Ligase chain reaction

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The ligase chain reaction (LCR) is a method of DNA amplification. The ligase chain reaction (LCR) is an amplification process that differs from polymerase chain reaction (PCR) in that it involves a thermostable ligase to join two probes or other molecules together which can then be amplified by standard PCR cycling. [1] Each cycle results in a doubling of the target nucleic acid molecule. A key advantage of LCR is greater specificity as compared to PCR. [2] Thus, LCR requires two completely different enzymes to operate properly: ligase, to join probe molecules together, and a thermostable polymerase (e.g., Taq polymerase) to amplify those molecules involved in successful ligation. The probes involved in the ligation are designed such that the 5′ end of one probe is directly adjacent to the 3′ end of the other probe, thereby providing the requisite 3′-OH and 5′-PO4 group substrates for the ligase.

Contents

LCR was originally developed to detect point mutations; a single base mismatch at the junction of the two probe molecules is all that is needed to prevent ligation. By performing the ligation right at the Tm of the oligonucleotide probe, only perfectly matched primer:template duplexes will be tolerated. LCR can also be used to amplify template molecules that have been successfully ligated for the purpose of assessing ligation efficiency and producing a large amount of product with even greater specificity than PCR. Thus, LCR is not necessarily an alternative, but rather a complement, to PCR.

Target conditions

It has been widely used for the detection of single base mutations, as in certain genetic diseases. [1]

LCR and PCR may be used to detect gonorrhea and chlamydia, and may be performed on first-catch urine samples, providing easy collection and a large yield of organisms. Endogenous inhibitors limit the sensitivity, but if this effect could be eliminated, LCR and PCR would have clinical advantages over any other methods of diagnosing gonorrhea and chlamydia. [3] Among these methods, LCR is emerging as the most sensitive method with high specificity for known single-nucleotide polymorphism (SNP) detection (20). LCR was first developed in 1989 by multiple groups, [4] who used thermostable DNA ligase to discriminate between normal and mutant DNA and to amplify the allele-specific product. A mismatch at the 3′ end of the discriminating primer prevents the DNA ligase from joining the two fragments together. By using both strands of genomic DNA as targets for oligonucleotide hybridization, the products generated from two sets of adjacent oligonucleotide primers, complementary to each target strand in one round of ligation, can become the targets for the next round. The amount of the products can thus be increased exponentially by repeated thermal cycling. [1]

See also

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<span class="mw-page-title-main">Primer (molecular biology)</span> Short strand of RNA or DNA that serves as a starting point for DNA synthesis

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<span class="mw-page-title-main">Reverse transcription polymerase chain reaction</span> Laboratory technique to multiply an RNA sample for study

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<i>Taq</i> polymerase Thermostable form of DNA polymerase I used in polymerase chain reaction

Taq polymerase is a thermostable DNA polymerase I named after the thermophilic eubacterial microorganism Thermus aquaticus, from which it was originally isolated by Chien et al. in 1976. Its name is often abbreviated to Taq or Taq pol. It is frequently used in the polymerase chain reaction (PCR), a method for greatly amplifying the quantity of short segments of DNA.

<span class="mw-page-title-main">Real-time polymerase chain reaction</span> Laboratory technique of molecular biology

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<span class="mw-page-title-main">Inverse polymerase chain reaction</span>

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The selector technique is a method to amplify and multiplex genomic DNA.

TaqMan probes are hydrolysis probes that are designed to increase the specificity of quantitative PCR. The method was first reported in 1991 by researcher Kary Mullis at Cetus Corporation, and the technology was subsequently developed by Hoffmann-La Roche for diagnostic assays and by Applied Biosystems for research applications.

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<span class="mw-page-title-main">Oligomer restriction</span>

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<span class="mw-page-title-main">History of polymerase chain reaction</span>

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The versatility of polymerase chain reaction (PCR) has led to modifications of the basic protocol being used in a large number of variant techniques designed for various purposes. This article summarizes many of the most common variations currently or formerly used in molecular biology laboratories; familiarity with the fundamental premise by which PCR works and corresponding terms and concepts is necessary for understanding these variant techniques.

A primer dimer (PD) is a potential by-product in the polymerase chain reaction (PCR), a common biotechnological method. As its name implies, a PD consists of two primer molecules that have attached (hybridized) to each other because of strings of complementary bases in the primers. As a result, the DNA polymerase amplifies the PD, leading to competition for PCR reagents, thus potentially inhibiting amplification of the DNA sequence targeted for PCR amplification. In quantitative PCR, PDs may interfere with accurate quantification.

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References

  1. 1 2 3 Barany, F (Jan 1991). "Genetic disease detection and DNA amplification using cloned thermostable ligase". Proc Natl Acad Sci U S A. 88 (1): 189–93. Bibcode:1991PNAS...88..189B. doi: 10.1073/pnas.88.1.189 . PMC   50775 . PMID   1986365.
  2. ↑ Wiedmann, M; Wilson, WJ; Czajka, J; Luo, J; Barany, F; Batt, CA (Feb 1994). "Ligase chain reaction (LCR)--overview and applications". PCR Methods and Applications. 3 (4): S51–64. doi: 10.1101/gr.3.4.s51 . PMID   8173509.
  3. ↑ Gregory, J. Locksmith MD. (1997). "New diagnostic tests for gonorrhea and chlamydia". Primary Care Update for OB/GYNS. 4 (5): 161–167. doi:10.1016/S1068-607X(97)00044-9.
  4. ↑ Shimer Jr, George H.; Backman, Keith C. (2000). "Ligase Chain Reaction". In Kessler, Christoph (ed.). Nonradioactive Analysis of Biomolecules. Springer Lab Manuals (Second ed.). Springer. doi:10.1007/978-3-642-57206-7_32. ISBN   978-3-642-57206-7.

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