Many molecular diagnostics tests are based on the polymerase chain reaction (PCR) and use a thermostable DNA polymerase, commonly Taq DNA polymerase. Hot-start PCR is the preferred option due to its ability to reduce nonspecific amplification. Hot-start PCR is commonly performed using a hot-start DNA polymerase where the polymerase activity is temporarily blocked by an antibody or aptamer or by chemical modification; activity is restored by heating the polymerase at high temperatures
. In this article, we discuss a few considerations when choosing the right hot-start DNA polymerase for molecular diagnostic test development.
Promega offers GoTaq® MDx Hot Start Polymerase(a–c) (Cat.# D6001) as a general purpose reagent that can be used as a component in molecular diagnostic applications and laboratory-developed tests without paying royalties. The GoTaq® MDx Hot Start Polymerase is licensed for use in human diagnostics and is designed and manufactured under Promega's stringent Quality Management System, which has been certified to ISO 9001 and ISO 13485 standards and ensures the consistently high product quality required by clinical laboratories. The enzyme meets strict performance specifications consistent with its intended use.
The GoTaq® MDx Hot Start Polymerase contains GoTaq® DNA Polymerase bound to a proprietary antibody that blocks polymerase activity. Polymerase activity is restored during the initial denaturation step when amplification reactions are heated at 94–95°C for 2 minutes. GoTaq® MDx Hot Start Polymerase products use a proprietary cationic detergent for superior stabilization under PCR conditions
, and the products are subjected to extensive stability studies to ensure reliability. GoTaq® MDx Hot Start Polymerase is licensed for use in human diagnostic applications and can be used as a component of laboratory-developed molecular diagnostic tests.
To meet your unique laboratory requirements, GoTaq® MDx Hot Start Polymerase can be manufactured in a custom format with a modified concentration, volume, buffer formulations or format, such as glycerol-free product.
Promega scientists monitor and control the quality of GoTaq® MDx Hot Start Polymerase at every step of the manufacturing process to ensure the highest level of product quality. Standard quality control tests include but are not limited to:
- Functional tests for amplification activity and hot-start performance.
- Mammalian DNA contamination assay as a contamination control.
- Purity analyses of the GoTaq® enzyme and inhibitory antibody.
- Assays for contaminating endonuclease and exonuclease activities.
- Lot-to-lot consistency tests to ensure reproducibility between lots.
The GoTaq® MDx Hot Start Polymerase is manufactured by Promega Corporation under U.S. Patent Numbers 6,242,235 and 5,587,287 and their corresponding foreign patents. Rights to use these patents for clinical applications is conveyed through the purchase of the product. No additional royalties are required for use of this product. This product can be used as a component of molecular diagnostic assays where applicable country law allows. This product by itself does not provide any diagnostic result.
Here we present a vendor benchmark of GoTaq® MDx Hot Start Polymerase to investigate the following PCR criteria: sensitivity, hot-start performance and compatibility with multiplex PCR. The performance of GoTaq® MDx Hot Start Polymerase in PCR was compared to that of other commercially available hot-start Taq DNA polymerase products.
To examine sensitivity, we compared the performance of GoTaq® MDx Hot Start Polymerase in PCR to that of eight other commercially available hot-start Taq DNA polymerases. We amplified a 2.4kb target of the ademonatosis polyposis coli (APC) gene from 104, 103 and 102 copies of Human Genomic DNA (Cat.# G3041) using PCR conditions recommended by the manufacturer. GoTaq® MDx Hot Start Polymerase amplified the APC gene from 102 copies of genomic DNA under these test conditions, whereas the other DNA polymerases required 103 copies (Figure 1), demonstrating greater sensitivity under low-template conditions than the other enzymes tested.
Hot-Start PCR Performance
To demonstrate hot-start performance, we amplified the chemokine (C-C motif) receptor 5 (CCR5) target from 103 copies of human genomic DNA using GoTaq® MDx Hot Start Polymerase and other commercially available hot-start DNA polymerases following the manufacturers’ recommended conditions. Amplification of CCR5 without synthesis of primer-dimer and secondary products is a challenging test to characterize the efficiency of hot-start DNA polymerases
. The GoTaq® MDx Hot Start Polymerase did not produce nonspecific primer-dimer at 50–150bp, but other hot-start Taq DNA polymerases had significant primer-dimer formation (Figure 2). When the same target was amplified without hot start, there was significant primer-dimer synthesis (Figure 2, lane 12).
The doublet at 500bp is the expected product. The 1,000bp band is a secondary product commonly seen under these experimental conditions. The GoTaq® MDx Hot Start Polymerase had minimal amounts of secondary amplification products. Many of the other vendors had significantly more secondary products in their amplifications.
Another test for hot-start function is amplification of the Corynephage omega gene from a plasmid template. A 1.5kb fragment of the Corynephage omega gene was amplified using GoTaq® MDx Hot Start Polymerase and other hot-start Taq DNA polymerases from other manufacturers. Without hot-start PCR, a secondary 400bp amplification product was synthesized (Figure 3). GoTaq® MDx Hot Start Polymerase performed as well as or better than the other hot-start enzymes with this target, with robust target amplification and no nonspecific amplification products.
Compatibility with Multiplex PCR
To compare the performance of GoTaq® MDx Hot Start Polymerase and AmpliTaq Gold® DNA polymerase in a complex multiplex PCR assay, we amplified seven loci using fluorescently labeled primers and compared the amount of product synthesized. GoTaq® MDx Hot Start Polymerase showed robust performance and performed as well as or better than AmpliTaq Gold® DNA polymerase under the same conditions (Figure 4).
GoTaq® MDx Hot Start Polymerase is the ideal choice for laboratories selecting a general purpose reagent for molecular diagnostic assays. The enzyme is manufactured under a stringent quality management system that ensures consistent product quality. The enzyme also is licensed for use in human diagnostic applications and can be used as a component of laboratory-developed molecular diagnostic tests without paying royalties. GoTaq® MDx Hot Start Polymerase has similar or better amplification sensitivity and higher specificity than other commercially available hot-start DNA polymerases. With custom ordering options, you can tailor the format of the GoTaq® MDx Hot Start Polymerase to meet your specific requirements.