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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

Bacterial RNA Polymerase

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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

Free-Radical Chain Reaction and Polymerization of Alkenes

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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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Hot Start Polymerase Chain Reaction (PCR).

Michael R Green, Joseph Sambrook

    Cold Spring Harbor Protocols
    |May 3, 2018
    PubMed
    Summary

    Hot start polymerase chain reaction (PCR) optimizes amplified DNA yield by preventing premature enzyme or primer activity. This method suppresses unwanted DNA products and primer dimers for more accurate results.

    Area of Science:

    • Molecular Biology
    • Biochemistry

    Background:

    • Polymerase chain reaction (PCR) is a fundamental technique for DNA amplification.
    • Standard PCR protocols can suffer from low specificity and yield due to premature component interactions.

    Purpose of the Study:

    • To explain the principle and benefits of hot start PCR.
    • To detail how hot start PCR enhances amplification specificity and yield.

    Main Methods:

    • Hot start PCR involves delaying the activation of a key reaction component, such as DNA polymerase or primers.
    • Activation is triggered by heating the reaction mixture to a specific high temperature.
    • This prevents primer binding to non-target sequences or primer dimer formation at lower temperatures.

    Main Results:

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    • Hot start PCR significantly optimizes the yield of the desired amplified DNA product.
    • Nonspecific amplification and the formation of primer dimers are effectively suppressed.
    • Improved specificity leads to cleaner and more accurate PCR results.

    Conclusions:

    • Hot start PCR is a crucial modification for improving the efficiency and specificity of DNA amplification.
    • By controlling component activation, this technique ensures reliable PCR outcomes for various applications.