:Plants, Genetically Modified; D016133:Polymerase Chain Reaction / Q000379:methods*; D013698:Templates, Genetic
D036022:Capsid Proteins; D004269:DNA, Bacterial; C036483:T-DNA; C071650:S8 protein, rice gall dwarf virus; D004247:DNA; D004262:DNA Restriction Enzymes