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Related Concept Videos

PCR01:32

PCR

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Overview
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Translesion DNA Polymerases02:10

Translesion DNA Polymerases

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Translesion (TLS) polymerases rescue stalled DNA polymerases at sites of damaged bases by replacing the replicative polymerase and installing a nucleotide across the damaged site. Doing so, TLS allows additional time for the cell to repair the damage before resuming regular DNA replication.
TLS polymerases are found in all three domains of life - archaea, bacteria, and eukaryotes. Of the different classes of TLS polymerases, members of the Y family are fitted with specialized structures that...
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Bacterial RNA Polymerase00:43

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Unlike eukaryotes, bacteria use a single RNA Polymerase (RNAP) to transcribe all genes. The different subunits of bacterial RNAPhave distinct functions. The multisubunit structure of the bacterial RNAP helps the enzyme to maintain catalytic function, facilitate assembly, interact with DNA and RNA, and self-regulate its activity.
In most genes, the transcription site is a single base present upstream of the coding sequence. Though RNAP is a catalytically efficient enzyme, it does not recognize...
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Free-Radical Chain Reaction and Polymerization of Alkenes02:35

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The conversion of alkenes to macromolecules called polymers is a reaction of high commercial importance. The structure of the polymer is defined by a repeating unit, while the terminal groups are considered insignificant. The average degree of polymerization represents the number of repeating units in the polymer molecule and is denoted by the subscript n.
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Eukaryotic RNA Polymerases00:58

Eukaryotic RNA Polymerases

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RNA Polymerase (RNAP) is conserved in all animals, with bacterial, archaeal, and eukaryotic RNAPs sharing significant sequence, structural, and functional similarities. Among the three eukaryotic RNAPs, RNA Polymerase II is most similar to bacterial RNAP in terms of both structural organization and folding topologies of the enzyme subunits. However, these similarities are not reflected in their mechanism of action.
All three eukaryotic RNAPs require specific transcription factors, of which the...
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RNA Polymerase II Accessory Proteins02:36

RNA Polymerase II Accessory Proteins

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Proteins that regulate transcription can do so either via direct contact with RNA Polymerase or through indirect interactions facilitated by adaptors, mediators, histone-modifying proteins, and nucleosome remodelers. Direct interactions to activate transcription is seen in bacteria as well as in some eukaryotic genes. In these cases, upstream activation sequences are adjacent to the promoters, and the activator proteins interact directly with the transcriptional machinery. For example, in...
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Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies
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Inverse Polymerase Chain Reaction (PCR).

Michael R Green, Joseph Sambrook

    Cold Spring Harbor Protocols
    |February 3, 2019
    PubMed
    Summary

    Inverse PCR amplifies unknown DNA flanking a known sequence by circularizing digested DNA fragments. This method enables amplification when primers for the target region are unavailable, using primers specific to the known sequence.

    Area of Science:

    • Molecular Biology
    • Genetics

    Background:

    • Standard Polymerase Chain Reaction (PCR) amplifies DNA between known primer sites.
    • Inverse PCR addresses limitations where flanking DNA sequences lack available primers.

    Purpose of the Study:

    • To describe the methodology of Inverse PCR for amplifying unknown flanking DNA sequences.
    • To detail a technique for generating DNA amplicons when primers are not readily available.

    Main Methods:

    • DNA digestion using restriction enzymes to create fragments.
    • Intramolecular ligation to form circularized DNA templates.
    • Utilizing specific primers binding to the known sequence to amplify flanking regions via PCR.

    Main Results:

    • Successful amplification of DNA sequences flanking a known region.

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  • Generation of linear DNA fragments containing a single restriction enzyme site at the junction of known and unknown sequences.
  • Amplicon size is determined by restriction site distribution.

    • Conclusions:

    • Inverse PCR is a valuable technique for sequencing DNA flanking known regions.
    • This method overcomes primer limitations in standard PCR applications.
    • It facilitates the characterization of unknown genomic regions adjacent to known sequences.