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Related Experiment Videos

Linked linear amplification: a new method for the amplification of DNA.

A A Reyes1, L A Ugozzoli, J D Lowery

  • 1Molecular Systems Division, Bio-Rad Laboratories, 5500 East Second St., Benicia, CA 94510, USA.

Clinical Chemistry
|January 10, 2001
PubMed
Summary

Linked Linear Amplification (LLA) offers comparable yields to PCR for nucleic acid amplification. This novel method significantly reduces carryover contamination, enhancing diagnostic accuracy.

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Genetics

Background:

  • Linked Linear Amplification (LLA) is an innovative nucleic acid amplification technique.
  • It utilizes multiple primer extension cycles with nonreplicable elements to achieve linear product accumulation.
  • Nested primers enable "linked" linear reactions, yielding results comparable to Polymerase Chain Reaction (PCR).

Purpose of the Study:

  • To evaluate the amplification yield of LLA with varying primer numbers.
  • To assess the carryover amplification efficiency of LLA compared to PCR.
  • To demonstrate the clinical applicability of LLA for mutation detection.

Main Methods:

  • The study employed 10-, 14-, and 18-primer LLA to amplify the human ss-globin gene, comparing yields with PCR.

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  • Carryover contamination was assessed by reamplifying serial dilutions of LLA and PCR products.
  • LLA combined with allele-specific oligonucleotide (ASO) capture was used to detect the factor V Leiden mutation in 111 DNA samples.

    • Main Results:

    • Amplification yields from 14- and 18-primer LLA were comparable to PCR.
    • LLA exhibited a four-orders-of-magnitude lower carryover amplification efficiency than PCR.
    • The LLA-ASO assay accurately identified the factor V Leiden genotype in all tested samples.

    Conclusions:

    • LLA is a robust nucleic acid amplification method with yields equivalent to PCR.
    • LLA demonstrates superior resistance to false positives from carryover amplicon contamination.
    • LLA offers a reliable alternative to PCR, particularly in settings sensitive to contamination.