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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

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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Touchdown Polymerase Chain Reaction (PCR).

Michael R Green, Joseph Sambrook

    Cold Spring Harbor Protocols
    |May 3, 2018
    PubMed
    Summary

    Touchdown polymerase chain reaction (PCR) enhances specificity by starting annealing at high temperatures and gradually decreasing them. This method minimizes off-target priming, ensuring accurate amplification of target DNA sequences.

    Area of Science:

    • Molecular Biology
    • Genetics
    • Biotechnology

    Background:

    • Polymerase chain reaction (PCR) is a fundamental technique for DNA amplification.
    • Standard PCR can suffer from low specificity due to off-target primer binding.
    • Increasing PCR specificity is crucial for accurate genetic analysis.

    Purpose of the Study:

    • To introduce and explain the touchdown polymerase chain reaction (PCR) method.
    • To highlight the benefits of touchdown PCR in increasing amplification specificity.
    • To detail the conditions and applications where touchdown PCR is most effective.

    Main Methods:

    • Touchdown PCR involves an initial high annealing temperature, gradually decreasing over cycles.
    • High stringency in early cycles favors perfect primer-template hybridization.

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  • Subsequent cycles at lower temperatures amplify the dominant target sequence.
  • Often combined with hot start protocols to further minimize mispriming.

    • Main Results:

    • Touchdown PCR significantly reduces non-specific amplification and primer-dimer formation.
    • Achieves higher specificity compared to standard PCR protocols.
    • Enables successful amplification even with imperfect primer matches or complex templates.

    Conclusions:

    • Touchdown PCR is an effective strategy for enhancing PCR specificity and yield.
    • It is particularly valuable when primer design is challenging or template DNA is diverse.
    • This method improves the reliability of PCR-based applications in various research fields.