Introduction
Ligase chain reaction (LCR) is a thermostable DNA ligase-dependent DNA amplification which can be initiated by repetitive cycles of the ligation of adjacent hybridized DNA probes for the achievement of exponential amplification of target DNA (Cao, 2004; Barany, 1991). LCR is a technique for detecting specific nucleic acid sequences through amplification. It has significant applications in both genotyping (e.g., for identifying genetic disorders like sickle cell disease) and infectious disease detection, especially for pathogens difficult to detect with traditional methods. Notably, LCR is highly sensitive for diagnosing infections such as Chlamydia trachomatis, outperforming other methods like cell culture or antigen-detection assays.
Mechanism of Ligase chain reaction (LCR)
LCR involves using two pairs of complementary oligonucleotides that are complementary to adjacent sequences on the target DNA. Unlike PCR, which uses only two primers, LCR requires four oligonucleotides each 20–35 nucleotides long. The adjacent oligonucleotide pairs are ligated together after hybridizing to their complementary sequences on the DNA strand. DNA ligase plays a central role by joining two adjacent oligonucleotide probes that have hybridized to a target DNA sequence. Key points of the LCR mechanism:
- Ligation occurs in the 3′ to 5′ orientation on the same DNA strand. The 5′ ends of the primers must be phosphorylated to enable ligation.
- Exponential Amplification: After ligation, these joined probes become templates in subsequent cycles of the reaction. The thermocycling process—repeated cycles of denaturation, hybridization, and ligation—facilitates the exponential accumulation of the ligated products.
- The use of thermostable ligases and high-fidelity, such as Thermus thermophilus ligase, allows LCR to operate at high temperatures. This enhances the specificity by using high-melting-temperature probes and stringent hybridization conditions. This minimizes the chances of non-specific binding, making LCR highly effective in differentiating single-base mismatches.
Additionally, although the two oligonucleotide pairs can occasionally ligate bluntly at the ends, allowing for amplification even without a target sequence, the ligation is significantly more efficient when the sequence at the ligation junction is perfectly matched.
LCR provides information on two key aspects: (1) whether target DNA sequences are adjacent, confirming their presence, and (2) whether there is perfect sequence complementarity at the junction, ensuring high specificity.
Ligase Chain Reaction (LCR) principle
Ligase Specificity and fidelity
DNA ligases catalyze the formation of phosphodiester bonds between adjacent nucleotides, sealing nicks in the DNA backbone: 5´-phosphorylated DNA termini to 3´-OH DNA termini. Their specificity and fidelity are influenced by the nucleotide sequence at the ligation site, the presence of mismatches, and the structural properties of the ligase.
High-fidelity ligases, such as T4 DNA Ligase, are designed to preferentially ligate correctly paired nucleotides while discriminating against mismatches, thanks to the architecture of their active sites. Conversely, Taq DNA Ligase can tolerate certain mismatches, potentially reducing fidelity. Reaction conditions, including temperature and buffer composition, also impact ligase performance.
Selecting the appropriate ligase based on its specificity, fidelity, and optimal conditions is essential for maintaining the integrity of amplified products and ensuring reliable results in downstream applications. [5]
Comparison with PCR
The Ligase Chain Reaction (LCR) is a nucleic acid amplification method that differs from the Polymerase Chain Reaction (PCR) in its mechanism. While PCR uses thermal cycling and DNA polymerase for amplification, LCR employs thermostable DNA ligase to covalently join adjacent oligonucleotide probes that hybridize to their complementary sequences. This mechanism enhances specificity, as successful ligation requires perfect base-pairing at the junction, reducing the risk of primer-dimer artifacts and non-specific products common in PCR. LCR is particularly valuable for applications needing high discrimination, such as detecting point mutations, single nucleotide polymorphisms (SNPs), and rare allelic variants, making it ideal for diagnostics requiring precise accuracy.
Difference between LCR and PCR
| Feature | Ligase Chain Reaction (LCR) | Polymerase Chain Reaction (PCR) |
|---|---|---|
| Enzyme Required | DNA ligase | DNA polymerase (e.g., Taq polymerase) |
| Mechanism | Probe hybridization and ligation of adjacent probes | Primer annealing, extension, and DNA synthesis |
| Amplification Type | Exponential accumulation through repeated ligation cycles | Exponential DNA synthesis by polymerase extension |
| Sensitivity to Base Mismatches | High; ligase requires perfect complementarity for ligation | Lower; polymerase can tolerate some mismatches during extension |
| Primer/Probe Requirement | Four probes targeting adjacent sequences | Two primers targeting flanking regions of the target |
| Temperature Cycles | Denaturation, hybridization, and ligation cycles (high stringency) | Denaturation, annealing, and extension cycles |
| Primary Applications | Mutation detection, SNP analysis, pathogen identification | General DNA amplification, cloning, diagnostics |
| Specificity | Very high, due to ligation requirement for perfect probe alignment | Moderate to high, depending on primer design |
| Complexity | Higher; requires precise probe design and stringent conditions | Moderate; less stringent probe design required |
| Limitations | Complex setup, sensitive to probe mismatches, cost of ligase enzyme | Potential for non-specific amplification, primer-dimer formation |
Modifications of LCR
Enhanced LCR techniques have optimized ligation efficiency through modified ligase enzymes with higher fidelity and optimized buffer conditions, resulting in more stable and accurate target amplification. Probe design improvements, such as the incorporation of locked nucleic acids (LNAs) and peptide nucleic acids (PNAs), increase binding affinity and specificity, while dual-probe systems further reduce non-specific ligation. Additionally, multiplexing capabilities allow simultaneous detection of multiple targets using unique probe sets and fluorescent tagging, enabling complex assays in a single reaction and broadening the utility of LCR in genotyping and diagnostics.
Ligase Detection Reaction (LDR)
LDR is a modified ligase chain reaction (LCR) technique that utilizes a pair of probes complementary to one strand of the target DNA, along with a high-fidelity thermostable ligase, such as Taq DNA Ligase. Unlike traditional LCR, which achieves exponential amplification, LDR facilitates linear amplification of the ligation product through cycling. This method is particularly useful for confirming the presence of specific single nucleotide polymorphisms (SNPs) in a target sequence previously amplified by techniques such as PCR [4].
Nested LCR
Nested LCR is a modified dual LCR technique that uses two separate tubes for primary amplification. Each tube contains four primers flanking the allele-specific base pair. Two primers bind on the sense strand, while the other two anneal to the antisense strand. After multiple cycles of LCR, each adjacent primer pair ligates to form approximately 30-nucleotide products. The tubes are then combined with fresh ligase for a new round of amplification, resulting in full-length products only in the presence of the correct DNA target allele. Nested LCR demonstrates a superior signal-to-noise ratio and reduced non-specific binding compared to traditional LCR, as the primary amplification products serve as primers for the secondary amplification [4].
Advanced LCR Techniques
- Real-Time LCR (RT-LCR): Incorporates real-time monitoring with fluorescent indicators, enabling continuous, quantitative measurement of target DNA.
- Digital LCR (dLCR): Uses microfluidics to partition reactions into droplets, achieving high-precision quantification of low-abundance targets through droplet counting.
- CRISPR/Cas Integration: Utilizes Cas proteins to cut DNA at target sites, guided by custom gRNAs, increasing specificity and minimizing off-target binding.
Summary of Modifications
| Modification | Technique | Benefit |
|---|---|---|
| Enhanced Ligation | Improved ligase/buffer systems | Higher efficiency & specificity |
| Probe Design | LNAs, PNAs, dual-probe systems | Increased specificity & stability |
| Multiplexing | Multiplex probe sets & fluorescent tagging | Simultaneous detection of targets |
| Real-Time LCR | Real-time monitoring with fluorescence | Quantitative analysis |
| Digital LCR | Microfluidic droplet partitioning | Absolute quantification |
| CRISPR Integration | CRISPR/Cas pre-selection and target enhancement | Higher accuracy in complex samples |
Applications of LCR
LCR has diverse applications, including:
- Genetic diagnostics, where it can be used to detect specific mutations associated with diseases.
- Genotyping for SNP identification and analysis.
- Detection of pathogens in clinical samples, which can assist in the diagnosis of infectious diseases.
- Research applications for studying gene expression and molecular interactions.
References
- Benjamin, W. H., Smith, K. R., & Waites, K. B. (2003). Ligase chain reaction. Humana Press eBooks, 135–150. https://doi.org/10.1385/1-59259-384-4:135
- Wee, E. J. H., Rauf, S., Shiddiky, M. J. A., Dobrovic, A., & Trau, M. (2014). DNA Ligase-Based Strategy for Quantifying Heterogeneous DNA Methylation without Sequencing. Clinical Chemistry, 61(1), 163–171. https://doi.org/10.1373/clinchem.2014.227546
- Lapitan, L. D. S., Jr, Guo, Y., & Zhou, D. (2015). Nano-enabled bioanalytical approaches to ultrasensitive detection of low abundance single nucleotide polymorphisms. The Analyst, 140(12), 3872–3887. https://doi.org/10.1039/c4an02304h
- Gibriel, A. A., & Adel, O. (2017). Advances in ligase chain reaction and ligation-based amplifications for genotyping assays: Detection and applications. Mutation Research/Reviews in Mutation Research, 773, 66–90. https://doi.org/10.1016/j.mrrev.2017.05.001
- New England Biolabs. (n.d.). Substrate specificity and mismatch discrimination in DNA ligases. Retrieved from NEB.
