23 public questions tagged with this topic.
DNA polymerase is the enzyme responsible for synthesizing new DNA strands during replication. This follows from NCERT principle where the relation explains the outcome clearly for students in simple steps.
Ref: NCERT Biology Textbook for Class XI and XII (Zoology section), Chapter: Principles of Inheritance, Molecular Basis of Inheritance and Biotechnology, Topic: Genetics, DNA techniques and applications.
Predominant advantage of EdU assay over BrdU is preservation of cellular architecture because it avoids harsh DNA denaturation. BrdU detection requires unwinding double helix using strong acid, heat, or enzymatic digestion to expose concealed bromo-deoxyuridine epitope for anti-BrdU antibody of large size 150 kDa that cannot access double-stranded DNA. These treatments hydrolyze fluorescent proteins such as GFP, destroy conformational epitopes of surface antigens CD markers, fragment histology, alter light scatter properties in flow cytometry, and distort cell cycle distributions. EdU click detection employs small organic azide dye around 1 kDa that diffuses freely into intact duplex forming covalent triazole without strand separation. Consequently morphology remains intact under phase-contrast, immunophenotype retains antigenicity enabling co-detection of stem markers, and compatibility with DNA content dyes propidium iodide or DAPI remains high without acid-induced artifacts. Sensitivity improves due to quantitative triazole formation, protocol duration reduces significantly, and multiplex capability expands making EdU preferred for tissue sections, whole mount embryos, and high-content imaging.
Ref: Buck et al Biotechniques 2008 EdU advantage no denaturation preserves antigens; Thermo Fisher Click-iT EdU vs BrdU no DNA denaturation needed.
EdU, 5-ethynyl-2'-deoxyuridine, retains Watson-Crick pairing but bears terminal alkyne at 5 position instead of bromine. Detection utilizes bioorthogonal click chemistry specifically copper(I)-catalyzed azide-alkyne cycloaddition first described by Sharpless. In this reaction, alkyne on EdU incorporated into DNA reacts with fluorescent azide forming stable 1,2,3-triazole covalent linkage, catalyzed by CuSO4 reduced to Cu(I) by sodium ascorbate, proceeding rapidly in aqueous buffer at room temperature with exquisite specificity. Because fluorescent azide small molecule penetrates duplex DNA, harsh denaturation required for BrdU antibody access unnecessary. Standard BrdU assays need 2M hydrochloric acid, 95°C heat, or DNase digestion to unwind helix exposing brominated base for antibody binding, steps that destroy protein epitopes and fluorescent proteins. Click reaction eliminates antibody step, shortens protocol from overnight incubation to less than one hour, preserves morphology, and allows multiplexing with surface markers. Unlike radioactive thymidine labeling, EdU non-radioactive and bright. This advance revolutionized proliferation assessment. Nobel Prize in Chemistry 2022 recognized click chemistry applications transforming biological labeling and drug discovery. This knowledge strengthens laboratory safety, protocol reproducibility, and regulatory compliance critical for translational research and clinical applications, ensuring reliable data and workforce protection.
Ref: Salic A & Mitchison TJ PNAS 2008 EdU click chemistry; Kolb et al Click chemistry concept Nobel Prize 2022 CuAAC azide-alkyne.
BrdU, 5-bromo-2'-deoxyuridine, is synthetic halogenated analog of thymidine where methyl group at 5 position replaced by bromine atom similar in size allowing Watson-Crick pairing with adenine during DNA synthesis. During S phase, DNA polymerases incorporate BrdU triphosphate from nucleotide pool into nascent strand in place of thymidine without halting polymerase at low micromolar concentrations. Incorporated BrdU detectable by specific anti-BrdU monoclonal antibodies after DNA denaturation exposes epitope, or by mass spectrometry. Assay quantifies fraction of cells actively replicating genome termed labeling index and S phase duration, monitors cell cycle kinetics, measures proliferation inhibition by cytotoxic agents via decreased incorporation, and tracks lineage in developmental biology through pulse-chase retaining label in slow-cycling stem cells while rapidly dividing progenitors dilute signal over successive divisions. Unlike puromycin incorporation marking protein synthesis or BrUTP marking nascent RNA transcripts, BrdU specifically reports DNA replication providing S phase specific information central to cancer and stem cell research.
Ref: Gratzner HG Science 1982 BrdU thymidine analog DNA replication detection; Alberts MBoC Ch.17 DNA synthesis S phase BrdU incorporation.
Aminopterin classifies as antifolate antimetabolite structurally analogous to folic acid with pteridine ring substitution preventing enzymatic reduction. Dihydrofolate reductase normally catalyzes NADPH dependent reduction of dihydrofolate to tetrahydrofolate, central one-carbon carrier crucial for biosynthesis. Tetrahydrofolate derivatives required at two distinct steps: 10-formyl tetrahydrofolate donates formyl groups to glycinamide ribonucleotide transformylase and aminoimidazole carboxamide ribonucleotide transformylase in de novo purine pathway synthesizing inosine monophosphate precursor to adenine and guanine nucleotides, and 5,10-methylene tetrahydrofolate provides methyl group to thymidylate synthase converting deoxyuridine monophosphate to thymidine monophosphate essential for DNA replication. Aminopterin competitive inhibition depletes all tetrahydrofolate pools, halts purine and thymidine triphosphate production, cellular ATP drops, AMPK activated, p53 stabilization triggers apoptosis within hours. Rapidly proliferating lymphocytes depend heavily on de novo route because salvage alone insufficient during S phase. Provision of hypoxanthine and thymidine allows salvage-competent cells to circumvent block via HGPRT and TK, explaining selective toxicity exploited in hybridoma selection and historically in cancer chemotherapy similar to methotrexate.
Ref: Goodman & Gilman Pharmacol 13th ed DHFR antifolate; Lodish MBoC 9th ed Fig 7-32 aminopterin blocks de novo purine thymidylate.
Primers bind to specific sequences and initiate DNA synthesis in the PCR process.
Ref: NCERT Class 12 Biology Chapter 10: Biotechnology and its Applications Biosafety and Overview of Applications
Reverse transcription is the process of making complementary DNA (cDNA) from an RNA template.
Ref: NCERT Class 12 Biology Chapter 10: Biotechnology and its Applications Ethical Issues - GEAC and Animal Ethics
Reverse transcriptase synthesizes complementary DNA (cDNA) from an RNA template.
Ref: NCERT Class 12 Biology Chapter 10: Biotechnology and its Applications Transgenic Animals - Models and Biological Products
Reverse transcriptase synthesizes complementary DNA (cDNA) from an RNA template.
Ref: NCERT Class 12 Biology Chapter 10: Biotechnology and its Applications Transgenic Animals - Models and Biological Products
DNA polymerase is used to synthesize new DNA strands by adding nucleotides to a growing DNA chain, making it essential for DNA replication and PCR.
Ref: NCERT Class 12 Biology Chapter 9: Biotechnology: Principles and Processes Polymerase Chain Reaction - PCR and Applications
DNA replication requires an RNA primer synthesized by primase to initiate the process.
Ref: NCERT Class 12 Biology Chapter 5: Molecular Basis of Inheritance DNA Replication - Semiconservative and Machinery
DNA polymerase is the enzyme responsible for synthesizing new DNA strands during replication.
Ref: NCERT Class 11 Biology Chapter 9: Biomolecules Enzymes - Properties Classification and Cofactors
