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PubMed1994

Effects of bile duct ligation on calcium excretion in the rat.

In an earlier study we showed that bile duct-ligated rats were highly susceptible to gentamicin nephrotoxicity and that oral calcium administration had a pronounced protective effect. The mechanism by which this occurs is unclear. Because cations compete with gentamicin for tubule binding sites, it has been suggested that the increased susceptibility of the kidney to gentamicin after bile duct ligation might result from decreased cation excretion. The aim of this study was to determine the effect of bile duct ligation on calcium excretion in relation to overall renal function. Male Sprague-Dawley rats underwent bile duct ligation and division. Pair-fed sham-operated rats served as controls. Metabolic and clearance studies were carried out at 3, 5, 7, 14 and 21 days after surgery. Urine output was higher in bile duct-ligated rats, and they excreted more calcium at 3, 5, 7 and 14 days, while excreting less sodium than controls. We conclude that after bile duct ligation, there is increased calcium excretion, which is independent of the abnormality in sodium excretion. Enhanced nephrotoxicity with aminoglycosides in the bile duct-ligated rat model cannot be explained by decreased calcium excretion.

Animals↗
PubMed1997

Experimental study in bile duct-ligated rats of vasopressin and preoperative volume loading to prevent hypotensive crises.

BACKGROUND: Systemic hypotension may result in postoperative renal failure in jaundiced patients. Attenuated responsiveness to catecholamines and hypovolaemia has been reported in jaundiced animals and may be a mechanism contributing to the increased susceptibility of jaundiced patients to haemorrhagic shock. This suggests that an alternative to vasoactive amines to control perioperative hypotension could be desirable. METHODS: This study evaluated the pressor response to vasopressin in normovolaemic 3-day bile duct-ligated rats and in 3-day bile duct-ligated rats after an acute controlled haemorrhage. It also evaluated the response after volume loading with 0.9 per cent saline, 7.5 per cent saline, colloid and mannitol before controlled haemorrhage. In addition, blood volume was measured using radiolabelled albumin. All the data obtained from bile duct-ligated rats were compared with data from sham-operated animals. RESULTS: Attenuated pressor responses to vasopressin were not observed in either normotensive bile duct-ligated rats or in the bile duct-ligated rats subjected to controlled haemorrhage. Volume loading with the four fluids over the dosing range 2.5-7.5 microliters per g body-weight in bile duct-ligated rats reversed the susceptibility to haemorrhagic hypotension. CONCLUSION: Although no reduction in blood volume was demonstrated, bile duct-ligated rats may have a reduced effective blood volume manifesting itself as a latent hypovolaemia and/or tendency to hypotension. Preoperative fluid loading could be beneficial because it corrects hypovolaemia and improves cardiovascular function, as well as improving the cardiovascular response to haemorrhage.

Animals↗
PubMed1999

Orthogonal ligation strategies for peptide and protein.

This review focuses on the concept, criteria, and methods of an orthogonal amide ligating strategy suitable for syntheses of peptides, peptide mimetics, and proteins. Utilizing unprotected peptides or proteins derived from chemical or biosynthetic sources, this ligation strategy has been shown to be general and exceptionally mild. Its orthogonality in ligating two unprotected segments with free N-terminal (NT)-amines at a specific NT-amine is achieved through a chemoselective capture step and then an intramolecular acyl transfer reaction. Both coupling reagents for enthalpic activation and protection schemes therefore become unnecessary. More than a dozen orthogonal ligation methods based on either imine or thioester captures have been developed to afford native and unusual amino acids at ligation sites of linear, branched, or cyclic peptides. Because unprotected peptides and proteins of different sizes and forms can be obtained from either chemical or recombinant sources, orthogonal ligation removes the size limitation imposed on the chemical synthesis of a protein with a native or non-native structure. Furthermore, by using building blocks from biosynthetic sources, orthogonal ligation provides a unifying operational concept for both total and semisynthesis of peptides and proteins.

Amino Acids↗
PubMed1983

Studies on anticancer treatment with an oily anticancer drug injected into the ligated feeding hepatic artery for liver cancer.

In six adult patients with nonresectable liver cancer, as well as in mature New Zealand white rabbits with implanted VX2 carcinoma in the liver, the artery feeding the hepatic lobe with the malignant lesion was ligated, and an oily contrast medium (Lipiodol Ultra-Fluid) was injected into the hepatoproximal lumen of the ligated artery of the liver with carcinoma. The oily contrast medium was detected in all the branches of the artery injected, and thereafter was found only in tumor tissue for 7 days experimentally and for 16 months clinically. Taking advantage of this phenomenon, the therapeutic effect of the injection of an oily anticancer drug (bleomycin oil suspension) into the hepatoproximal lumen of the ligated hepatic artery was investigated in rabbits with VX2 carcinoma of the liver. The mean concentration level of bleomycin in the tumor tissue was 2.4 +/- 0.4 microgram/g 1 week after the injection of bleomycin oil suspension (1.5 mg potency/kg) in three rabbits. However, its concentration level in nontumorous tissue of the liver was undetectably low in two rabbits, but 0.6 microgram/g in the third rabbit. The group of rabbits receiving an injection of bleomycin oil suspension into the ligated artery had a significantly longer mean survival time than those of the experimental group receiving an injection of saline solution of bleomycin into the ligated artery as well as the three other groups treated (P less than 0.02, N = 5 for each group). It may be concluded that an oily anticancer drug injected into the hepatoproximal lumen of the ligated hepatic artery can intensify the anticancer effects of a ligation of the hepatic artery for liver cancer.

Animals↗
PubMed2001

Methods and strategies of peptide ligation.

This review focuses on the concept, methods, and strategies of orthogonal peptide ligation. It updates our previous review in 1999 on the same subject matter in Biopolymers (Peptide Science, 1999, Vol. 51, p. 311). Orthogonal peptide ligation is an amino terminal specific method to couple chemically unprotected peptides or proteins derived from synthetic or biosynthetic sources. Unlike conventional chemical methods, peptide ligation methods do not require coupling reagents or protection schemes, but are achieved through a variable chemoselective capture step and then an invariable intramolecular acyl transfer reaction. It is also a convergent method with the fewest steps. More than a dozen orthogonal ligation methods have been developed based on captures by either imine or thioester chemistries to afford native and unusual amino acids at ligation sites of linear, branched, or cyclic peptides. The ligation strategies for multiple segments including sequential and tandem ligations are also discussed.

Amino Acids↗
PubMed1998

Ligation of CR1 attenuates Fc receptor-mediated myeloperoxidase release and HOCl production by neutrophils.

Surface adherent immunoglobulins are potent stimuli for inducing neutrophil release of myeloperoxidase and production of hypochlorous acid (HOCl), an oxidant that promotes activity of neutrophil proteases. Opsonization of surface adherent IgG (SAIgG) by complement results in attenuation of these responses, despite augmenting neutrophil production of superoxide and release of specific granule enzymes. The role of complement receptor ligation in modulating Fc receptor-triggered myeloperoxidase release and HOCl production by neutrophils was determined by incubating neutrophils with SAIgG in the presence of complement receptor ligating antibody reagents. Ligation of CR1 by F(ab')2 derived from CR1 specific monoclonal antibody (mAb 543) resulted in significant attenuation of surface-associated IgG (SAIgG)-induced release of myeloperoxidase and HOCl production but did not result in attenuation of SAIgG-induced superoxide or hydrogen peroxide production; ligation of CR1 by mAb 543 F(ab')2 also attenuated surface adherent IgA-induced myeloperoxidase release and HOCl production. HOCl production was not significantly attenuated when neutrophils were activated with SAIgG in the presence of surface adherent C1q or when CR3 was ligated by F(ab')2 derived from mAb having specificity for the CD11b (mAb M1/70) or CD18 (mAb TS1) subunits of CR3. These results indicate that ligation of CR1 on neutrophils by C3b fixed to IgG may alter signal transduction events linking ligation of neutrophil Fc receptors to cellular events required for release of myeloperoxidase and generation of HOCl.

Antibodies, Monoclonal↗
PubMed1998

Non-histone chromosomal proteins HMG1 and 2 enhance ligation reaction of DNA double-strand breaks.

DNA ligase IV in a complex with XRCC4 is responsible for DNA end-joining in repair of DNA double-strand breaks (DSB) and V(D)J recombination. We found that non-histone chromosomal high mobility group (HMG) proteins 1 and 2 enhanced the ligation of linearized pUC119 DNA with DNA ligase IV from rat liver nuclear extract. Intra-molecular and inter-molecular ligations of cohesive-ended and blunt-ended DNA were markedly stimulated by HMG1 and 2. Recombinant HMG2-domain A, B, and (A + B) polypeptides were similarly, but non-identically, effective for the stimulation of DSB ligation reaction. Ligation of single-strand breaks (nicks) was only slightly activated by the HMG proteins. The DNA end-binding Ku protein singly or in combination with the catalytic component of DNA-dependent protein kinase (DNA-PK) as the DNA-PK holoenzyme was ineffective for the ligation of linearized pUC119 DNA. Although the stimulatory effect of HMG1 and 2 on ligation of DSB in vitro was not specific to DNA ligase IV, these results suggest that HMG1 and 2 are involved in the final ligation step in DNA end-joining processes of DSB repair and V(D)J recombination.

Animals↗
PubMedDate not supplied

Tandem peptide ligation for synthetic and natural biologicals.

We describe the concept and methods of peptide ligation and tandem peptide ligation for preparing synthetic and natural biologicals. Peptide ligation is a segment coupling method for free peptides or proteins through an amide bond without the use of a coupling reagent or a protecting group scheme. Because unprotected peptides or proteins prepared from either a chemical or biochemical source are being used as building blocks, the ligation removes the size limitation for peptide and protein synthesis. A key feature of the peptide ligation is that the coupling reaction is orthogonal, i.e. it is specific to a particular alpha-amino terminus (NT). This NT-amino acid-specific feature permits the development of a tandem peptide ligation method employing three unprotected peptide segments containing different NT-amino acids to form consecutively two amide bonds, an Xaa-SPro (thiaproline) and then an Xaa-Cys. This strategy was tested in peptides ranging from 28 to 70 amino acid residues, including analogues of somatostatins and two CC-chemokines MIP-1alpha and MIP-1beta. The thiaproline replacements in these peptides and proteins did not result in altered biological activity. By eliminating the protecting group scheme and coupling reagents, tandem ligation of multiple free peptide segments in aqueous solutions enhances the scope of protein synthesis and may provide a useful approach for preparing protein biologicals and synthetic vaccines.

Amino Acid Sequence↗
PubMed1994

Intramolecular and intermolecular DNA ligation mediated by topoisomerase II.

The ability of topoisomerase II to mediate a number of DNA rearrangements was examined at the molecular level. For this purpose a new type of defined donor and acceptor substrate have been developed, and used for studies of the intramolecular and intermolecular DNA ligation reactions of topoisomerase II. Intramolecular ligation occurred only to single-stranded acceptor molecules with the ability to base-pair to the donor substrate, while the intermolecular ligation reaction displayed a strong preference for double-stranded acceptor molecules with a short four base, single-stranded region. The efficiency of the intermolecular ligation reaction was highly dependent on base-pairing between the acceptor molecule and the DNA donor cleaved by topoisomerase II. Thus, acceptor molecules containing a blunt end or a four base 5' overhang without base-pairing ability ligated with an approximately eightfold reduced efficiency, as compared with the base-pairing control. Experiments demonstrated that the enzyme can ligate DNA molecules, where nucleotides were either removed or inserted in the employed acceptor molecules. The results indicate that topoisomerase II might be responsible for similar rearrangements in vivo, since gapped and nicked DNA structures appear as intermediates in processes such as replication and repair. The reaction is, however, likely to be constrained by the requirement of base-pairing for ligation.

Base Composition↗
PubMed1990

Linear DNA introduced into carrot protoplasts by electroporation undergoes ligation and recircularization.

The integrated DNA in stable transformants formed by direct gene transfer often shows complex restriction patterns. One cause of these complex restriction patterns could be the ligation of plasmid fragments prior to their integration. This paper provides evidence for the ligation of plasmid fragments by plant cells. Carrot protoplasts were electroporated in the presence of pCaMVCATM and assayed for chloramphenicol actyltransferase (CAT) activity 24 h later. Linear and supercoiled forms of pCaMVCATM supported similar levels of CAT expression. Surprisingly, digestion of the plasmid at a site between the CaMV 35S promoter and the CAT coding region reduced expression by only 40-50%. Electroporation carried out in the presence of isolated plasmid fragments suggested that this result was due to ligation of the linearized plasmid by the protoplasts. CAT expression was obtained with a mixture of isolated CaMV 35S promoter and the CAT coding region; neither fragment alone supported expression. Further evidence of ligation was provided by electroporation of protoplasts in the presence of a mixture of linearized pGEM and the 1.5-kb Hind III fragment of pCaMVCATM. DNA isolated from nuclei of the protoplasts was used to transform competent cells of Escherichia coli, and colonies were recovered that carried pGEM with Hind III-CaMVCAT inserts. Electroporation of protoplasts in the presence of linear and supercoiled pGEM and use of DNA isolated from nuclei to transform E. coli yielded an estimate of the frequency of plasmid ligation. A maximum of only 4% of the input linear DNA was recovered as circular molecules. This result suggests the frequency of ligation is low, but examination of the plasmid DNA in the plant nuclei by electrophoresis indicates extensive degradation of the plasmid and preferential loss of the circular forms. Thus, the ligated plasmids may be converted to the linear form and hence rendered unrecoverable by cloning into E. coli.

Chloramphenicol O-Acetyltransferase↗
PubMed1994

In vitro selection of optimal DNA substrates for ligation by a water-soluble carbodiimide.

We have used in vitro selection to investigate the sequence requirements for efficient template-directed ligation of oligonucleotides at 0 degrees C using a water-soluble carbodiimide as condensing agent. We find that only 2 bp at each side of the ligation junction are needed. We also studied chemical ligation of substrate ensembles that we have previously selected as optimal for ligation by RNA ligase or by DNA ligase. As anticipated, we find that substrates selected with DNA ligase ligate efficiently with a chemical ligating agent, and vice versa. Substrates selected using RNA ligase are not ligated by the chemical condensing agent and vice versa. The implications of these results for prebiotic chemistry are discussed.

Base Sequence↗
PubMed1992

Primary somatosensory cortex in rats with pain-related behaviours due to a peripheral mononeuropathy after moderate ligation of one sciatic nerve: neuronal responsivity to somatic stimulation.

Single-unit recordings were made under moderate gaseous anaesthesia in the hindpaw representation area of the two primary somatosensory motor cortices (SmI) of rats (n = 58) rendered mononeuropathic by four loose ligatures placed around one common sciatic nerve 2-3 weeks beforehand. The rats exhibited clear hyperalgesia and allodynia from the paw with the ligated sciatic nerve, to both mechanical and thermal stimuli. From the tested neuronal population (n = 640), about the same proportion could be activated by somatic stimuli in each cortex: 165/362 (45%) in the cortex contralateral to the ligated sciatic nerve (Cc), 105/278 (37%) in the cortex ipsilateral to the ligated sciatic nerve (Ci). Neurones driven by light touch, exhibited RFs strictly contralateral to the recording sites. Their proportion and response characteristics were similar regardless of recording side. However, the number of neurones with RFs in the sciatic nerve territory was above 95% in the Ci, and was dramatically reduced to 43% in the Cc. By contrast, the number of neurones with RFs supplied by the saphenous nerve reached 57% on this side. Although the RF size of all the neurones appeared roughly normal, there were fewer Cc than Ci neurones with RFs located on the paw itself and with RFs of extremely small size in the sciatic nerve territory. The proportion of neurones responding to a joint stimulus was significantly higher in the Cc than in the Ci. The neuronal responses to joint stimuli of the paw with the ligated sciatic nerve were significantly more sustained than those recorded in the Ci and elicited from the normal paw. The proportion of neurones driven by mechanical stimulation which gave rise to nociceptive reactions in freely moving animals, i.e. "nociceptive" neurones, was comparable in each cortex. However, half of the Cc neurones exhibited paroxysmal discharges occurring without intentional stimulation and of long duration (1 min to several minutes). Only 66% of Cc but 93% of Ci "nociceptive" neurones were exclusively activated by pinch. The remaining Cc neurones were also activated by applying moderate pressure to the paw with the ligated nerve. Pinch responses from the paw with the ligated nerve were often more intense and of longer duration than responses elicited from the intact paw. The "nociceptive" Cc neurones were especially sensitive to thermal stimuli of 39-44 degrees C when the stimuli were applied to the paw with the ligated nerve. They also responded vigorously to a 10 degrees C stimulus applied to this paw.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2006

Identification of zinc-ligated cysteine residues based on 13Calpha and 13Cbeta chemical shift data.

Although a significant number of proteins include bound metals as part of their structure, the identification of amino acid residues coordinated to non-paramagnetic metals by NMR remains a challenge. Metal ligands can stabilize the native structure and/or play critical catalytic roles in the underlying biochemistry. An atom's chemical shift is exquisitely sensitive to its electronic environment. Chemical shift data can provide valuable insights into structural features, including metal ligation. In this study, we demonstrate that overlapped 13Cbeta chemical shift distributions of Zn-ligated and non-metal-ligated cysteine residues are largely resolved by the inclusion of the corresponding 13Calpha chemical shift information, together with secondary structural information. We demonstrate this with a bivariate distribution plot, and statistically with a multivariate analysis of variance (MANOVA) and hierarchical logistic regression analysis. Using 287 13Calpha/13Cbeta shift pairs from 79 proteins with known three-dimensional structures, including 86 13Calpha and 13Cbeta shifts for 43 Zn-ligated cysteine residues, along with corresponding oxidation state and secondary structure information, we have built a logistic regression model that distinguishes between oxidized cystines, reduced (non-metal ligated) cysteines, and Zn-ligated cysteines. Classifying cysteines/cystines with a statistical model incorporating all three phenomena resulted in a predictor of Zn ligation with a recall, precision and F-measure of 83.7%, and an accuracy of 95.1%. This model was applied in the analysis of Bacillus subtilis IscU, a protein involved in iron-sulfur cluster assembly. The model predicts that all three cysteines of IscU are metal ligands. We confirmed these results by (i) examining the effect of metal chelation on the NMR spectrum of IscU, and (ii) inductively coupled plasma mass spectrometry analysis. To gain further insight into the frequency of occurrence of non-cysteine Zn ligands, we analyzed the Protein Data Bank and found that 78% of the Zn ligands are histidine and cysteine (with nearly identical frequencies), and 18% are acidic residues aspartate and glutamate.

Bacillus subtilis↗
PubMed1979

Experimental ischemic myopathy. Effects of aortic ligation and serotonin.

Support for the hypothesis that biogenic amines are involved in the production of muscle diseases comes from a report that rat muscle is damaged by combining distal aortic ligation with serotonin injection. Our studies explore the role of serotonin in the production of the myopathic changes in the aortic ligation-serotonin model. Twenty-six young Sprague-Dawley rats were subjected either to aortic ligation alone, aortic ligation followed by injection of serotonin (40 mg/kg, i.p.), or injection of serotonin alone. Following sacrifice 7--14 days later, 10 micrometer frozen sections of the soleus muscle were stained by trichrome, NADH-TR, and ATPase methods. Focal necrosis and phagocytosis or focal regeneration were seen after aortic ligation with, or without, subsequent serotonin injection. Serotonin alone produced only occasional mild changes in muscle. Therefore, we conclude that the significant damage to muscle in the ligation-serotonin model is provided by the aortic ligation, not the serotonin injection.

Animals↗
PubMed2004

Utero-ovarian morphology and blood flow after tubal ligation by the Pomeroy technique.

OBJECTIVES: The objectives of this study were to evaluate ovarian sonographic morphology and pelvic blood flow in patients who had undergone bilateral tubal ligation by the Pomeroy technique. DESIGN: Twenty women who had undergone bilateral tubal ligation during cesarean section by the Pomeroy techniques were compared to 20 matched controls who had undergone cesarean section alone. Ovarian morphology as well as ovarian and uterine pulsatility index (PI), resistance index and peak systolic velocity were evaluated using transvaginal sonography and Doppler velocimetry. RESULTS: Patients post tubal ligation by the Pomeroy technique were found to have a significantly higher rate of polycystic appearance of the ovaries (60% vs. 5%; p < 0.005). Six patients (30%) had post tubal ligation symptoms, as compared to none of the controls. Five of the six symptomatic tubal ligation patients (83%) had ovarian polycystic appearance, which was not significantly different than asymptomatic patients. Ovarian artery PI was significantly higher in symptomatic patients (2.7 +/- 1.9) compared to asymptomatic patients (1.5 +/- 0.9) (p < 0.05). CONCLUSIONS: We therefore conclude that patients post bilateral tubal ligation by the Pomeroy technique may acquire an ovarian polycystic appearance and increased ovarian PI, which may be associated with a tendency to develop post tubal ligation symptoms.

Adult↗
PubMed2004

Reduction of neoreflux after correctly performed ligation of the saphenofemoral junction. A randomized trial.

BACKGROUND: Neoreflux at the sapheno-femoral junction (SFJ) is an important cause of recurrent great saphenous varicose veins. This study compares four surgical methods of ligating the SFJ with the aim to reduce the rate of neoreflux. METHOD: In a prospective study, 379 patients (500 SFJ ligations) were randomised to one of four surgical procedures at the SFJ (125 groins each). In group A (control group) the SFJ was ligated in standard fashion with Vicryl (absorbable ligature); in group B, after Vicryl ligation continuous Prolene (non-absorbable) was sutured over the stump endothelium to prevent any contact with surrounding tissue; in group C. SFJ ligation was done with Ethibond (non-absorbable); in group D Ethibond ligation was followed by Prolene oversewing. The final study group included 114 patients (152 groins) who were all known to be free from recurrent groin reflux 3 months postoperatively and had colour duplex venous imaging 2 years after operation. RESULTS: Duplex imaging identified neoreflux at the SFJ in 10 out of 114 groins after 2 years (7%). There were differences in the rates between the four groups: Group A 3/31 (10%), Group B 0/32, Group C 5/44 (11%) and Group D 2/45 (4%). Neoreflux was significantly reduced in the two groups with endothelial closure (B and D): 2/70 (3%) versus 8/75 (11%, p<0.025). CONCLUSION: Recurrent reflux in the groin was reduced by over sewing the ligated SFJ in patients having varicose vein surgery. This adds weight to the theory of neovascularisation as a cause of recurrent veins and offers a means to reduce clinical recurrence rates.

Female↗
PubMed2007

Patent ductus arteriosus ligation in premature infants: who really benefits, and at what cost?

PURPOSE: Patent ductus arteriosus (PDA) ligation in premature infants has been shown to have low surgical morbidity and mortality. Ligation goals include prompt improvement in cardiorespiratory failure, with rapid wean from mechanical ventilation; less risk of prolonged mechanical ventilation and subsequent chronic lung disease (CLD); and survival to discharge. This study was designed to examine true morbidity after ligation and elucidate which preoperative factors might predict favorable outcomes. METHODS: Institutional review board-approved retrospective review of 197 infants less than 38 weeks of gestational age (GA), undergoing PDA ligation via thoracotomy between January 1, 1992, and January 1, 2004. Chronic lung disease defined as need for supplemental oxygen at 36 weeks corrected GA. Student t and chi2 tests were used. RESULTS: Mean GA was 27 weeks (range, 23-35 weeks), birth weight was 957 g (range, 440-3170 g); infants underwent ligation at 16 days of life (range, 1-132 days). Duration of surgery was 50.5 minutes (range, 13-150 minutes). Mean postoperative times were 27 days to extubation, 60 days to wean from supplemental oxygen, and 84 days to discharge. Early extubation (within 10 days of ligation) occurred in only 54 patients (30%). Only 44 (22%) survived to discharge without CLD. Forty patients (20%) died, with respiratory failure the most common cause (70%). In general, early extubation, survival without CLD and survival to discharge were associated with greater GA and birth weight, higher Apgar scores, greater age and weight at surgery, no preoperative intraventricular hemorrhage, lack of ventilator dependence, and lower ventilator settings (P < .05). Preoperative amount and duration of indomethacin use, chest x-ray findings, and echocardiographic assessment of ductus size did not predict favorable outcomes (all P > .05). CONCLUSIONS: Most premature infants currently undergoing PDA ligation at our institution do not experience the anticipated rapid improvements in cardiorespiratory status and go on to develop CLD. Few preoperative variables (including radiographic and echocardiographic assessments) definitively predict outcomes.

Ductus Arteriosus, Patent↗
PubMed2002

High-efficiency DNA ligation for clamp attachment without polymerase chain reaction.

We coupled ligation with mass action to achieve high-efficiency clamp attachment without polymerase chain reaction (PCR). Using a 10-fold molar excess of a GC-rich clamp of synthesized and hybridized oligonucleotides, we achieved the maximum clamp-ligation efficiency in which the clamp was ligated to >95% of 10(10)-10(12) restriction ends of a PCR-amplified fragment. The maximum efficiency was confirmed by ligating the clamp to 10(11)-10(12) restriction ends of human genomic DNA. Our approach can be added to a constant denaturant capillary electrophoresis (CDCE)-based method of analyzing rare point mutants at fractions as low as 10(-6); such mutants appear as small copy numbers in the initial samples. This CDCE-based method alone is applicable to only those DNA sequences juxtaposed with an internally occurring clamp of a higher melting temperature in genomic DNA. Since such sequences represent 9% of the human genome, the addition of clamp ligation significantly increases the scanning range for the human genome without reducing the initial mutant copy numbers. Furthermore, clamp ligation/attachment without PCR prevents PCR-created mutants from interfering with rare mutational analysis. In addition to those applications seeking high-efficiency DNA ligation, our approach can be generally applied to ligation of restriction ends.

Base Composition↗