Construction of gapped circular DNA from phage M13 by in vitro hybridization

Courage-Tebbe, U.; Kemper, Börries

doi:10.1016/0167-4781(82)90037-9

Cited by 10 publications

(5 citation statements)

References 10 publications

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“…IPTG was from Serva. For agarose gels "Seakem-Agarose" from Marine Colloids was used, for polyacrylamide gels premixed "Acrylamide/BIS" 29 d(pGGTTTTCCCAGTCACG) (for reversion of the amber mutation) were synthesized by the phosphoramidite method20) manually21) or using a DNA synthesizer (Applied Biosystems). The oligonucleotides were purified by high pressure liquid chromatography22) or preparative gel electrophoresis23).…”

Section: Materials and Methods Bacterial Strainsmentioning

confidence: 99%

The gapped duplex DNA approach to oligonucleotide-directed mutation construction

Kramer¹,

Drutsa²,

Jansen³

et al. 1984

Nucl Acids Res

514

246

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A simple and efficient method is described to introduce structurally pre-determined mutations into recombinant genomes of filamentous phage M13. The method rests on gapped duplex DNA (gdDNA) molecules of the phage M13 genome as the key intermediate. In this gdDNA, the (+) and the (shorter) (-) strand carry different genetic markers in such a way, that a rigorous selection can be applied for phage carrying the markers of the (-) strand. For introduction of the mutation, a synthetic oligonucleotide with partial homology to a target site within the single stranded DNA region is annealed to the gdDNA. The oligonucleotide subsequently becomes part of the (-) strand by enzymatic DNA gap filling and sealing. This physical linkage is preserved at the genetic level after transfection of a recipient E.coli strain deficient in DNA mismatch correction, so that the synthetic marker can be selected from the phage progeny independent from its potential phenotype. It is demonstrated that by this method mutants can be constructed with marker yields in excess of 70%.

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Section: Materials and Methods Bacterial Strainsmentioning

confidence: 99%

The gapped duplex DNA approach to oligonucleotide-directed mutation construction

Kramer¹,

Drutsa²,

Jansen³

et al. 1984

Nucl Acids Res

514

246

View full text Add to dashboard Cite

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“…A version carrying the nonsense mutation of mutant DU9 was constructed by cloning a 2.7-kbp EcoRI fragment spanning exon 3 from the cosmid pDU9 into pUC8 to yield pDU92E. From this plasmid, a 77-bp HpaIISstI fragment from exon 3 that contained the DU9 mutation was used to mutagenize pDCH0 by a modified gappedsynthesis method (17,32). The DU9 mutation has destroyed a TaqI site at cDNA position 196; the absence of this site made it possible to confirm the presence of the mutation in constructs and in transfectants.…”

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confidence: 99%

Nonsense mutations in the dihydrofolate reductase gene affect RNA processing.

et al. 1989

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Steady-state dihydrofolate reductase (dhfr) mRNA levels were decreased as a result of nonsense mutations in the dhfr gene. Thirteen DHFR-deficient mutants were isolated after treatment of Chinese hamster ovary cells with UV irradiation. The positions of most point mutations were localized by RNA heteroduplex mapping, the mutated regions were isolated by cloning or by enzymatic amplification, and base changes were determined by DNA sequencing. Two of the mutants suffered large deletions that spanned the entire dhfr gene. The remaining 11 mutations consisted of nine single-base substitutions, one double-base substitution, and one single-base insertion. All of the single-base substitutions took place at the 3' position of a pyrimidine dinucleotide, supporting the idea that UV mutagenesis proceeds through the formation of pyrimidine dimers in mammalian cells. Of the 11 point mutations, 10 resulted in nonsense codons, either directly or by a frameshift, suggesting that the selection method favored a null phenotype. Biol., in press) showed that translation termination mutations in any of the internal exons of the gene gave rise to a low-RNA phenotype, whereas missense mutations in these exons or terminations in exon 6 (the final exon) did not affect dhfr mRNA levels. Nuclear run-on experiments showed that transcription of the mutant genes was normal. The stability of mature dhfr mRNA also was not affected, since (i) decay rates were the same in wild-type and mutant cells after inhibition of RNA synthesis with actinomycin D and (ii) intronless minigene versions of cloned wild-type and nonsense mutant genes were expressed equally after stable transfection. We conclude that RNA processing has been affected by these nonsense mutations and present a model in which both splicing and nuclear transport of an RNA molecule are coupled to its translation. Curiously, the low-RNA mutant phenotype was not exhibited after transfer of the mutant genes, suggesting that the transcripts of transfected genes may be processed differently than are those of their endogenous counterparts.In an effort to identify and understand those aspects of gene structure that play a role in gene expression in mammalian cells, we have been carrying out a detailed mutational analysis of the dihydrofolate reductase (dhfir) gene in Chinese hamster ovary (CHO) cells. dlifr exhibits many typical characteristics of mammalian housekeeping genes: it is 25 kilobase pairs (kbp) in size, contains five variously sized introns, and has a promoter region with no TATA box but with a very high G+C content and an Spl binding site (10,22,40,53). The basic strategy has been to isolate mutants that are deficient in DHFR enzyme activity; such mutants can be readily selected from a CHO cell line that is hemizygous at this locus (58, 59). To focus on lesions that may affect transcription or RNA processing, mutants can be screened for alterations that have affected dimfi-mRNA, either qualitatively or quantitatively.We have previously described spontaneous point mutations in th...

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“…The use of a psoralen derivative to label a cloned plasmid DNA was achieved by making the inserted sequences partly single-stranded and keeping the vector region double-stranded. Other approaches to obtain ss DNA inserts use M13 clones by making them ds in the vector region (30,31). This is more time-consuming.…”

Section: Discussionmentioning

confidence: 99%

Sensitive non-radioactive dot-blot hybridization using DNA probes labelled with chelate group substituted psoralen and quantitative detection by europium ion fluorescence

Oser¹,

Roth

Valet³

1988

Nucl Acids Res

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A new labelling method for cloned DNA probes used in hybridization assays is described. The DNA insert of recombinant plasmid DNA was made partially single-stranded for the labelling reaction by a restriction enzyme digest, followed by a controlled exonuclease III incubation. A thiol-containing psoralen derivative was covalently bound through irradiation with UV-light to the remaining double-stranded region of the plasmid DNA. The psoralen-SH groups were labelled with a large number of metal chelators (diethylentriamine pentaacetic acid, DTPA) using poly-L-lysine as a macromolecular carrier. The main advantage of the labelling procedure is that a high degree of labelling is achieved without modification of the single-stranded DNA hybridizing sequences. The specific hybrids were labelled after filter hybridization with europium ions through the chelating groups of DTPA. The europium ions were quantitatively detected by time-resolved fluorometry. The sensitivity of the assay for target DNA detection was in the low picogram range, comparable to radioactively labelled DNA probes.

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Construction of gapped circular DNA from phage M13 by in vitro hybridization

Cited by 10 publications

References 10 publications

The gapped duplex DNA approach to oligonucleotide-directed mutation construction

The gapped duplex DNA approach to oligonucleotide-directed mutation construction

Nonsense mutations in the dihydrofolate reductase gene affect RNA processing.

Sensitive non-radioactive dot-blot hybridization using DNA probes labelled with chelate group substituted psoralen and quantitative detection by europium ion fluorescence

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