Objective: To develop a simplified polymerase chain reaction (PCR) protocol on single cells for the purpose of preimplantation genetic diagnosis. Also to evaluate a new thermal cycler, RoboCycler 40 (Stratagene, La Jolla, CA), for reducing the time to complete PCR amplification.
Design: PCR amplification without DNA purification or reamplification of a 149 base pair (bp) segment of the human Y chromosome was used as a model. The assay was tested in human fetal cells, single lymphocytes and single human blastomeres.
Results: Amplification of the 149 bp segment using fetal cells was 100% correct. Results on single lymphocytes were concordant in all but one of the 15 male cases. However, 2 of the 25 female cases were identified as male suggesting the occurrence of DNA contamination. Analysis of 61 blastomeres were concordant in 57 cases (93%); results for male blastomeres showed 12% of false negatives. No false positives were detected for female cells. Amplification using the simplified PCR protocol in combination with the RoboCycler was completed in 2 hours.
Conclusion: These data show that this PCR assay performed directly, without DNA extraction or purification and without re-amplification is a practical and effective approach for amplification of specific DNA sequences in single cells. Furthermore, the simplified PCR protocol significantly reduced the time to complete DNA amplification. The reduced time is expected to facilitate the management of a routine program for preimplantation genetic diagnosis.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.1016/s0015-0282(16)57719-x | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
AptaFluorescence: An aptamer-based fluorescent imaging protocol for biomolecule visualization.
PLoS One
December 2024
School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.
Immunofluorescence is highly dependent on antibody-antigen interactions for accurate visualization of proteins and other biomolecules within cells. However, obtaining antibodies with high specificity and affinity for their target proteins can be challenging, especially for targets that are complex or naturally present at low levels. Therefore, we developed AptaFluorescence, a protocol that utilizes fluorescently labeled aptamers for in vitro biomolecule visualization.
View Article and Find Full Text PDFThe combined use of scRNA-seq and network propagation highlights key features of pan-cancer Tumor-Infiltrating T cells.
PLoS One
December 2024
Servier, Research & Development, Gif-sur-Yvette, France.
Improving the selectivity and effectiveness of drugs represents a crucial issue for future therapeutic developments in immuno-oncology. Traditional bulk transcriptomics faces limitations in this context for the early phase of target discovery as resulting gene expression levels represent the average measure from multiple cell populations. Alternatively, single cell RNA sequencing can dive into unique cell populations transcriptome, facilitating the identification of specific targets.
View Article and Find Full Text PDFPotential role of circulating tumor cells and cell-free DNA as biomarkers in oral squamous cell carcinoma: A prospective single-center study.
PLoS One
December 2024
Department of Oral Oncology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
Metastasis in patients with oral squamous cell carcinoma has been associated with a poor prognosis. However, sensitive and reliable tests for monitoring their occurrence are unavailable, with the exception of PET-CT. Circulating tumor cells and cell-free DNA have emerged as promising biomarkers for determining treatment efficacy and as prognostic predictors in solid tumors such as breast cancer and colorectal cancer.
View Article and Find Full Text PDFThe role of KRT18 in lung adenocarcinoma development: integrative bioinformatics and experimental validation.
Discov Oncol
December 2024
School of Pharmacy, Shaoyang University, Shaoyang, 422000, Hunan, China.
Lung adenocarcinoma (LUAD) represents one of the most common subtypes of lung cancer with high rates of incidence and mortality, which contributes to substantial health and economic demand across the globe. Treatment today mainly consists of surgery, radiotherapy, and chemotherapy, but their efficacy in advanced stages is often suboptimal and emphasizes the clear need for new biomarkers and therapeutic targets. Using comprehensive bioinformatics analyses consisting of the Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), Human Protein Atlas (HPA) and Clinical Proteomic Tumor Analysis Consortium (CPTAC), immune infiltration analysis and functional enrichment analysis, and single-cell analysis, we examined the potential of keratin 18 (KRT18) as a candidate biomarker in advanced LUAD.
View Article and Find Full Text PDFEfficient radiolabeling of mesoporous silica nanoparticles for single-cell PET imaging.
Eur J Nucl Med Mol Imaging
December 2024
Department of Radiation Oncology, Stanford University, Stanford, CA, USA.
Purpose: Nanoparticles are highly efficient vectors for ferrying contrast agents across cell membranes, enabling ultra-sensitive in vivo tracking of single cells with positron emission tomography (PET). However, this approach must be fully characterized and understood before it can be reliably implemented for routine applications.
Methods: We developed a Langmuir adsorption model that accurately describes the process of labeling mesoporous silica nanoparticles (MSNP) with Ga.
Want AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!