• Journal of the Korean Society of Tobacco Science
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• v.37 no.1
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• pp.25-33
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• 2015

Genome walking is a par ticular application for identifying sequences of unknown genomic regions adjacent to a known region. Many genome walking methods based on polymerase chain reaction (PCR) are available. Even if earlier techniques suffer from low reproducibility, inefficiency, and non-specificity, improved strategies have been developed. In this study, we present an alternative strategy: the genomic DNA is digested with restriction enzymes. After cytosine overhangs at 5' ends, the fragments are ligated to linker adaptor s had guanine overhang at 3' ends. Then nested PCR is performed. The improvements in this strategy focus on two points. The first is the C tailing method using Pfu polymerase instead of the A tailing method based on nontemplate-dependent terminal transferase activity of Taq polymerase. Therefore unintended modification of target DNA can be prevented without A tailing error. The second point is the use of C/G-specific ligation had advantage in the ligation efficiency compared with A/T-specific ligation. Therefore, the C-G linker PCR method increases ligation efficiency between digested genomic DNA and adaptor DNA. As a result, the quantity of target DNA to amplify by PCR is enriched. We successfully used G-C linker PCR to retrieve flanking regions bordering the phophinothricin resistance gene in genetically modified tobacco (GMO).

• Journal of Microbiology and Biotechnology
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• v.4 no.3
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• pp.233-234
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• 1994

A general improved procedure for preparation of a cosmid library based on the use of a pBLcosT vectorwas described. The vector was modified to contain 2 tandem Xcml sites and was digested with Xcml to yield 2 terminal 3 T overhangs, capable of ligation with the insert that contains 2 terminal complementary 3 A overhangs. The resultant ligation mixture was packaged and a cosmid library in Escherichia coli was established.

• BMB Reports
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• v.34 no.1
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• pp.59-65
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• 2001

In subtractive hybridization, target sequences in the tester are enriched by hybridizing with an excess amount of driver, followed by removing the tester hybridized with the driver. All of existing subtractive cloning methods are designed to remove the tester/driver hybrid. The removal of hybrid, however, is often unsatisfactory For various reasons. In this study we developed a subtractive enrichment protocol in which the tester/driver can be completely removed by selecting only the tester/tester after hybridization. In this protocol both the tester and driver DNAs are ligated with same linker DNAs and amplified by polymerase chain reaction (PCR). The tester DNA is then digested with two different enzymes and used in subsequent hybridization with an excess driver. After hybridization, the DNA is ligated with the adaptor that is only compatible with the tester/tester. Since only the tester/tester can have the new adaptor, no tester/driver can be amplified by PCR in this protocol. Unlike other methods, a 100% subtraction efficiency can be achieved even though the enzymatic treatments used in the enrichment procedure are incomplete. Furthermore, only the hybridized tester DNA can have the new adaptor and be amplified by PCR, resulting in 100% denaturation in effect. The efficacy of this novel method was verified with the model system in which a known amount of the target sequence is included.

• Korean Journal of Veterinary Research
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• v.39 no.4
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• pp.797-805
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• 1999

T sergenti cDNA library were constructed to get a more broad information about the structural, functional or antigenic properties of the proteins, and analyzes for their partial cDNA sequences and expression sequences tags(ESTg). The mRNA were purified from T sergenti isolates to identify the information of antigen gene, then first and second strand cDNA was synthesized. EcoR I adaptor ligation and Xho I enzyme restriction were used to the synthesized cDNA, and ligated into a Uni-ZAP XR vector. T sergenti cDNA library was constructed with packaging and amplification in vitro. Antibody screening was performed with constructed T sergenti cDNA library using antisera against T sergenti. Among those clones, eight phagemids were rescued from the recombinant in vivo excision with f1 helper phage. Using the analysis of endonuclease restriction and PCR, the recombinant cDNA were proved having a 0.5-3.0kb of inserts. The eight of partial cDNA clones' sequences were obtained and examined for their homology using BLASTN and BLASTX. The eight of sequenced clones were classified into three groups according to the basis of database searches. A total 3,045bp of partial cDNA sequence were determined from six clones. The putatively identified clones contain a cytochrome c gene, a heat shock protein gene, a cyclophilin gene, and a ribosomal protein gene. The unidentified clones have a homology to ATP-binding protein(mtrA) gene of S argillaceus, DNA-binding protein(DBP) gene of Pseudorabies virus 85kDa merozoite protein gene of B bovis, mRNA spm1 protein of T annulata and glycine-rich RNA-binding protein mRNA of O sativa etc.