Specificity of the nick-closing activity of bacteriophage T4 DNA ligase

Wu, Dong; Rb, Wallace

doi:10.1016/0378-1119(89)90165-0

Cited by 159 publications

(132 citation statements)

References 19 publications

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“…2). Our result on discriminating different 3′-side mismatches with Tth DNA ligase are similar to those reported for T4 DNA ligase (9), although Tth DNA ligase does not require high salt, spermidine or very low enzyme concentrations to suppress mismatch ligations (7,9).…”

Section: Fidelity Of Nick Closure By Wild-type Tth Dna Ligasesupporting

confidence: 87%

“…The apparent fidelity of T4 DNA ligase may be improved in the presence of spermidine, high salt and very low ligase concentrations, where only T-G or G-T mismatch ligations were detected (7,9). DNA ligase from Saccharomyces cerevisiae discriminates 3′-hydroxyl and 5′-phosphate termini separated by a 1 nt gap and 3′ A-G or T-G mismatches, however 5′ A-C, T-C, C-A or G-A mismatches had very little effect on ligation efficiency (10).…”

Section: Or 3′ C-a C-t T-g T-t T-c A-c G-g or G-t Mismatches (9)mentioning

confidence: 99%

“…The fidelity of T4 DNA ligase has been expressed as a specificity ratio, defined as the ratio of ligation product formed in the presence of matched versus mismatched template in the presence of 100 nM template at 30_C with 1 U enzyme (7). At high salt concentrations (200 mM) the specificity ratio increased from 6 to 60 for T-T mismatches and from 1.5 to 40 for A-A mismatches, although these more stringent conditions also increased the K m 4-fold and decreased the V max ∼30-fold (7).…”

Section: Improving the Fidelity Of Tth Dna Ligasementioning

confidence: 99%

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Improving the Fidelity of Thermus Thermophilus DNA Ligase

Luo

Bergstrom

Barany

1996

Nucleic Acids Research

103

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The DNA ligase from Thermus thermophilus (Tth DNA ligase) seals single-strand breaks (nicks) in DNA duplex substrates. The specificity and thermostability of this enzyme are exploited in the ligase chain reaction (LCR) and ligase detection reaction (LDR) to distinguish single base mutations associated with genetic diseases. Herein, we describe a quantitative assay using fluorescently labeled substrates to study the fidelity of Tth DNA ligase. The enzyme exhibits significantly greater discrimination against all single base mismatches on the 3′-side of the nick in comparison with those on the 5′-side of the nick. Among all 12 possible single base pair mismatches on the 3′-side of the nick, only T-G and G-T mismatches generated a quantifiable level of ligation products after 23 h incubation. The high fidelity of Tth DNA ligase can be improved further by introducing a mismatched base or a universal nucleoside analog at the third position of the discriminating oligonucleotide. Finally, two mutant Tth DNA ligases, K294R and K294P, were found to have increased fidelity using this assay.

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Section: Fidelity Of Nick Closure By Wild-type Tth Dna Ligasesupporting

confidence: 87%

Section: Or 3′ C-a C-t T-g T-t T-c A-c G-g or G-t Mismatches (9)mentioning

confidence: 99%

Section: Improving the Fidelity Of Tth Dna Ligasementioning

confidence: 99%

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Improving the Fidelity of Thermus Thermophilus DNA Ligase

Luo

Bergstrom

Barany

1996

Nucleic Acids Research

103

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“…The polymerase chain reaction (PCR) developed by Saiki et al (5) provided a method to rapidly amplify small amounts of a particular target DNA. The amplified DNA could then be readily analyzed for the presence of DNA sequence variation (e.g., the sickle cell mutation) by allelespecific oligonucleotide hybridization (6), restriction enzyme cleavage (5,7), ligation of oligonucleotide pairs (8,9), or ligation amplification (10 MATERIALS AND METHODS Oligonucleotide Synthesis. Oligonucleotides were synthesized on an Applied Biosystems 380B DNA synthesizer by the phosphoramidite method.…”

mentioning

confidence: 99%

Allele-specific enzymatic amplification of beta-globin genomic DNA for diagnosis of sickle cell anemia.

Ugozzoli

Pal

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

553

260

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A rapid nonradioactive approach to the diagnosis of sickle cell anemia is described based on an allelespecific polymerase chain reaction (ASPCR). This method allows direct detection of the normal or the sickle cell g3-globin allele in genomic DNA without additional steps of probe hybridization, ligation, or restriction enzyme cleavage. Two allele-specific oligonucleotide primers, one specific for the sickle cell allele and one specific for the normal allele, together with another primer complementary to both alleles were used in the polymerase chain reaction with genomic DNA templates. The allele-specific primers differed from each other in their terminal 3' nucleotide. Under the proper annealing temperature and polymerase chain reaction conditions, these primers only directed amplification on their complementary allele. In a single blind study of DNA samples from 12 individuals, this method correctly and unambiguously allowed for the determination of the genotypes with no false negatives or positives. If ASPCR is able to discriminate all allelic variation (both transition and transversion mutations), this method has the potential to be a powerful approach for genetic disease diagnosis, carrier screening, HLA typing, human gene mapping, forensics, and paternity testing.Sickle cell anemia is the prototype ofa genetic disease caused by a single base-pair mutation, an A -* T transversion in the sequence encoding codon 6 of the human f3-globin gene. In homozygous sickle cell anemia, the substitution of a single amino acid (Glu -* Val) in the B-globin subunit of hemoglobin results in a reduced solubility of the deoxyhemoglobin molecule and erythrocytes assume irregular shapes. The sickled erythrocytes become trapped in the microcirculation and cause damage to multiple organs.Kan and Dozy (1) were the first to describe the diagnosis of sickle cell anemia in the DNA of affected individuals based on the linkage of the sickle cell allele to an Hpa I restriction fragment length polymorphism. Later, it was shown that the mutation itself affected the cleavage site of both Dde I and Mst II and could be detected directly by restriction enzyme cleavage (2, 3). Conner et al. (4) described a more general approach to the direct detection of single nucleotide variation by the use of allele-specific oligonucleotide hybridization. In this method, a short synthetic oligonucleotide probe specific for one allele only hybridizes to that allele and not to others under appropriate conditions. All of the above approaches are technically challenging, require a reasonably large amount of DNA, and are not very rapid. The polymerase chain reaction (PCR) developed by Saiki et al. (5) provided a method to rapidly amplify small amounts of a particular target DNA. The amplified DNA could then be readily analyzed for the presence of DNA sequence variation (e.g., the sickle cell mutation) by allelespecific oligonucleotide hybridization (6), restriction enzyme cleavage (5, 7), ligation of oligonucleotide pairs (8, 9), or ligation amplificatio...

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“…resulting from the low-fidelity of T4 DNA ligase under some conditions (18,19,20) Conventional cloning procedures such as blunt-end cloning require that the vector be dephosphorylated to prevent a high nonrecombinant background. PCR does not provide sufficiently blunt-ended molecules, necessitating additional polymerase or exonuclease treatments to polish the ends of PCR products (6,7).…”

Section: Discussionmentioning

confidence: 99%

L igation‐ i ndependent c loning of PCR products (LIC‐PCR)

2015

The Dictionary of Genomics, Transcriptomics and Proteomics

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Specificity of the nick-closing activity of bacteriophage T4 DNA ligase

Cited by 159 publications

References 19 publications

Improving the Fidelity of Thermus Thermophilus DNA Ligase

Improving the Fidelity of Thermus Thermophilus DNA Ligase

Allele-specific enzymatic amplification of beta-globin genomic DNA for diagnosis of sickle cell anemia.

L igation‐ i ndependent c loning of PCR products (LIC‐PCR)

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