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TriLink's CleanAmp™ Primers have allowed us to develop a triplex one-step reverse transcription-PCR (RT-PCR) assay for detection of HIV-1, HSV, and HCV. This approach is unique from other Hot Start approaches, since it allows for control of which primers are extended during the RT step. The ability to control the RT step provided a high level of sensitivity and specificity for our in-house nucleic acid test.”

Xinglong Xiao, Ph.D.
South China University; Taitaigen, Inc.

 

Our work, in part, involves development of high sensitivity tests for mutations present in only a small fraction of cells under test. This requires high specificity also and it has proven to be the case that the TriLink CleanAmp™ Primers provided the specificity needed to capitalize on qPCR’s inherent high sensitivity. Using your primers we can routinely identify the presence of mutation fractions as low as 0.001%.”

John Owen M.D.
Wake Forest University

 

We designed a quadruplex PCR assay to simultaneously amplify four herpesviruses, including herpes simplex virus (HSV), cytomegalovirus (CMV), Epstein-Barr virus (EBV) and varicella zoster virus (VZV). We found that the use of CleanAmp™ Turbo Primers enhanced amplification efficacies in the quadruplex herpesvirus PCR assay where normal multiplex techniques previously failed.

Yi-Wei Tang
University of Vanderbilt

 

A Hot Start Assay for Less

1CleanAmp™ Primers are similar to CleanAmp™ dNTPs in that they contain a thermolabile chemical modification that allows Hot Start activation in PCR. This novel modification also serves to prevent primer extension at the lower temperatures of PCR setup and manipulation. A Hot Start thermal activation step removes the modification generating the corresponding unmodified primer, which supports amplification of the desired target. CleanAmp™ Primers specifically amplify the target by eliminating formation of primer dimer (Figure 1A) and mis-priming (Figure 1B) events. Furthermore, CleanAmp™ Primers eliminate the need for Hot Start DNA polymerases because they are compatible with a number of standard DNA polymerases, such as Taq.

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CleanAmp™ Primers are a key choice for diagnostic kits and other established assays. In these assays CleanAmp™ Primers can be as little as $0.03 per reaction at our standard starting scale. TriLink guarantees at least 10 ODs per primer synthesis, which is enough material for approximately 6,250 reactions. TriLink offers two types of CleanAmp™ Primers that vary in the rate of thermal activation: Turbo and Precision. The different rates of release of CleanAmp™ Turbo and Precision Primer modifications can be exploited for improved results in advanced applications, such as multiplex PCR (Figure 2), reverse-transcription PCR, low copy number detection (Figure 4) and fast PCR. The performance of CleanAmp™ Primers can be applied to critical applications such as molecular diagnostics, forensics, detection of infectious agents and gene expression validation.

Maximize Your Multiplex

If you are seeking a Hot Start solution for a problematic multiplex system, CleanAmp™ Primers may be your answer. Like CleanAmp™ dNTPs, CleanAmp™ Primers will improve yield and specificity in multiplex systems. However, CleanAmp™ Primers offer higher specificity with a greater number of targets. In the systems shown in Figure 2, CleanAmp™ Turbo Primers greatly improve formation of the desired products in multiplex PCR for seven and nine targets from genomic DNA sources. The development of a robust multiplex PCR assay typically requires an iterative design process to discover primer pairs that are both specific for the targets of interest and exhibit a low level of off-target amplicon formation. With CleanAmp™ Primers minimal design optimization is required.

The reduction or elimination of nonspecific amplifications achieved with the use of CleanAmp™ Primers makes them a powerful solution for improved one-step reverse-transcription (RT) PCR performance (Figure 3). By introducing CleanAmp™ Primers only the RT Primer can elongate during the reverse transcription step of the protocol, resulting in specific amplification in a one tube, single step protocol.

Figures 2 & 3

Reaching New Limits of Detection

CleanAmp™ Primers also offer an advantage over CleanAmp™ dNTPs in systems hindered by low template concentrations. A 10-100 fold increase in correct amplicon formation is seen when standard primers are replaced with CleanAmp™ Precision Primers (Figure 4). CleanAmp™ Primers greatly improve these systems by reducing or eliminating the off-target amplifications that compete with the desired amplicon.

CleanAmp™ Primers have utility in many high-sensitivity downstream applications, such as single molecule detection.

Figure 4

Product Details

CleanAmp™ Primers are available in two forms that differ in the rate of thermal activation. See table below for application based benefits. CleanAmp™ Primers are supplied cartridge purified. However, for those applications where PCR performance is of the utmost importance, we can RP-HPLC purify your CleanAmp™ Primers on a custom quote basis. Each primer undergoes quality control analysis by PAGE, MS and RP-HPLC to ensure high quality. CleanAmp™ Primers 15-40 bases in length are $250/pair. We guarantee at least a 10 OD final yield from every synthesis.

CleanAmp™ Turbo Primers CleanAmp™ Precision Primers
Fast cycling Standard cycling
Multiplex PCR One-step reverse-transcription PCR
Improves amplicon yield Improves specificity and limit of detection
Reduces mis-priming/primer dimer formation Greatest reduction in mis-priming/primer dimer formation

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CleanAmp™ Amidites

CleanAmp™ modified phosphoramidites are available for in-house synthesis of CleanAmp™ Primers.

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