NGS Oligos

Tsingke ensures sample uniformity by isolating monoclonal clones. Each clone is validated by Sanger sequencing, covering the entire gene and at least 50 bp of the vector backbone upstream and downstream, which ensures the gene is correct and properly placed in the vector. The plasmid then undergoes NGS to verify the accuracy of the entire plasmid and detect any contamination. Before shipment, a strict QC review is conducted.

Capture probes are commonly classified based on their application areas, including:

* Cancer research probes

* Basic research probes

* Hematology-related probes

* Pathogen detection probes

* Genetic disease probes

* Methylation analysis probes

Successful multiplex PCR primer design requires careful consideration of the following factors:

(1) Primer length: Primers are typically designed to be 18-24 nucleotides long to ensure specificity while minimizing the risk of primer-dimer formation.

(2) Melting temperature (Tm): All primers included in a multiplex reaction should have similar Tm values to enable uniform annealing and efficient amplification under a single set of cycling conditions.

(3) 3'-End complementarity: Complementarity between primers, especially at the 3' ends, should be avoided to reduce the likelihood of primer-dimer formation and non-specific amplification.

(4) Stepwise optimization: Each primer pair should be optimized individually before being combined. Once pooled, primers should be mixed and further optimized incrementally to achieve balanced amplification.

Adhering to these principles helps improve specificity, efficiency, and reproducibility in multiplex PCR assays.

In multiplex PCR, multiple primer pairs are used simultaneously, making optimization of reaction conditions critical for balanced and specific amplification.

(1) Annealing temperature: Each primer pair has its own optimal annealing temperature. For multiplex reactions, the annealing temperature is typically set based on the lowest melting temperature (Tm) among all primers to ensure efficient binding and amplification across targets.

(2) Cycle number: Using the minimum number of PCR cycles required to obtain sufficient product helps maintain amplification efficiency and specificity, while reducing nonspecific products and amplification bias between targets.

Careful optimization of both annealing temperature and cycle number is essential for reliable and reproducible multiplex PCR performance.

In multiplex PCR, multiple targets are amplified simultaneously, increasing competition for key reagents such as DNA polymerase, dNTPs, and primers. If reagent concentrations are not properly optimized, amplification efficiency and balance between targets may be compromised.

Additionally, the simultaneous synthesis of multiple amplicons increases the overall demand on the polymerase, often requiring longer extension times than single-target PCR to ensure complete and efficient synthesis of all products.

Optimizing reagent concentrations and extension time is therefore essential to achieve uniform amplification, high specificity, and reliable results in multiplex PCR assays.

Multiplex PCR primers should be 18-24 bases long. Longer primers are more likely to form primer dimers.

This issue is most commonly associated with the inherent limitations of chemical oligonucleotide synthesis. Synthetic primers are not 100% pure, and trace amounts of truncated or failure sequences may be present. These minor impurities can occasionally participate in PCR amplification and be carried through cloning and sequencing.

We recommend the following actions:

(1) Sequence additional clones: Re-pick and sequence one or more additional colonies. In most cases, the initially sequenced clone originates from a PCR product amplified by a minor impurity, while other clones contain the correct sequence.

(2) Contact technical support if the issue persists: If the same primer-region error is observed consistently in 2-3 independent clones, the primer batch itself may be the source. Please contact our technical support team for further assistance. We will arrange prompt resynthesis or replacement of the primer at no additional cost.