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#DNA synthesis

23 public questions tagged with this topic.

ZOOLOGY

Which enzyme is responsible for synthesizing DNA from a template strand?

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.

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Animal Tissue Culture 3

An advantage of EdU assay is:

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 de

Ref: Buck et al Biotechniques 2008 EdU advantage no denaturation preserves antigens; Thermo Fisher Click-iT EdU vs BrdU no DNA denaturation needed.

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Animal Tissue Culture 3

EdU assay differs from BrdU assay because it:

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, ha

Ref: Salic A & Mitchison TJ PNAS 2008 EdU click chemistry; Kolb et al Click chemistry concept Nobel Prize 2022 CuAAC azide-alkyne.

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Animal Tissue Culture 3

BrdU is a thymidine analog used to detect:

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 genom

Ref: Gratzner HG Science 1982 BrdU thymidine analog DNA replication detection; Alberts MBoC Ch.17 DNA synthesis S phase BrdU incorporation.

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Immunological Applications

Aminopterin in HAT medium functions by:

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

Ref: Goodman & Gilman Pharmacol 13th ed DHFR antifolate; Lodish MBoC 9th ed Fig 7-32 aminopterin blocks de novo purine thymidylate.

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