General method for direct cloning of DNA fragments generated by the polymerase chain reaction

Kovalic, David; Kwak, Jin‐Hwan; Weisblum, Bernard

doi:10.1093/nar/19.16.4560

Cited by 125 publications

(64 citation statements)

References 5 publications

(1 reference statement)

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“…The PCR products were directly inserted into a Ttailed derivative of pGEM5tZ(+) [kindly provided by Bernard Weisblum (Kovalic et al, 1991)]. This T-tail vector provides a single 3' T-overhang at the insertion site, which can be ligated with 3' A-overhangs characteristically remaining on most Taq polymerase PCR products.…”

Section: Fluorescence In Situ Hybridization (Fish)mentioning

confidence: 99%

“…The negative control (no DNA) was blank after PCR using the universal primer (Telenius et al, 1992), while both a positive control (5 ng total human DNA) and dissected DNA resulted in a smear ranging from ~100-900 bp. PCR products were then directly inserted into a T-tailed derivative of pGEM5fZ(+) (Kovalic et al, 1991), generating a library of 20,000 clones. Examples of 12 clones from this library are shown in Fig.…”

Section: I C R O D I S S E C T I O N a N D F I S H A N A L Y S I S mentioning

confidence: 99%

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Rapid generation of region-specific genomic clones by chromosome microdissection: Isolation of DNA from a region frequently deleted in malignant melanoma

et al. 1992

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Malignant melanoma is frequently characterized by the deletion of the long arm of chromosome 6 (usually encompassing 6q16-q21). In an effort to saturate this region with DNA markers, microdissection and molecular cloning of DNA from banded human metaphases have been performed. This work was facilitated by the recent development of a novel chromosome microdissection scheme that omits microchemical manipulation of DNA. Microdissection was targeted on band 6q21. Direct PCR amplification of dissected DNA was first used as a probe in chromosomal in situ hybridization of normal metaphases to confirm the specificity of material excised for cloning. A genomic library of 20,000 clones, which is highly enriched for sequences encompassing 6q21, was then constructed. Clones from this library have been mapped against a human-rodent somatic cell hybrid mapping panel that divides chromosome 6 into seven regions, confirming the localization of probes within the target region. Direct PCR amplification of DNA excised by microdissection greatly simplifies and facilitates this chromosome band-specific cloning strategy. The isolation of microclones from this region of chromosome 6 should assist in establishing a physical map of the melanoma deletion region.

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Section: Fluorescence In Situ Hybridization (Fish)mentioning

confidence: 99%

Section: I C R O D I S S E C T I O N a N D F I S H A N A L Y S I S mentioning

confidence: 99%

Rapid generation of region-specific genomic clones by chromosome microdissection: Isolation of DNA from a region frequently deleted in malignant melanoma

et al. 1992

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“…adding a single thymidine at the 3# blunt ends of a linearized vector (Holton and Graham, 1991;Marchuk et al, 1991) and generating single 3#-T overhangs of a linearized vector by restriction endonuclease digestion (Kovalic et al, 1991;Mead et al, 1991;Ichihara and Kurosawa, 1993;Chen et al, 2006a). Although the former has been used to produce commercial cloning kits like the pGEM-T system, we selected the restriction endonuclease digestionmediated strategy to develop a TA cloning vector system because this approach is easy to use for individual laboratories.…”

Section: Construction Of the Zebata Systemmentioning

confidence: 99%

“…Although the former has been used to produce commercial cloning kits like the pGEM-T system, we selected the restriction endonuclease digestionmediated strategy to develop a TA cloning vector system because this approach is easy to use for individual laboratories. Previous publications have described the use of restriction enzyme XcmI (Kovalic et al, 1991;Mead et al, 1991) or AhdI/Eam1105I (Ichihara and Kurosawa, 1993;Chen et al, 2006a) to produce intermediate T-vectors. We chose XcmI as the digestion enzyme to develop the ZeBaTA cloning system because it had a better digestion efficiency than AhdI.…”

Section: Construction Of the Zebata Systemmentioning

confidence: 99%

A Versatile Zero Background T-Vector System for Gene Cloning and Functional Genomics

et al. 2009

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With the recent availability of complete genomic sequences of many organisms, high-throughput and cost-efficient systems for gene cloning and functional analysis are in great demand. Although site-specific recombination-based cloning systems, such as Gateway cloning technology, are extremely useful for efficient transfer of DNA fragments into multiple destination vectors, the two-step cloning process is time consuming and expensive. Here, we report a zero background TA cloning system that provides simple and high-efficiency direct cloning of PCR-amplified DNA fragments with almost no self-ligation. The improved T-vector system takes advantage of the restriction enzyme XcmI to generate a T-overhang after digestion and the negative selection marker gene ccdB to eliminate the self-ligation background after transformation. We demonstrate the feasibility and flexibility of the technology by developing a set of transient and stable transformation vectors for constitutive gene expression, gene silencing, protein tagging, protein subcellular localization detection, and promoter fragment activity analysis in plants. Because the system can be easily adapted for developing specialized expression vectors for other organisms, zero background TA provides a general, cost-efficient, and high-throughput platform that complements the Gateway cloning system for gene cloning and functional genomics.

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“…A synthetic oligonucleotide linker was introduced into the EcoRI site of the vector which introduced an NcoI site. A synthetic linker was then cloned into the new NcoI site which contained two XcmI sites [23]. Digestion with XcmI yields a vector with T overhangs at either terminus.…”

Section: Cdna Synthesismentioning

confidence: 99%

Characterization of a marsupial sperm protamine gene and its transcripts from the North American opossum (Didelphis marsupialis)

Winkfein

Nishikawa

Connor

et al. 1993

European Journal of Biochemistry

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A synthetic oligonucleotide primer, designed from marsupial protamine protein-sequence data [Balhorn, R., Corzett, M., Matrimas, J. A., Gummins, J. & Faden, B. (1989) Analysis of protamines isolated from two marsupials, the ring-tailed wallaby and gray short-tailed opossum, J. Cell. Biol. 107 was used to amplify, via the polymerase chain reaction, protamine sequences from a North American opossum (Didelphis marsupialis) cDNA. Using the amplified sequences as probes, several protamine cDNA clones were isolated. The protein sequence, predicted from the cDNA sequences, consisted of 57 amino acids, contained a large number of arginine residues and exhibited the sequence ARYR at its amino terminus, which is conserved in avian and most eutherian mammal protamines. Like the true protamines of trout and chicken, the opossum protamine lacked cysteine residues, distinguishing it from placental mammalian protamine 1 (P1 or stable) protamines. Examination of the protamine gene, isolated by polymerase-chain-reaction amplification of genomic DNA, revealed the presence of an intron dividing the protamine-coding region, a common characteristic of all mammalian P1 genes. In addition, extensive sequence identity in the 5' and 3' flanking regions between mouse and opossum sequences classify the marsupial protamine as being closely related to placental mammal P1. Protamine transcripts, in both birds and mammals, are present in two size classes, differing by the length of their poly(A) tails (either short or long). Examination of opossum protamine transcripts by Northern hybridization revealed four distinct mRNA species in the total RNA fraction, two of which were enriched in the poly(A)-rich fraction. Northern-blot analysis, using an intron-specific probe, revealed the presence of intron sequences in two of the four protamine transcripts. If expressed, the corresponding protein from intron-containing transcripts would differ from spliced transcripts by length (49 versus 57 amino acids) and would contain a cysteine residue.Protamine genes encode small basic proteins whose replacement of histones and other chromosomal proteins results in extreme nuclear condensation during spermatogenesis in many, but not all, organisms [2]. Protamine 1 (Pl) is found in all eutherian (placental) mammals examined to date, while in certain eutherians (primates and certain rodents) an additional protein, protamine 2 (P2), is also involved in nuclear condensation. Both P1 and P2 are highly basic proteins and the genes of both possess a single intron. Unlike P1, P2 is rich in histidine residues and is synthesized as a precursor protein, being cleaved to yield the mature protein [ 3 ] . However, recent studies indicate that other mammals, which do not express P2, possess P2 genes which are nonfunctional due to mutations within the P2 gene [4, 51.

show abstract

General method for direct cloning of DNA fragments generated by the polymerase chain reaction

Cited by 125 publications

References 5 publications

Rapid generation of region-specific genomic clones by chromosome microdissection: Isolation of DNA from a region frequently deleted in malignant melanoma

Rapid generation of region-specific genomic clones by chromosome microdissection: Isolation of DNA from a region frequently deleted in malignant melanoma

A Versatile Zero Background T-Vector System for Gene Cloning and Functional Genomics

Characterization of a marsupial sperm protamine gene and its transcripts from the North American opossum (Didelphis marsupialis)

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