Performance of ChatGPT and Bard in self-assessment questions for nephrology board renewal.

BACKGROUND: Large language models (LLMs) have impacted advances in artificial intelligence. While LLMs have demonstrated high performance in general medical examinations, their performance in specialized areas such as nephrology is unclear. This study aimed to evaluate ChatGPT and Bard in their potential nephrology applications. METHODS: Ninety-nine questions from the Self-Assessment Questions for Nephrology Board Renewal from 2018 to 2022 were presented to two versions of ChatGPT (GPT-3.5 and GPT-4) and Bard. We calculated the correct answer rates for the five years, each year, and question categories and checked whether they exceeded the pass criterion. The correct answer rates were compared with those of the nephrology residents. RESULTS: The overall correct answer rates for GPT-3.5, GPT-4, and Bard were 31.3% (31/99), 54.5% (54/99), and 32.3% (32/99), respectively, thus GPT-4 significantly outperformed GPT-3.5 (p < 0.01) and Bard (p < 0.01). GPT-4 passed in three years, barely meeting the minimum threshold in two. GPT-4 demonstrated significantly higher performance in problem-solving, clinical, and non-image questions than GPT-3.5 and Bard. GPT-4's performance was between third- and fourth-year nephrology residents. CONCLUSIONS: GPT-4 outperformed GPT-3.5 and Bard and met the Nephrology Board renewal standards in specific years, albeit marginally. These results highlight LLMs' potential and limitations in nephrology. As LLMs advance, nephrologists should understand their performance for future applications.

The temporal balance between self-renewal and differentiation of human neural stem cells requires the amyloid precursor protein.

Neurogenesis in the developing human cerebral cortex occurs at a particularly slow rate owing in part to cortical neural progenitors preserving their progenitor state for a relatively long time, while generating neurons. How this balance between the progenitor and neurogenic state is regulated, and whether it contributes to species-specific brain temporal patterning, is poorly understood. Here, we show that the characteristic potential of human neural progenitor cells (NPCs) to remain in a progenitor state as they generate neurons for a prolonged amount of time requires the amyloid precursor protein (APP). In contrast, APP is dispensable in mouse NPCs, which undergo neurogenesis at a much faster rate. Mechanistically, APP cell-autonomously contributes to protracted neurogenesis through suppression of the proneurogenic activator protein-1 transcription factor and facilitation of canonical WNT signaling. We propose that the fine balance between self-renewal and differentiation is homeostatically regulated by APP, which may contribute to human-specific temporal patterns of neurogenesis.

RevGel-seq: instrument-free single-cell RNA sequencing using a reversible hydrogel for cell-specific barcoding.

Progress in sample preparation for scRNA-seq is reported based on RevGel-seq, a reversible-hydrogel technology optimized for samples of fresh cells. Complexes of one cell paired with one barcoded bead are stabilized by a chemical linker and dispersed in a hydrogel in the liquid state. Upon gelation on ice the complexes are immobilized and physically separated without requiring nanowells or droplets. Cell lysis is triggered by detergent diffusion, and RNA molecules are captured on the adjacent barcoded beads for further processing with reverse transcription and preparation for cDNA sequencing. As a proof of concept, analysis of PBMC using RevGel-seq achieves results similar to microfluidic-based technologies when using the same original sample and the same data analysis software. In addition, a clinically relevant application of RevGel-seq is presented for pancreatic islet cells. Furthermore, characterizations carried out on cardiomyocytes demonstrate that the hydrogel technology readily accommodates very large cells. Standard analyses are in the 10,000-input cell range with the current gelation device, in order to satisfy common requirements for single-cell research. A convenient stopping point after two hours has been established by freezing at the cell lysis step, with full preservation of gene expression profiles. Overall, our results show that RevGel-seq represents an accessible and efficient instrument-free alternative, enabling flexibility in terms of experimental design and timing of sample processing, while providing broad coverage of cell types.

Minimally Invasive Spinal Fusion Using Percutaneous Pedicle Screw Instrumentation Can Provide a Better Health-Related QOL in Early Stage Than Conventional Methods in the Treatment of Single-Level Degenerative Lumbar Spinal Diseases.

Purpose: To investigate whether the minimally invasive spinal fusion can provide the better outcome than conventional fusion surgery in the treatment of degenerative lumbar spinal diseases. Patients and Methods: One hundred and thirteen patients who had undergone single-level fusion surgery for degenerative lumbar spinal diseases were examined with a minimum of one-year follow-up. There were 56 men and 57 women with a median age of 70s ranging 47-88. The following three-types of fusion surgery were performed; minimally invasive transforaminal interbody fusion after microscopic decompression through a unilateral approach with percutaneous pedicle screwing (MTLIF), transforaminal interbody fusion after microscopic decompression through a unilateral approach (TLIF), and posterior lumbar interbody fusion with posterolateral fusion after open decompression through a bilateral approach (PLIF). The purpose for limiting on single level degenerative spinal disease was that it would be easy to compare the surgical outcomes among the three groups. Results: There were no statistically significant differences among three groups in terms of VAS scores, RDQ scores, and all of the domains in the JOABPEQ scores at the baseline. The JOABPEQ score for pain-related disorders at 6 months after surgery was statistically significantly higher in MTLIF group than the other two groups (P = 0.023). There were no statistically significant differences in the scores of the other outcome measures among three groups in whole follow-up period. Conclusion: The current study demonstrated that the JOABPEQ score for pain-related disorders at 6 months postoperatively was significantly better in MTLIF group than in the other groups. Since lumbar degenerative diseases mostly consisted in elderly patients, less invasive surgeries are desirable. MTLIF resulted in a better health-related QOL at 6 months after surgery, and its outcomes at the final follow-up were non-numerical inferiority. The results strongly indicate that MTLIF is desirable surgery especially for elderly patients with degenerative spinal diseases.

Clinical outcomes of lumbar diseases specific test in patients who undergo endoscopy-assisted tubular surgery with lumbar herniated nucleus pulposus: an analysis using the Japanese Orthopaedic Association Back Pain Evaluation Questionnaire (JOABPEQ).

PURPOSE: This study was to evaluate clinical outcomes using a patient-oriented test that scores health-related quality of life (HRQOL) for patients after minimally invasive surgery using microendoscopic discectomy (MED) for lumbar disc hernia. Few studies regarding MED in terms of disease-specific quality of life measures using Japanese Orthopaedic Association Back Pain Evaluation Questionnaire (JOABPEQ) have been published. METHODS: Retrospective analysis of the surgical and clinical outcomes with regard to reducing pain and improving the functional status for 31 patients who underwent MED for lumbar disc hernia was conducted. These patients were evaluated at 3-year follow-up. The evaluations were based on a visual analogue scale (VAS), the Japanese Orthopaedic Association (JOA) scoring system, and the JOABPEQ, which is an objective, patient-oriented test that assesses HRQOL in patients with lumbar disorders. RESULTS: A low rate of improvement was seen only in mental health until 1 year, the low rate of improvement in mental health and was independently correlated with body mass index (BMI), pre-operative scores on the Brief Scale for Psychiatric problems in Orthopaedic Patients (BS-POP), and scores on the BS-POP at 12 months post-operatively. CONCLUSIONS: All categories of VAS, JOA scores, and all domains of JOABPEQ were significantly higher over 3 years than those obtained pre-operatively. But only mental health domain showed mild improvement until 1 year. Moreover, BMI showed a negative correlation with improvements in the mental health domain post-operatively. As patients may be mentally exhausted from lumbar disc herniation, pre-operative mental health may be improved by surgical treatment.

Seven-year follow-up for malrotation of a radial diaphysis fracture in a child corrected by osteotomy for loss of motion: A case report.

A greenstick fracture is an incomplete fracture where the compressive side of the cortex is still intact but plastically deformed. The incidence of poor results following the closed treatment of greenstick fractures in children >10 years of age is seriously underestimated. Therefore, fixing the position of the forearm is important for initial treatment. In cases of greenstick fracture, the possibility of inadequate remodeling of angulated deformities during growth, and in particular, the lack of correction between rotational malalignment and growth when the diaphyseal forearm is involved in the fracture, should be considered. A male, 10-year-old, left hand-dominant, Asian patient fell while playing in the garden and was immediately assessed by an orthopedic doctor at an Emergency Orthopaedic Clinic. Initial examination revealed a deformity of his dominant left forearm and an angulated greenstick fracture of the radius. However, after 3 months, he developed loss of supination of his left forearm and complained of limitation of left forearm supination. Radiography demonstrated a volar angulation of 20°. The patient underwent open reduction, internal fixation and 10° bending with a plate for correction without corrective rotation. At 12 months after injury, the patient did not exhibit pain or limitation of the elbow and wrist. After follow-up for 7 years, the patient was able to perform normal day-to-day activities with no adverse symptoms. The present case indicated that corrective osteotomy is required following the loss of supination after a greenstick fracture of the diaphysis of the radius. The patient of the current study exhibited rotation due to the central band of the interosseous membrane. In the treatment of greenstick fractures, a radius apex angulation of 20° must be corrected via osteotomy due to loss of rotation. The present case indicated that corrective osteotomy of the radius apex alone without rotational correction, in combination with plate bending improved the loss of forearm rotation.

Recovery of paraplegia following postoperative epidural hematomas in lumbar canal stenosis surgery by closed kinetic chain (CKC) exercises: A case report.

RATIONALE: A postoperative spinal epidural hematoma (PSEH) is among the most devastating complications following spine and spinal cord surgery, and it should be considered before performing microendoscopic decompression as part of minimally invasive surgery, since early recovery is one of the advantages of this procedure. PATIENT CONCERNS: A 70-year-old woman with lumbar spinal stenosis at L4-5 underwent tubular surgery with the assistance of endoscopic laminectomy (MEL), but 2 days after the surgery, the patient noticed decreased lower limb sensation and power of the right leg, and she developed numbness from the level of L5 and weakness from the L4, 5 myotome distally. DIAGNOSES: An epidural hematoma at the L4-5 surgical site was found on magnetic resonance imaging of the lumbar spine and evacuated operatively. This rare complication appears to be the result of a PSEH. In the present case, complete neurological recovery was not achieved, despite rapid surgery. INTERVENTIONS: The patient's course and physical therapy, which focused on attitude maintenance practice and muscle-strengthening exercise of the closed kinetic change (CKC) type from the early stage of paraplegia, were specifically examined. OUTCOMES: The patient recovered complete motor function with attitude maintenance practice and muscle strengthening exercises after 3 months. CKC exercise in particular may contribute to improving caudal muscle weakness, including the L4, 5 innervated area (e.g., tibialis anterior [TA], extensor hallucis longus [EHL], flexor hallucis longus [FHL], gastrocnemius [GC], etc.). LESSONS: Prevention of PSEH is needed to not only avoid neurological deterioration, but also avoid delaying the patient's recovery. CKC exercise may contribute to improving the prolonged paralysis associated with a PSEH. Future studies should involve larger numbers of patients to evaluate the clinical features of PSEH and treatment by rehabilitation with more effective muscle exercises and stretches.

A complete posterior tibial stress fracture that occurred during a middle-distance running race: a case report.

Posterior tibial stress fractures are more frequent than anterior tibial stress fractures, and they are considered to have a good prognosis for returning to sports; cases leading to a complete fracture are rare. A 17-year-old male involved in high school athletics middle-distance running had a 3-week history of pain with training. He was running up to 300 km/week on streets and cross-country in an even distribution. He had posterior tibial stress fractures, but despite the lower leg pain, he continued running. One year later, he was brought to the emergency department after having sustained an injury to the right lower leg while running in a middle-distance race; bilateral tibial stress fractures, with one side complete and the opposite side incomplete, had developed simultaneously. This relatively rare case of bilateral posterior stress fractures, with one side a complete fracture and the opposite side an incomplete fracture, that was treated surgically via exchange intramedullary nailing is reported. The patient could begin light jogging from 3 months after surgery and was without symptoms at 5 months after surgery. He could resume middle-distance racing after 1 year. Posterior tibial cortical fractures are more common and respond better to conservative treatment than anterior tibial stress fractures, and they are a common fracture type in runners. We believe that close, careful follow-up is necessary if patients continue excessive training.

Tubular surgery with the assistance of endoscopic surgery via a paramedian or midline approach for lumbar spinal canal stenosis at the L4/5 level.

BACKGROUND: Lumbar spinal canal stenosis surgery has recently improved with the use of minimally invasive techniques. Less invasive procedures have emerged, and microendoscopic decompression through smaller incisions is frequently performed. Tubular surgery with the assistance of endoscopic surgery procedures has led to particularly remarkable changes in surgery, with reduced tissue trauma and morbidity. PURPOSE: The purpose of this study was to compare the clinical outcomes of two different minimally invasive decompressive surgical techniques (microendoscopic bilateral decompression surgery using the unilateral approach [microendoscopic laminectomy (MEL)] and microendoscopy-assisted muscle-preserving interlaminar decompression (MILD; ME-MILD)) using spinal endoscopy for lumbar spinal canal stenosis measured using a visual analog scale (VAS), the Japanese Orthopedic Association (JOA) score, and the JOA Back Pain Evaluation Questionnaire (JOABPEQ), which is based on a patient-oriented scoring system. STUDY DESIGN: This study was a retrospective review of prospectively collected surgical data. METHODS: The study included 81 patients (MEL 39 patients, 20 men and 19 women, mean age 68.9 years; and ME-MILD 42 patients, 22 men and 20 women, mean age 73.1 years) with lumbar spinal stenosis (LSS). The indications for surgery were moderate-to-severe stenosis, persistent neurological symptoms, and failure of conservative treatment over 3 months, with a JOA score under 15 points or intermittent claudication at 100 m. This study included patients having LSS at a single vertebral level (L4/5). RESULTS: Low back pain, buttock-leg pain, and numbness were significantly improved in terms of the VAS score from 3 months with both MEL and ME-MILD. In all periods, JOA scores over 3 years of follow-up were significantly higher than those obtained before surgery with both MEL and ME-MILD, and there were improvements of low back pain and walking function. CONCLUSIONS: These observations demonstrate that ME-MILD is a safe and very effective minimally invasive technique for degenerative LSS, similar to MEL.

Effect of intermittent administration of teriparatide on the mechanical and histological changes in bone grafted with ß-tricalcium phosphate using a rabbit bone defect model.

Grafting ß-tricalcium phosphate (TCP) is a well-established method for restoring bone defects; however, there is concern that the mechanical stability of the grafted ß-TCP is not maintained during bone translation. Teriparatide has an anabolic effect, stimulating bone formation and increasing bone mineral density for the treatment of osteoporosis. The aim of the present study was to evaluate the effect of intermittent teriparatide treatment on changes in bone grafted with ß-TCP using a rabbit bone defect model. Bone defects (5×15 mm) were created in the distal femoral condyle of Japanese white rabbits, and ß-TCP granules of two different total porosities were manually grafted. Teriparatide (40 µg/kg) or 0.2% rabbit serum albumin solution as a vehicle control was subcutaneously injected three times per week following the surgery. At 4 or 8 weeks post-surgery, serum samples were obtained and the levels of γ-carboxylated osteocalcin (Gla-OC) were quantified using ELISA. Histomorphometry was also performed using sections of graft sites following staining for tartrate resistant acid phosphatase. Activity and mechanical strength (maximum shear strength, maximum shear stiffness and total energy absorption) were evaluated using an axial push-out load to failure test. Teriparatide treatment significantly increased (P<0.05) the serum levels of Gla-OC, a specific marker for bone formation, suggesting that teriparatide enhances bone formation in ß-TCP-grafted rabbits. Furthermore teriparatide increased the degradation of ß-TCP by bone remodeling (P<0.05) and promoted the formation of new bone following application of the graft compared with the control group (P<0.01). Furthermore, teriparatide suppressed the reduction in mechanical strength (P<0.05) during bone translation in bone defects grafted with ß-TCP. The results of the present study demonstrate that teriparatide is effective in maintaining the mechanical stability of grafted ß-TCP, possibly by promoting new bone formation.

Molecular combing compared to Southern blot for measuring D4Z4 contractions in FSHD.

We compare molecular combing to Southern blot in the analysis of the facioscapulohumeral muscular dystrophy type 1 locus (FSHD1) on chromosome 4q35-qter (chr 4q) in genomic DNA specimens sent to a clinical laboratory for FSHD testing. A de-identified set of 87 genomic DNA specimens determined by Southern blot as normal (n = 71), abnormal with D4Z4 macrosatellite repeat array contractions (n = 7), indeterminate (n = 6), borderline (n = 2), or mosaic (n = 1) was independently re-analyzed by molecular combing in a blinded fashion. The molecular combing results were identical to the Southern blot results in 75 (86%) of cases. All contractions (n = 7) and mosaics (n = 1) detected by Southern blot were confirmed by molecular combing. Of the 71 samples with normal Southern blot results, 67 (94%) had concordant molecular combing results. The four discrepancies were either mosaic (n = 2), rearranged (n = 1), or borderline by molecular combing (n = 1). All indeterminate Southern blot results (n = 6) were resolved by molecular combing as either normal (n = 4), borderline (n = 1), or rearranged (n = 1). The two borderline Southern blot results showed a D4Z4 contraction on the chr 4qA allele and a normal result by molecular combing. Molecular combing overcomes a number of technical limitations of Southern blot by providing direct visualization of D4Z4 macrosatellite repeat arrays on specific chr 4q and chr 10q alleles and more precise D4Z4 repeat sizing. This study suggests that molecular combing has superior analytical validity compared to Southern blot for determining D4Z4 contraction size, detecting mosaicism, and resolving borderline and indeterminate Southern blot results. Further studies are needed to establish the clinical validity and diagnostic accuracy of these findings in FSHD.

Effects of the combination of vitamin K and teriparatide on the bone metabolism in ovariectomized rats.

The purpose of the present study was to evaluate the combined effects of vitamin K (VK) and teriparatide (TPTD) on bone mineral density (BMD), mechanical strength and other parameters for bone metabolism using a rat ovariectomized osteoporosis model. Ovariectomized female Sprague-Dawley rats were administered with VK (an oral dose of 30 mg/kg/day), TPTD (a subcutaneous dose of 30 µg/kg, three times a week) or a combination for 8 weeks. Thereafter, serum levels of γ-carboxylated osteocalcin (Gla-OC) were quantitated by ELISA; BMD and mechanical strength were measured by computed tomography and biomechanical testing, respectively at the femoral metaphysis. Additionally, histomorphometry was performed using the toluidine blue-stained coronal sections of distal femur. The combination of VK and TPTD clearly increased the serum levels of Gla-OC (a specific marker for bone formation) and osteoblast surface (the number of osteoblasts attaching with the surface of cancellous bone), compared to VK or TPTD alone. In addition, the combination of the two agents improved the BMD and bone strength of the femur in the ovariectomized rats, compared to VK or TPTD alone. Taken together, these findings suggest that the treatment with VK and TPTD may have a therapeutic advantage over VK or TPTD monotherapy for postmenopausal osteoporosis, possibly by enhancing the bone formation through the actions on OC and osteoblasts.

Active human nucleolar organizer regions are interspersed with inactive rDNA repeats in normal and tumor cells.

AIM: The synthesis of rRNA is a key determinant of normal and malignant cell growth and subject to epigenetic regulation. Yet, the epigenomic features of rDNA arrays clustered in nucleolar organizer regions are largely unknown. We set out to explore for the first time how DNA methylation is distributed on individual rDNA arrays. MATERIALS & METHODS: Here we combined immunofluorescence detection of DNA modifications with fluorescence hybridization of single DNA fibers, metaphase immuno-FISH and methylation-sensitive restriction enzyme digestions followed by Southern blot. RESULTS: We found clustering of both hypomethylated and hypermethylated repeat units and hypermethylation of noncanonical rDNA in IMR90 fibroblasts and HCT116 colorectal carcinoma cells. Surprisingly, we also found transitions between hypo- and hypermethylated rDNA repeat clusters on single DNA fibers. CONCLUSION: Collectively, our analyses revealed co-existence of different epialleles on individual nucleolar organizer regions and showed that epi-combing is a valuable approach to analyze epigenomic patterns of repetitive DNA.

Primary microcephaly, impaired DNA replication, and genomic instability caused by compound heterozygous ATR mutations.

Ataxia telangiectasia-mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) kinases are two key regulators of DNA-damage responses (DDR) that are mainly activated in response to DNA double-strand breaks and single-stranded DNA damages, respectively. Seckel syndrome, a rare genetic disorder characterized by a microcephaly and a markedly reduced body size, has been associated with defective ATR-dependent DNA damage signaling. However, the only human genetic ATR defect reported so far is a hypomorphic splicing mutation identified in five related individuals with Seckel syndrome. Here, we report the first case of primary microcephaly with compound heterozygous mutations in ATR: a 540 kb genomic deletion on one allele and a missense mutation leading to splice dysregulation on the other, which ultimately lead to a sharp decrease in ATR expression. DNA combing technology revealed a profound spontaneous alteration of several DNA replication parameters in patient's cells and FISH analyses highlighted the genomic instability caused by ATR deficiency. Collectively, our results emphasize the crucial role for ATR in the control of DNA replication, and reinforce the complementary and nonredundant contributions of ATM and ATR in human cells to face DNA damages and warrant genome integrity.

Spi-1/PU.1 oncogene accelerates DNA replication fork elongation and promotes genetic instability in the absence of DNA breakage.

The multistage process of cancer formation is driven by the progressive acquisition of somatic mutations. Replication stress creates genomic instability in mammals. Using a well-defined multistep leukemia model driven by Spi-1/PU.1 overexpression in the mouse and Spi-1/PU.1-overexpressing human leukemic cells, we investigated the relationship between DNA replication and cancer progression. Here, using DNA molecular combing and flow cytometry methods, we show that Spi-1 increases the speed of replication by acting specifically on elongation rather than enhancing origin firing. This shortens the S-phase duration. Combining data from Spi-1 knockdown in murine cells with Spi-1 overexpression in human cells, we provide evidence that inappropriate Spi-1 expression is directly responsible for the replication alteration observed. Importantly, the acceleration of replication progression coincides with an increase in the frequency of genomic mutations without inducing DNA breakage. Thus, we propose that the hitherto unsuspected role for spi-1 oncogene in promoting replication elongation and genomic mutation promotes blastic progression during leukemic development.

Recovery of intact yeast chromosomal DNA from agarose gel plugs using coil-globule transition.

In the current studies, we designed a new approach for sizing and isolating chromosomal DNA using coil-globule transition, which avoids fragmentation of giant DNA due to mechanical stress. Although coil-globule transition is reversible and globular DNA is tolerant to mechanical stress, globular DNA cannot be manipulated by an electric field because of the loss of its negative charges. In our system, however, DNA is extracted from an agarose gel in a coiled state into a solution of PEG, and coil-globule transition is induced by cations generated at the anode. This method achieves buffer exchange without stirring, which is the main cause of mechanical stress. Real-time analysis of T4dc viral DNA molecules revealed that they change immediately from a coiled to a globular form when the cation concentration is sufficiently high. This method was used to prepare yeast chromosomal DNA in a globular state without fragmentation.

Adaptor polymerase chain reaction for single molecule amplification.

The adaptor polymerase chain reaction (PCR) permits the amplification of DNA fragments with arbitrary sequences. In this paper, we describe the successful amplification of plasmid-derived single molecule DNAs digested by a restriction enzyme. By using adaptors made of short and long oligonucleotides, nonspecific interactions during PCR were suppressed. The method will be applicable to the detection of single molecule DNA fragments even if their sequence is unknown.

Single-molecule reverse transcription polymerase chain reaction using water-in-oil emulsion.

A new method based on a combination of reverse transcription polymerase chain reaction (RT-PCR) and a water-in-oil (W/O) emulsion was developed. Reverse transcription and initial thermal cycles were carried out in droplets of the W/O emulsion. Then, the droplets were united, followed by remaining secondary PCR cycles. This two-step method succeeded in detecting a single RNA molecule.

Activation of restriction enzyme by electrochemically released magnesium ion.

Observation and cutting of DNA molecules at intended positions permit several new experimental methods that are completely different from conventional molecular biology methods; therefore several cutting methods have been proposed and studied. In this paper, a new cutting method for a DNA molecule by localizing the activity of a restriction enzyme is presented. Since most restriction enzymes require magnesium ions for their activation, local restriction enzyme activity can be controlled by the local concentration of magnesium ions. Applying a direct current (dc) voltage to a needle electrode of metallic magnesium made it possible to control the local magnesium ion concentration at the tip of the needle. The restriction enzyme was activated only when magnesium ions were electrochemically supplied.

Single-molecule PCR using water-in-oil emulsion.

Polymerase chain reaction (PCR) using a single molecule of DNA is very useful for analysis, detection and cloning of the desired DNA fragment. We developed a simple PCR method utilizing a water-in-oil (W/O) emulsion that included numerous droplets of reaction mixture in bulk oil phase. These droplets, which were stable even at high temperatures, functioned as micro-reactors. This allows the effective concentration of template DNA to be increased, even for low concentrations of template DNA. The present method consists of a two-step thermal cycle. The first step was carried out using the W/O emulsion. During this step, the template DNA was amplified in the limited volume of the droplets in the W/O emulsion. The W/O emulsion was broken and the second PCR step was carried out. This method can be easily applied to amplify a single DNA molecule.