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PubMed1984

[Incidents and hazards in the use of blank metal clamps with venous catheters].

The use of blank metal clamps resulted in severe damaging of infusion systems in two cases. In one case the silicone tube of a Shaldon catheter was damaged by the clamps and thus became detached from the Luer-Lock syringe connecting piece, whereupon the patient bled to death. In another case the too forcible screwing while fastening a Luer-Lock coupling by means of a metal clamp produced a tear in the connecting piece. The female patient, who sat upright, collapsed and died under the clinical suspicion of air embolism, which, however, could not be confirmed at autopsy. Such incidents are avoidable only if doctors and nurses are carefully trained in the handling of vascular catheters and infusion systems and if they are expressly informed not to use blank metal clamps.

Embolism, Air↗
PubMed2001

Interaction of the beta sliding clamp with MutS, ligase, and DNA polymerase I.

The beta and proliferating cell nuclear antigen (PCNA) sliding clamps were first identified as components of their respective replicases, and thus were assigned a role in chromosome replication. Further studies have shown that the eukaryotic clamp, PCNA, interacts with several other proteins that are involved in excision repair, mismatch repair, cellular regulation, and DNA processing, indicating a much wider role than replication alone. Indeed, the Escherichia coli beta clamp is known to function with DNA polymerases II and V, indicating that beta also interacts with more than just the chromosomal replicase, DNA polymerase III. This report demonstrates three previously undetected protein-protein interactions with the beta clamp. Thus, beta interacts with MutS, DNA ligase, and DNA polymerase I. Given the diverse use of these proteins in repair and other DNA transactions, this expanded list of beta interactive proteins suggests that the prokaryotic beta ring participates in a wide variety of reactions beyond its role in chromosomal replication.

Adenosine Triphosphatases↗
PubMed1985

Ca2+ entry in squid axons during voltage-clamp pulses is mainly Na+/Ca2+ exchange.

Intact squid axons were injected with aequorin and bathed in 3 mM Ca seawater (a concentration close to that of squid blood). Sodium and potassium currents were pharmacologically blocked and repetitive voltage-clamp pulses of a duration of 1.5 ms were applied (to simulate the duration of an action potential) at amplitudes of +30 to +90 mV and at frequencies of 100/s. In a very fresh axon (low internal Na concentration) no detectable change in aequorin glow resulted from this treatment, whether the axons were in Na-containing or in Na-free seawater. In axons subjected to modest Na loading, repetitive voltage-clamp pulsing did not result in an increased aequorin glow when the pulses were delivered in Na seawater, whereas in Na-free seawater there was an easily measurable increase in aequorin light emission during repetitive pulsing. The increase in aequorin photons emitted per voltage-clamp pulse was e-fold for 22 mV of depolarization, and the process showed no signs of saturating at pulse amplitudes of +180 mV (i.e., at a membrane potential close to ECa). The aequorin light emission per voltage-clamp pulse increased linearly with pulse duration (at constant amplitude).

Action Potentials↗
PubMed2001

Stepwise unfolding of titin under force-clamp atomic force microscopy.

Here we demonstrate the implementation of a single-molecule force clamp adapted for use with an atomic force microscope. We show that under force-clamp conditions, an engineered titin protein elongates in steps because of the unfolding of its modules and that the waiting times to unfold are exponentially distributed. Force-clamp measurements directly measure the force dependence of the unfolding probability and readily captures the different mechanical stability of the I27 and I28 modules of human cardiac titin. Force-clamp spectroscopy promises to be a direct way to probe the mechanical stability of elastic proteins such as those found in muscle, the extracellular matrix, and cell adhesion.

Animals↗
PubMed2004

Increased dNTP binding affinity reveals a nonprocessive role for Escherichia coli beta clamp with DNA polymerase IV.

Replication forks often stall at undamaged or damaged template sites in Escherichia coli. Subsequent resumption of DNA synthesis occurs by replacing DNA polymerase III, which is bound to DNA by the beta-sliding clamp, with one of three damage-induced DNA polymerases II, IV, or V. The principal role of the beta clamp is to tether the normally weakly bound polmerases to DNA thereby increasing their processivities. DNA polymerase IV binds dNTP substrates with about 10-fold lower affinity compared with the other E. coli polymerases, which if left unchecked could hinder its ability to synthesize DNA in vivo. Here we report a new property for the beta clamp, which when bound to DNA polymerase IV results in a large increase in dNTP binding affinity that concomitantly increases the efficiency of nucleotide incorporation at normal and transiently slipped mispaired primer/template ends. Primer-template DNA slippage resulting in single nucleotide deletions is a biological hallmark of DNA polymerase IV infidelity responsible for enhancing cell fitness in response to stress. We show that the increased DNA polymerase IV-dNTP binding affinity is an intrinsic property of the DNA polymerase IV-beta clamp interaction and not an indirect consequence of an increased binding of DNA polymerase IV to DNA.

Base Pair Mismatch↗
PubMed2000

Cross-utilization of the beta sliding clamp by replicative polymerases of evolutionary divergent organisms.

Chromosomal replicases are multiprotein machines comprised of a DNA polymerase, a sliding clamp, and a clamp loader. This study examines replicase components for their ability to be switched between Gram-positive and Gram-negative organisms. These two cell types diverged over 1 billion years ago, and their sequences have diverged widely. Yet the Escherichia coli beta clamp binds directly to Staphylococcus aureus PolC and makes it highly processive, confirming and extending earlier results (Low, R. L., Rashbaum, S. A. , and Cozzarelli, N. R. (1976) J. Biol. Chem. 251, 1311-1325). We have also examined the S. aureus beta clamp. The results show that it functions with S. aureus PolC, but not with E. coli polymerase III core. PolC is a rather potent polymerase by itself and can extend a primer with an intrinsic speed of 80-120 nucleotides per s. Both E. coli beta and S. aureus beta converted PolC to a highly processive polymerase, but surprisingly, beta also increased the intrinsic rate of DNA synthesis to 240-580 nucleotides per s. This finding expands the scope of beta function beyond a simple mechanical tether for processivity to include that of an effector that increases the intrinsic rate of nucleotide incorporation by the polymerase.

Amino Acid Sequence↗
PubMed2002

Ligand and coactivator identity determines the requirement of the charge clamp for coactivation of the peroxisome proliferator-activated receptor gamma.

The activation function 2 (AF-2)-dependent recruitment of coactivator is essential for gene activation by nuclear receptors. We show that the peroxisome proliferator-activated receptor gamma (PPARgamma) (NR1C3) coactivator-1 (PGC-1) requires both the intact AF-2 domain of PPARgamma and the LXXLL domain of PGC-1 for ligand-dependent and ligand-independent interaction and coactivation. Although the AF-2 domain of PPARgamma is absolutely required for PGC-1-mediated coactivation, this coactivator displayed a unique lack of requirement for the charge clamp of the ligand-binding domain of the receptor that is thought to be essential for LXXLL motif recognition. The mutation of a single serine residue adjacent to the core LXXLL motif of PGC-1 led to restoration of the typical charge clamp requirement. Thus, the unique structural features of the PGC-1 LXXLL motif appear to mediate an atypical mode of interaction with PPARgamma. Unexpectedly, we discovered that various ligands display variability in terms of their requirement for the charge clamp of PPARgamma for coactivation by PGC-1. This ligand-selective variable requirement for the charge clamp was coactivator-specific. Thus, distinct structural determinants, which may be unique for a particular ligand, are utilized by the receptor to recognize the coactivator. Our data suggest that even subtle differences in ligand structure are perceived by the receptor and translated into a unique display of the coactivator-binding surface of the ligand-binding domain, allowing for differential recognition of coactivators that may underlie distinct pharmacological profiles observed for ligands of a particular nuclear receptor.

Amino Acid Motifs↗
PubMed2003

Mechanism of loading the Escherichia coli DNA polymerase III sliding clamp: I. Two distinct activities for individual ATP sites in the gamma complex.

The Escherichia coli DNA polymerase III gamma complex loads the beta clamp onto DNA, and the clamp tethers the core polymerase to DNA to increase the processivity of synthesis. ATP binding and hydrolysis promote conformational changes within the gamma complex that modulate its affinity for the clamp and DNA, allowing it to accomplish the mechanical task of assembling clamps on DNA. This is the first of two reports (Snyder, A. K., Williams, C. R., Johnson, A., O'Donnell, M., and Bloom, L. B. (2004) J. Biol. Chem. 279, 4386-4393) addressing the question of how ATP binding and hydrolysis modulate specific interactions with DNA and beta. Pre-steady-state rates of ATP hydrolysis were slower when reactions were initiated by addition of ATP than when the gamma complex was equilibrated with ATP and were limited by the rate of an intramolecular reaction, possibly ATP-induced conformational changes. Kinetic modeling of assays in which the gamma complex was incubated with ATP for different periods of time prior to adding DNA to trigger hydrolysis suggests a mechanism in which a relatively slow conformational change step (kforward = 6.5 s(-1)) produces a species of the gamma complex that is activated for DNA (and beta) binding. In the absence of beta, 2 of the 3 molecules of ATP are hydrolyzed rapidly prior to releasing DNA, and the 3rd molecule is hydrolyzed slowly. In the presence of beta, all 3 molecules of ATP are hydrolyzed rapidly. These results suggest that hydrolysis of 2 molecules of ATP may be coupled to conformational changes that reduce interactions with DNA, whereas hydrolysis of the 3rd is coupled to changes that result in release of beta.

Adenosine Triphosphate↗
PubMed2005

Stability of the topoisomerase II closed clamp conformation may influence DNA-stimulated ATP hydrolysis.

Type II DNA topoisomerases catalyze changes in DNA topology and use nucleotide binding and hydrolysis to control conformational changes required for the enzyme reaction. We examined the ATP hydrolysis activity of a bisdioxopiperazine-resistant mutant of human topoisomerase II alpha with phenylalanine substituted for tyrosine at residue 50 in the ATP hydrolysis domain of the enzyme. This substitution reduced the DNA-dependent ATP hydrolysis activity of the mutant protein without affecting the relaxation activity of the enzyme. A similar but stronger effect was seen when the homologous mutation (Tyr28 --> Phe) was introduced in yeast Top2. The ATPase activities of human TOP2alpha(Tyr50 --> Phe) and yeast Top2(Tyr28 --> Phe) were resistant to both bisdioxopiperazines and the ATPase inhibitor sodium orthovanadate. Like bisdioxopiperazines, vanadate traps the enzyme in a salt-stable closed conformation termed the closed clamp, which can be detected in the presence of circular DNA substrates. Consistent with the vanadate-resistant ATPase activity, salt-stable closed clamps were not detected in reactions containing the yeast or human mutant protein, vanadate, and ATP. Similarly, ADP trapped wild-type topoisomerase II as a closed clamp, but could not trap either the human or yeast mutant enzymes. Our results demonstrate that bisdioxopiperazine-resistant mutants exhibit a difference in the stability of the closed clamp formed by the enzyme and that this difference in stability may lead to a loss of DNA-stimulated ATPase. We suggest that the DNA-stimulated ATPase of topoisomerase II is intimately connected with steps that occur while the N-terminal domain of the enzyme is dimerized.

Adenosine Diphosphate↗
PubMed2006

The beta sliding clamp binds to multiple sites within MutL and MutS.

The MutL and MutS proteins are the central components of the DNA repair machinery that corrects mismatches generated by DNA polymerases during synthesis. We find that MutL interacts directly with the beta sliding clamp, a ring-shaped dimeric protein that confers processivity to DNA polymerases by tethering them to their substrates. Interestingly, the interaction of MutL with beta only occurs in the presence of single-stranded DNA. We find that the interaction occurs via a loop in MutL near the ATP-binding site. The binding site of MutL on beta locates to the hydrophobic pocket between domains two and three of the clamp. Site-specific replacement of two residues in MutL diminished interaction with beta without disrupting MutL function with helicase II. In vivo studies reveal that this mutant MutL is no longer functional in mismatch repair. In addition, the human MLH1 has a close match to the proliferating cell nuclear antigen clamp binding motif in the region that corresponds to the beta interaction site in Escherichia coli MutL, and a peptide corresponding to this site binds proliferating cell nuclear antigen. The current report also examines in detail the interaction of beta with MutS. We find that two distinct regions of MutS interact with beta. One is located near the C terminus and the other is close to the N terminus, within the mismatch binding domain. Complementation studies using genes encoding different MutS mutants reveal that the N-terminal beta interaction motif on MutS is essential for activity in vivo, but the C-terminal interaction site for beta is not. In light of these results, we propose roles for the beta clamp in orchestrating the sequence of events that lead to mismatch repair in the cell.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Replication protein A directs loading of the DNA damage checkpoint clamp to 5'-DNA junctions.

The heterotrimeric checkpoint clamp comprises the Saccharomyces cerevisiae Rad17, Mec3, and Ddc1 subunits (Rad17/3/1, the 9-1-1 complex in humans). This DNA damage response factor is loaded onto DNA by the Rad24-RFC (replication factor C-like complex with Rad24) clamp loader and ATP. Although Rad24-RFC alone does not bind to naked partial double-stranded DNA, coating of the single strand with single-stranded DNA-binding protein RPA (replication protein A) causes binding of Rad24-RFC via interactions with RPA. However, RPA-mediated binding is abrogated when the DNA is coated with RPA containing a rpa1-K45E (rfa1-t11) mutation. These properties allowed us to determine the role of RPA in clamp-loading specificity. The Rad17/3/1 clamp is loaded with comparable efficiency onto naked primer/template DNA with either a 3'-junction or a 5'-junction. Remarkably, when the DNA was coated with RPA, loading of Rad17/3/1 at 3'-junctions was completely inhibited, thereby providing specificity to loading at 5'-junctions. However, Rad17/3/1 loaded at 5'-junctions can slide across double-stranded DNA to nearby 3'-junctions and thereby affect the activity of proteins that act at 3'-termini. These studies show a unique specificity of the checkpoint loader for 5'-junctions of RPA-coated DNA. The implications of this specificity for checkpoint function are discussed.

Adenosine Triphosphatases↗
PubMed2002

Abnormalities of the attachment clamps of representatives of the family Diplozoidae.

A comparative study has been made of the haptoral morphology of four species of diplozoon (Monogenea: Diplozoidae) from the gills of fish exposed to different levels of water pollution in two river systems in eastern Europe. An examination of the haptors of Paradiplozoon homoion (Bychowsky & Nagibina 1959), Paradiplozoon ergensi (Pejcoch 1968) and Paradiplozoon megan (Bychowsky & Nagibina 1959) from chub caught in the River Morava, Czech Republic and of Diplozoon paradoxum (Nordmann 1832) from bream recovered from the River Volga, Russia has revealed abnormalities to the attachment clamps. Two abnormal conditions were found: structural alterations to the attachment clamps and changes in the number of attachment clamps; these occurred both singly and in combination. A higher frequency of abnormal attachment clamps was found in diplozoons from fish caught in the more polluted localities of both rivers. The abnormalities have been recorded and their morphology compared in the light of conditions of environmental stress.

Animals↗
PubMed1999

Operations that require supracoeliac aortic cross-clamping: perioperative monitoring with gastric tonometry.

OBJECTIVE: To assess the predictive value of a gastric intramucosal pH of less than 7.35 for mortality in surgical patients after supracoeliac aortic cross-clamping. DESIGN: Open prospective clinical study. SETTING: University hospital, The Netherlands. SUBJECTS: Six patients who required temporary supracoeliac, and four patients who required temporary infrarenal, cross-clamping of the aorta. MAIN OUTCOME MEASURES: Mortality and conventional measures of organ dysfunction correlated with gastric tonometry. RESULTS: All 6 patients who required supracoeliac cross-clamping underwent a steep, and 5 patients a prolonged, decrease in the gastric intramucosal pH. The mean lowest gastric intramucosal pH in the supracoeliac group was 7.05 and in the infrarenal group 7.28. All patients recovered completely. CONCLUSION: A pHig value below 7.35 does not seem to be a marker of mortality in patients who have undergone supracoeliac cross-clamping of the aorta.

APACHE↗
PubMed2003

Initial experience in laparoscopic partial nephrectomy for renal tumor with clamping of renal vessels.

PURPOSE: To describe our initial experience with laparoscopic partial nephrectomy (LPN) with clamping of the renal vessels before tumor excision and suturing of the renal parenchyma. PATIENTS AND METHODS: Between July 2001 and April 2002, 19 consecutive patients underwent transperitoneal LPN in our institution, 14 for tumors <4 cm with suspicion of renal-cell cancer and 5 for suspicion of angiomyolipoma at CT with one tumor confirmed histopathologically by percutaneous needle biopsy. We divided these patients into the first 10 cases (Group 1) and the last 9 cases (Group 2). One patient had end-stage renal disease but was not on dialysis; the remaining patients had elective partial nephrectomy. Initially, a ureteral catheter was placed. The partial nephrectomy was performed with clamping of the renal vessels, so that the tumor was excised with cold scissors. Intracorporeal cooling of the kidney was achieved by a ureteral catheter connected to a 4 degrees C solution flowing to the renal pelvis during the whole procedure until the clamps were released. Intracorporeal free-hand suturing was exclusively used to close the collecting system (when opened) and to approximate the renal parenchyma. RESULTS: All procedures were completed laparoscopically. The mean renal warm ischemia time was 28.5+/-7 minutes (range 15-47 minutes). The mean laparoscopic operating time was 125+/-37 minutes (range 90-390 minutes). The mean intraoperative blood loss was 290+/-276 mL (range 25-1200 mL). Two patients required blood transfusion, and four had complications. There was immediate deterioration in renal function (creatinine 1.42+/-0.56 mg/dL), but improvement was seen at 1 month (1.17+/-0.34 mg/dL). There were no statistically significant differences in operative features and outcomes in Groups 1 and 2, but there were improvements in the mean operating time by 30 minutes, the mean intraoperative blood loss by 113 mL without any transfusion, and the mean renal warm ischemia time by 6 minutes. There was only one patient in Group 2 with a complication. The surgical margin was negative for tumor for all patients. Postoperative pathology examination showed renal-cell cancer in 11 patients (pT1), oncocytoma in 3 patients, and angiomyolipoma in 5 patients. The mean tumor grade was 2. The mean tumor size was 25.8+/-11.6 mm with a mean tumor-free margin of 2.6+/-2.4 mm. The median follow-up is 3 months, so oncologic outcome cannot be assessed. CONCLUSION: The technique of LPN can be standardized and should be proposed for small tumors when they are not invading the hilum. Clamping the renal pedicle allows better vision for more accurate tumor excision with a safety margin and hemostatic suturing of the parenchymal defect, resulting in less blood loss and shorter operative time, parameters that improve with experience.

Adenoma, Oxyphilic↗
PubMed2004

Hand-assisted laparoscopic partial nephrectomy without hilar vascular clamping using a saline-cooled, high-density monopolar radiofrequency device.

BACKGROUND AND PURPOSE: Nephron-sparing surgery is now accepted as an alternative treatment option for small renal tumors. However, hemostasis during laparoscopic partial nephrectomy can be technically challenging, especially without hilar vascular clamping. The aim of our study was to evaluate the technique of hand-assisted laparoscopic partial nephrectomy using the TissueLink (TissueLink Medical, Dover, NH), a saline-cooled monopolar radiofrequency device, without hilar vascular clamping. PATIENTS AND METHODS: Using the hand-assisted laparoscopic approach, the kidney is mobilized transperitoneally, and the renal tumor with overlying perinephric fat is exposed. The tumor is excised with a 1-cm margin using a combination of the TissueLink device and endoscopic scissors. The tumor and a biopsy of the base of the tumor bed are sent for frozen-section examination. The bleeding vessels are controlled with digital compression and the TissueLink device. At the end of procedure, the tumor bed is covered with a hemostatic agent. Three female and four male patients ages 52 to 76 years (mean 66 years) were treated with this new device for incidental tumors detected during imaging studies (N = 6) or during work-up for gross hematuria (N = 1). Preoperative imaging studies included CT in six patients and MRI in three. The average tumor size was 2.2 cm (range 1.3-3 cm). Only peripheral tumors that did not approach the hilum or the collecting system were selected. RESULTS: All of the patients underwent a hand-assisted laparoscopic partial nephrectomy using the TissueLink device without hilar vascular clamping. There were no intraoperative complications or conversions to open surgery. The mean operative time was 175 minutes, with an estimated blood loss of 186 mL (range 100-300 mL). Histologic examination demonstrated renal-cell carcinoma in five cases, oncocytoma in one, and an angiomyolipoma in one. The dimensions of the normal tissue around the tumor ranged from 1 to 4 mm, and frozen-section analysis showed tumor-free margins in all cases. Postoperatively, all patients recovered well except one patient who developed transient atrial fibrillation, which was treated medically in the immediate postoperative period. All patients were discharged in good condition at an average of 3 days (range 2-6 days). CONCLUSION: Hand-assisted laparoscopic partial nephrectomy without vascular clamping using the TissueLink device is a safe and feasible technique for exclusion of small exophytic renal tissues.

Aged↗
PubMed1995

Structural and functional similarities of prokaryotic and eukaryotic DNA polymerase sliding clamps.

The remarkable processivity of cellular replicative DNA polymerases derive their tight grip to DNA from a ring-shaped protein that encircles DNA and tethers the polymerase to the chromosome. The crystal structures of prototypical 'sliding clamps' of prokaryotes (beta subunit) and eukaryotes (PCNA) are ring shaped proteins for encircling DNA. Although beta is a dimer and PCNA is a trimer, their structures are nearly superimposable. Even though they are not hexamers, the sliding clamps have a pseudo 6-fold symmetry resulting from three globular domains comprising each beta monomer and two domains comprising each PCNA monomer. These domains have the same chain fold and are nearly identical in three-dimensions. The amino acid sequences of 11 beta and 13 PCNA proteins from different organisms have been aligned and studied to gain further insight into the relation between the structure and function of these sliding clamps. Furthermore, a putative embryonic form of PCNA is the size of beta and thus may encircle DNA as a dimer like the prokaryotic clamps.

Amino Acid Sequence↗
PubMed2002

Acridine-modified, clamp-forming antisense oligonucleotides synergize with cisplatin to inhibit c-Myc expression and B16-F0 tumor progression.

The c-myc protooncogene plays a key role in the abnormal growth regulation of melanoma cells. We have targeted three polypurine sequences within the mouse myc mRNA with acridine-modified, clamp-forming antisense oligonucleotides (AS ODNs) in an effort to inhibit growth of murine melanoma cells. These ODNs are unique in that they hybridize to the target mRNA by both Watson-Crick and Hoogsteen hydrogen bond interactions, forming a triple-stranded structure. At a concentration of 3 microM E1C, E2C and E3C inhibit B16-F0 proliferation by 76, 66 and 78%, respectively. Both immunofluorescent staining and western blot analysis corroborate a proportional reduction in c-Myc expression by all three ODNs. There were clear distinctions in the ability of these ODNs to inhibit tumor progression in C57BL/6 mice as a function of Myc expression. There was no synergy demonstrated between ODN E1C with cisplatin (DDP), which inhibited tumor growth by 77% alone and 82% in combination. Although E2C inhibited growth by 54%, its effect was decreased to 32% with DDP, when compared with controls. E3C, on the other hand, demonstrated a synergistic effect with DDP, inhibiting growth by 72% in combination, but only by 1% as a single agent. Immunofluorescence analysis of tumors for each group revealed a concomitant reduction in c-Myc expression in tumors from mice treated with the most active clamp ODN alone (E1C) or clamp ODN + DDP (E1C/E3C + DDP). Western blot analysis confirmed this decrease in target protein expression. Our results document the growth-inhibitory activity of two myc-targeting antisense clamp ODNs; E1C, which has activity as a single agent, and E3C, which has in vivo synergy with DDP pretreatment. These data confirm the antiproliferative effects of these novel ODNs and document an interesting synergy with the chemotherapeutic agent DDP.

Acridines↗
PubMed2003

Ligation of high-melting-temperature 'clamp' sequence extends the scanning range of rare point-mutational analysis by constant denaturant capillary electrophoresis (CDCE) to most of the human genome.

Mutations cause or influence the prevalence of many diseases. In human tissues, somatic point mutations have been observed at fractions at or below 4/10,000 and 5/100,000 in mitochondrial and nuclear DNA, respectively. In human populations, fractions for the multiple alleles that code for recessive deleterious syndromes are not expected to exceed 5 x 10(-4). Both nuclear and mitochondrial point mutations have been measured in human cells and tissues at fractions approaching 10(-6) using constant denaturant capillary electrophoresis (CDCE) coupled with high-fidelity PCR (hifiPCR). However, this approach is only applicable to those target sequences (approximately 100 bp) juxtaposed with a 'clamp', a higher-melting-temperature sequence, in genomic DNA; such naturally clamped targets represent approximately 9% of the human genome. To open up most of the human genome to rare point-mutational analysis, a high-efficiency DNA ligation procedure was recently developed so that a clamp could be attached to any target of interest. We coupled this ligation procedure with prior CDCE/hifiPCR and achieved a sensitivity of 2 x 10(-5) in human cells for the first time using an externally attached clamp. At this sensitivity, somatic mutations, each representing an anatomically distinct cluster of cells (turnover unit) derived from a mutant stem cell, may be detected in a series of tissue samples, each containing as many as 5 x 10(4) turnover units. Additionally, rare inherited mutations may be scanned in pooled DNA samples, each derived from as many as 10(5) persons.

Cell Line↗