Restricted PCR: amplification of an individual sequence flanked by a highly repetitive element from total human DNA

Puskás, László G.; Fartmann, Berthold; Bottka, Sándor

doi:10.1093/nar/22.15.3251

Cited by 11 publications

(7 citation statements)

References 6 publications

(4 reference statements)

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“…Thus, convenient PCR-based methods have been developed instead of construction and screening of genome library. Some classical methods include inverse PCR [2,[4][5][6], PCR of various adapters (vectorett or linker) [7][8][9][10][11], restrictionsite PCR [12][13][14], and TAIL-PCR [15][16][17][18][19][20], which have extensive applications to genome walking in various organisms including microorganisms.…”

Section: Introductionmentioning

confidence: 99%

A Novel and Simple PCR Walking Method for Rapid Acquisition of Long DNA Sequence Flanking a Known Site in Microbial Genome

Luo

et al. 2010

Mol Biotechnol

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Acquisition of flanking sequence adjacent to a known DNA site is an important task in microbial genome-related research. In this study, we developed a new method containing two rounds of PCR followed by cloning and sequencing. Firstly, specific primer (SP) is added into the reaction system for primary locus-specific linear amplification, and then a complex long primer (CLP) is added into the cooled reaction system for only one cycle. Amplification products from the first round of PCR are directly purified without electrophoresis, diluted, and used as the templates of the second PCR. Secondly, one long specific primer (LSP) and one long base-fixed primer (LFP) are adopted. The amplicons are purified for cloning and sequencing. The achievement of specific amplification for long flanking region mainly depends on ingenious and precise settings of PCR programs, structure design of CLP primer, adding of CLP primer after specific linear amplification, concentration ratio of CLP and SP primer, applying long primers, etc. Through this method, we successfully performed the long PCR walkings (>1.5 Kb) on rpoB gene of Vibrio vulnificus, transposon-like gene of V. alginolyticus, and sto gene of V. cholerae. The method provides a robust and simple strategy for rapid amplification of long unknown DNA fragments from microbes.

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Section: Introductionmentioning

confidence: 99%

A Novel and Simple PCR Walking Method for Rapid Acquisition of Long DNA Sequence Flanking a Known Site in Microbial Genome

Luo

et al. 2010

Mol Biotechnol

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“…The first of these random priming PCR usually depends on priming highly repetitive sequences (e.g. ALU sequences) as well as the LTR [26,27]. By chance, if a junction is near an ALU repeat, it will be amplified.…”

Section: Discussionmentioning

confidence: 99%

“…Arbitrary primed PCR [28] is a variation of this technique where low initial annealing temperatures allow primers to anneal anywhere, while higher subsequent annealing temperatures then restrict the PCR to IHJ. Further manipulations are usually required to remove random fragments of genomic sequences amplified by the degenerate primers only [26,29,30]. A second basic approach, restriction digest and ligation-based techniques, has evolved from inverse PCR amplification [31], which requires circularization of digested fragments through complex cassettes designed to overcome cassette-cassette amplification (vectorette [32][33][34] and splinkterette [35,36] PCR), to the most utilized technique, LAM-PCR [14].…”

Section: Discussionmentioning

confidence: 99%

Flanking-sequence exponential anchored–polymerase chain reaction amplification: a sensitive and highly specific method for detecting retroviral integrant–host–junction sequences

et al. 2008

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Background-Retroviral vectors are regularly used to transduce stem cells and their derivatives for experimental and therapeutic purposes. Because these vectors integrate semi-randomly into the cellular genome, analysis of integranated retroviral DNA/host cell DNA junctions (IHJ) facilitates clonality studies of engrafted cells, allowing their differentiation, survival and fate to be tracked. In the case of any adverse events, IHJ analysis can allow the identification of potentially oncogenic integration sites. At present, most measures to assess IHJ are complex, insensitive and may be subject to IHJ selection bias inherent to the technology used.

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“…Hence, extending the applicability of Alu-PCR, the inserted foreign sequence can be directly amplified between the known inserted sequence and a human Alu consensus sequence and therefore identify the integrant-cellular junction sequence. Hence it is called 'novel Alu-PCR' [26]. The overall strategy is outlined in schematic form ( fig.…”

Section: Novel Alu-pcrmentioning

confidence: 99%

“…Two specific primers are needed in Alu-PCR: one primer annealing to the known integrant sequence and the other to human Alu repeat sequences. In order to avoid illegitimate products which are amplified from Alu sequences themselves, two techniques have been suggested [26]. First, the primers should be synthesized by incorporating deoxyuridine triphosphates (dUTPs).…”

Section: Novel Alu-pcrmentioning

confidence: 99%

Strategies for cloning unknown cellular flanking DNA sequences from foreign integrants

Hui¹,

Wang²,

Lo³

1998

Cellular and Molecular Life Sciences (CMLS)

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Many virus and transposon DNAs can integrate into the host genome. In this review, techniques, including inverse polymerase chain reaction (IPCR), novel Alu-PCR and vectorette- or splinkerette-PCR are introduced as possible strategies for cloning flanking DNA regions of the integrants. Targeted gene-walking PCR, restriction-site PCR, capture PCR, and panhandle PCR and boomerang DNA amplification are also described. The principles, advantages and limitations of each approach are discussed.

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Restricted PCR: amplification of an individual sequence flanked by a highly repetitive element from total human DNA

Cited by 11 publications

References 6 publications

A Novel and Simple PCR Walking Method for Rapid Acquisition of Long DNA Sequence Flanking a Known Site in Microbial Genome

A Novel and Simple PCR Walking Method for Rapid Acquisition of Long DNA Sequence Flanking a Known Site in Microbial Genome

Flanking-sequence exponential anchored–polymerase chain reaction amplification: a sensitive and highly specific method for detecting retroviral integrant–host–junction sequences

Strategies for cloning unknown cellular flanking DNA sequences from foreign integrants

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