Artistic rendition of strip of tubes containing lyophilized powder

Thermo Fisher Scientific offers a comprehensive portfolio of lyophilization-compatible (lyo-ready) reverse transcriptases (RTs) and thermostable DNA polymerases, providing:

  • Flexible assay designs that maximize the same functional enzyme performance as conventional formats, with glycerol content below 0.1-0.5%
  • Higher confidence in results with minimal risk of bacterial, human or plasmid DNA contamination
  • Assurance of ISO 13485 quality and lot-to-lot consistency
  • Custom solutions for your specific applications, including modified concentration, volume or packaging
     

Lyophilization: Changing the Game for Molecular Diagnostics

Molecular assay developer quotes

“It has been very useful for stabilizing the assay without a need to concentrate the enzyme in an effort to minimize glycerol level."

—R&D Manager, Small Business Health Care Company

“Providing robust glycerol-free enzymes that performed in our assays as required without the need for big changes in formulations/processes. Providing reliable technical support and flexible supply service.”

—R&D Manager, Medium Enterprise Health Care Company

“They have performed better than other enzymes, this has allowed us more robust amplification.”

—R&D Director, Small Business Consumer Products Company

Lyo-ready reverse transcriptases (RTs)

  Lyo-ready SuperScript IV RT Lyo-ready SuperScript III RT
Optimal reaction temperature 50–55°C 50°C
Reaction time 10 min 50 min
Sensitivity Superior Good
Inhibitor tolerance Superior Medium
Sample available Yes Yes

Sensitivity, or the ability of RTs to generate cDNA from low RNA input, is an important attribute for RTs. The high sensitivity of lyo-ready SuperScript IV RT is advantageous for assays where sample amount is limited or the target RNA is at a low concentration in the sample. The amplification plot (Figure 1) illustrates high sensitivity and linearity of three different formulations of SuperScript IV RT (with glycerol; lyo-ready; lyophilized and reconstituted) across a wide dynamic range of input GAPDH RNA from 1.25 ng to 0.125 fg.

High sensitivity and linearity of cDNA synthesis with lyo-ready, lyophilized and reconstituted SuperScript IV RT’s

Figure 1. High sensitivity and linearity of cDNA synthesis with lyo-ready, lyophilized and reconstituted SuperScript IV RTs. Two step RT-qPCR for glycerol-containing, lyo-ready, and lyophilized and reconstituted SuperScript IV RT, using 1.25 ng–0.125 fgof GAPDH RNA as template, were performed according to the recommended product protocols.

RNA sample quality is one of major factors determining accuracy and relevance of any downstream analysis. Difficult samples, such as paraffin-embedded samples, buccal swabs, and organ biopsies commonly used for diagnostics and biomedical analysis, present a challenge to obtain high-quality RNA. Integrity of RNA can be affected during handling of samples or through the nucleic acid extraction process.

An efficient RT will reverse transcribe a wide range of RNA targets including those that are degraded during the purification process. Data presented earlier have demonstrated SuperScript IV RT to be robust and efficient with degraded RNA as compared to other commercially available RTs. Here, we show lyo-ready SuperScript IV RT delivers equally efficient cDNA synthesis with degraded RNA as compared to the conventional formulation of SuperScript IV RT (Figure 2).

Highly efficient cDNA synthesis with challenging RNA samples

Figure 2. Highly efficient cDNA synthesis with challenging RNA samples. Two step RT-qPCR reactions of degraded RNA (RIN: 2-3) from human cells were performed with lyo-ready SuperScript IV RT (blue) and conventional SuperScript IV RT with glycerol (orange). Three RT reactions were performed for each input RNA, 10% of the cDNA product was added to TaqMan assays for GAPDH, PPIA, HPRT1 and TBP targets. Three qPCR reactions were performed and average Cq values for each RNA input were calculated.

Inhibitors, such as trace amounts of reagents used in RNA purification, can cause problems during reverse transcription. Inhibitors may also be inherent in the biological sample source such as hemin, found in blood. To test how chemical compounds affect reverse transcription efficiency, various inhibitors were added to Jurkat total RNA prior to oligo(dT)20 annealing step. Figure 3 demonstrates, how lyo-ready SuperScript IV RT performs better than other commercially available RTs in the presence of a variety of inhibitors and retains this ability even after lyophilization and reconstitution.

Resistance to inhibitors

Figure 3. Resistance to inhibitors. Jurkat total RNA spiked with different inhibitors was used in two -step RT-qPCR with PGK1 gene specific primers. cDNA reactions were performed according to the recommended protocols from each reverse transcriptase vendor. Inhibitors used: SDS, Guanidinium Hydrochloride (GuHCl) and Hemin. NIC – Non Inhibitor Control.

SuperScript IV and SuperScript III lyo-ready RTs offer enhanced sensitivity and reduced reaction times and therefore are recommended for your most demanding RT-qPCR assays.  However, our comprehensive portfolio of lyo-ready reverse transcriptases also includes RevertAid (M-MuLV) and Maxima reverse transcriptases. If you’re interested to learn more, please contact us at MDxenzymes@thermofisher.com.

Sensitivity, or the ability of RTs to generate cDNA from low RNA input, is an important attribute for RTs. The high sensitivity of lyo-ready SuperScript IV RT is advantageous for assays where sample amount is limited or the target RNA is at a low concentration in the sample. The amplification plot (Figure 1) illustrates high sensitivity and linearity of three different formulations of SuperScript IV RT (with glycerol; lyo-ready; lyophilized and reconstituted) across a wide dynamic range of input GAPDH RNA from 1.25 ng to 0.125 fg.

High sensitivity and linearity of cDNA synthesis with lyo-ready, lyophilized and reconstituted SuperScript IV RT’s

Figure 1. High sensitivity and linearity of cDNA synthesis with lyo-ready, lyophilized and reconstituted SuperScript IV RTs. Two step RT-qPCR for glycerol-containing, lyo-ready, and lyophilized and reconstituted SuperScript IV RT, using 1.25 ng–0.125 fgof GAPDH RNA as template, were performed according to the recommended product protocols.

RNA sample quality is one of major factors determining accuracy and relevance of any downstream analysis. Difficult samples, such as paraffin-embedded samples, buccal swabs, and organ biopsies commonly used for diagnostics and biomedical analysis, present a challenge to obtain high-quality RNA. Integrity of RNA can be affected during handling of samples or through the nucleic acid extraction process.

An efficient RT will reverse transcribe a wide range of RNA targets including those that are degraded during the purification process. Data presented earlier have demonstrated SuperScript IV RT to be robust and efficient with degraded RNA as compared to other commercially available RTs. Here, we show lyo-ready SuperScript IV RT delivers equally efficient cDNA synthesis with degraded RNA as compared to the conventional formulation of SuperScript IV RT (Figure 2).

Highly efficient cDNA synthesis with challenging RNA samples

Figure 2. Highly efficient cDNA synthesis with challenging RNA samples. Two step RT-qPCR reactions of degraded RNA (RIN: 2-3) from human cells were performed with lyo-ready SuperScript IV RT (blue) and conventional SuperScript IV RT with glycerol (orange). Three RT reactions were performed for each input RNA, 10% of the cDNA product was added to TaqMan assays for GAPDH, PPIA, HPRT1 and TBP targets. Three qPCR reactions were performed and average Cq values for each RNA input were calculated.

Inhibitors, such as trace amounts of reagents used in RNA purification, can cause problems during reverse transcription. Inhibitors may also be inherent in the biological sample source such as hemin, found in blood. To test how chemical compounds affect reverse transcription efficiency, various inhibitors were added to Jurkat total RNA prior to oligo(dT)20 annealing step. Figure 3 demonstrates, how lyo-ready SuperScript IV RT performs better than other commercially available RTs in the presence of a variety of inhibitors and retains this ability even after lyophilization and reconstitution.

Resistance to inhibitors

Figure 3. Resistance to inhibitors. Jurkat total RNA spiked with different inhibitors was used in two -step RT-qPCR with PGK1 gene specific primers. cDNA reactions were performed according to the recommended protocols from each reverse transcriptase vendor. Inhibitors used: SDS, Guanidinium Hydrochloride (GuHCl) and Hemin. NIC – Non Inhibitor Control.

SuperScript IV and SuperScript III lyo-ready RTs offer enhanced sensitivity and reduced reaction times and therefore are recommended for your most demanding RT-qPCR assays.  However, our comprehensive portfolio of lyo-ready reverse transcriptases also includes RevertAid (M-MuLV) and Maxima reverse transcriptases. If you’re interested to learn more, please contact us at MDxenzymes@thermofisher.com.

Lyo-ready Taq DNA polymerases

  Platinum II Taq DNA polymerase Platinum Taq DNA polymerase LibertyTaq DNA polymerase
Hot-start PCR Antibody-based Antibody-based Proprietary
Reactivation time 30 sec to 2 min 2 min 0 min
Sensitivity Superior Good Medium
Specificity Superior Good Medium
Sample availability Yes Yes Yes

Platinum Taq DNA polymerase uses stringent antibody-based hot-start technology, which enables detection of low-abundance DNA targets with high accuracy. Similarly, lyo-ready Platinum Taq CG DNA polymerase offers the same great performance as Platinum Taq DNA polymerase, but in a formulation compatible with lyophilization (Figure 4).

High sensitivity with lyo-ready Platinum Taq, CG DNA Polymerase

Figure 4. High sensitivity with lyo-ready Platinum Taq CG DNA Polymerase. Lyo-ready Platinum Taq CG and Platinum Taq DNA Polymerases were used to amplify a 199 bp fragment of human DNA (A) and a 639 bp fragment of E. coli DNA (B) from varying amounts of template DNA in duplicate assays. The lyo-ready enzyme formulation offered sensitivity, specificity, and yields comparable to those of the standard formulation of Platinum Taq polymerase. NTC = no-template control. DNA ladder: Thermo Scientific ZipRuler Express DNA Ladder.

Lyo-ready Platinum Taq and LibertyTaq Hot start DNA polymerases are a strong choice for qPCR based assays. These enzymes offer fast activation, robust, sensitive and specific amplification across a wide dynamic range.

Efficient and reproducible qPCR assays

Figure 5. Efficient and reproducible qPCR assays. Lyo-ready LibertyTaq (blue curves) and Invitrogen Platinum Taq (red curves) DNA Polymerases were evaluated for their performance in qPCR using Applied Biosystems TaqMan Assays for human PPP1CA and varying amounts of human input DNA. Equally efficient and sensitive amplification was achieved with both DNA polymerases. No amplification was observed in no-template controls, indicating that, based on this detection method, formulations are free of contaminating human DNA. qPCR reaction efficiency is in the range of 90-110% with a determination coefficient R2 ≥0.990.

Stringent manufacturing process and a proprietary low glycerol formulation allows lyo-ready Platinum Taq and LibertyTaq DNA polymerases to exhibit high stability even under non-optimal shipping and storage conditions. The stability of DNA polymerase after multiple freeze/thaw cycles ensures product quality and performance in nucleic acid based assays.

Stability of LibertyTaq DNA Polymerase under different shipping conditions

Figure 6. Stability of LibertyTaq DNA Polymerase under different shipping conditions. To mimic different shipping and storage conditions, Lyo-ready LibertyTaq DNA Polymerase was subjected to 20 freeze/thaw cycles prior to its use in E. coli 23S TaqMan Assays (blue curves). Performance was compared to that of lyo-ready LibertyTaq DNA Polymerase stored at –20°C (red curves). Multiple freezing and thawing cycles did not affect the enzyme’s performance.

Platinum Taq DNA polymerase uses stringent antibody-based hot-start technology, which enables detection of low-abundance DNA targets with high accuracy. Similarly, lyo-ready Platinum Taq CG DNA polymerase offers the same great performance as Platinum Taq DNA polymerase, but in a formulation compatible with lyophilization (Figure 4).

High sensitivity with lyo-ready Platinum Taq, CG DNA Polymerase

Figure 4. High sensitivity with lyo-ready Platinum Taq CG DNA Polymerase. Lyo-ready Platinum Taq CG and Platinum Taq DNA Polymerases were used to amplify a 199 bp fragment of human DNA (A) and a 639 bp fragment of E. coli DNA (B) from varying amounts of template DNA in duplicate assays. The lyo-ready enzyme formulation offered sensitivity, specificity, and yields comparable to those of the standard formulation of Platinum Taq polymerase. NTC = no-template control. DNA ladder: Thermo Scientific ZipRuler Express DNA Ladder.

Lyo-ready Platinum Taq and LibertyTaq Hot start DNA polymerases are a strong choice for qPCR based assays. These enzymes offer fast activation, robust, sensitive and specific amplification across a wide dynamic range.

Efficient and reproducible qPCR assays

Figure 5. Efficient and reproducible qPCR assays. Lyo-ready LibertyTaq (blue curves) and Invitrogen Platinum Taq (red curves) DNA Polymerases were evaluated for their performance in qPCR using Applied Biosystems TaqMan Assays for human PPP1CA and varying amounts of human input DNA. Equally efficient and sensitive amplification was achieved with both DNA polymerases. No amplification was observed in no-template controls, indicating that, based on this detection method, formulations are free of contaminating human DNA. qPCR reaction efficiency is in the range of 90-110% with a determination coefficient R2 ≥0.990.

Stringent manufacturing process and a proprietary low glycerol formulation allows lyo-ready Platinum Taq and LibertyTaq DNA polymerases to exhibit high stability even under non-optimal shipping and storage conditions. The stability of DNA polymerase after multiple freeze/thaw cycles ensures product quality and performance in nucleic acid based assays.

Stability of LibertyTaq DNA Polymerase under different shipping conditions

Figure 6. Stability of LibertyTaq DNA Polymerase under different shipping conditions. To mimic different shipping and storage conditions, Lyo-ready LibertyTaq DNA Polymerase was subjected to 20 freeze/thaw cycles prior to its use in E. coli 23S TaqMan Assays (blue curves). Performance was compared to that of lyo-ready LibertyTaq DNA Polymerase stored at –20°C (red curves). Multiple freezing and thawing cycles did not affect the enzyme’s performance.

Lyo-ready Proofreading DNA polymerases

Product Platinum SuperFi DNA Polymerase Phusion Hot Start II High-Fidelity DNA Polymerase
Fidelity vs. Taq polymerase >100X 52X
Hot-start PCR Antibody-based Antibody-based
Target length ≤20 kb ≤20 kb
Extension rate 15-30 sec/kb 15-30 sec/kb
TaqMan probe-compatible No No
Inhibitor tolerance ++++ +++
Multiplexing Yes Yes
Sample availability Yes Yes

Invitrogen Platinum SuperFi DNA polymerase is engineered with a DNA-binding domain exhibiting high processivity and increased resistance to common PCR inhibitors such as Guanidinium Hydrochloride and Urea. Lyo–ready Platinum SuperFi DNA polymerase retains reliable performance in presence of the inhibitors.

Reliable performance in presence of common PCR inhibitors

Figure 7. Reliable performance in presence of common PCR inhibitors. Amplification of ~2 kb E.coli gDNA fragment using lyo-ready Platinum SuperFi, standard (with glycerol) Platinum SuperFi DNA polymerases. 50 µL reaction mixes contained 1 ng gDNA and 2 – no inhibitor, 3 – 45 mM guanidine hydrochloride, 4 – 200 mM urea. 1 – no template control.

Lyo-ready Platinum SuperFi DNA polymerase combines performance properties attributed to standard glycerol formulation and offers that same performance in lyo-ready and lyophilized and reconstituted state.

Reliable performance across various formats

Figure 8. Reliable performance across various formats (glycerol, lyo-ready and lyophilized and reconstituted). Amplification of 666 bp E.coli gDNA fragment using lyo-ready Platinum SuperFi, lyophilized and reconstituted lyo-ready Platinum SuperFi, standard (with glycerol) Platinum SuperFi DNA polymerases. 50 µL reaction mixes contained 1 – 10 ng DNA, 2 – 1 ng DNA, 3 – 0.1 ng DNA.

Invitrogen Platinum SuperFi DNA polymerase is engineered with a DNA-binding domain exhibiting high processivity and increased resistance to common PCR inhibitors such as Guanidinium Hydrochloride and Urea. Lyo–ready Platinum SuperFi DNA polymerase retains reliable performance in presence of the inhibitors.

Reliable performance in presence of common PCR inhibitors

Figure 7. Reliable performance in presence of common PCR inhibitors. Amplification of ~2 kb E.coli gDNA fragment using lyo-ready Platinum SuperFi, standard (with glycerol) Platinum SuperFi DNA polymerases. 50 µL reaction mixes contained 1 ng gDNA and 2 – no inhibitor, 3 – 45 mM guanidine hydrochloride, 4 – 200 mM urea. 1 – no template control.

Lyo-ready Platinum SuperFi DNA polymerase combines performance properties attributed to standard glycerol formulation and offers that same performance in lyo-ready and lyophilized and reconstituted state.

Reliable performance across various formats

Figure 8. Reliable performance across various formats (glycerol, lyo-ready and lyophilized and reconstituted). Amplification of 666 bp E.coli gDNA fragment using lyo-ready Platinum SuperFi, lyophilized and reconstituted lyo-ready Platinum SuperFi, standard (with glycerol) Platinum SuperFi DNA polymerases. 50 µL reaction mixes contained 1 – 10 ng DNA, 2 – 1 ng DNA, 3 – 0.1 ng DNA.

Lyo ready 1-step RT-qPCR

The Invitrogen Lyo-ready SuperScript Reverse Transcriptase, 1-step RT-qPCR system is specifically designed for performance in 1-step qPCR. Specially formulated Lyo-ready SuperScript Reverse Transcriptase and Lyo-ready Platinum II Taq DNA polymerase are both highly processive, next generation enzymes that deliver the same product yield within a shorter reaction time.  Multiplexing capability allows testing of more analytes per reaction, while inhibitor-tolerant enzymes reduce time spent on tedious purification steps.  The RT-qPCR protocol for this 1-step RT-qPCR system with optimized reaction buffer allows for a quicker and more cost-effective assay

Lyo-ready Isothermal DNA Polymerases

Thermo Scientific isothermal DNA polymerases all exhibit strong strand displacement activity and processivity, making them the ideal choice for applications like loop-mediated isothermal amplification (LAMP), whole genome amplification (WGA), rolling circle amplification (RCA), and more.  These enzymes are perfect for applications where PCR and temperature cycling are not needed.

Thermo Scientific EquiPhi29 DNA Polymerase is a proprietary phi29 DNA Polymerase mutant developed through in vitro protein evolution. This enzyme is significantly improved over phi29 DNA Polymerase in protein thermostability, reaction speed, product yield, and amplification bias. This enzyme is useful in unbiased whole genome amplification (WGA), providing high yields from minimal template amount and highly accurate DNA synthesis in a shorter amount of time.

EquiPhi29 DNA polymerase demonstrates low GC bias when amplifying 3 whole genomes from bacteria

Figure 11. EquiPhi29 DNA Polymerase demonstrated low GC bias when amplifying 3 bacterial genomes. A mixture of bacterial genomes with low-GC (S. aureus, 33% GC), moderate-GC (E. coli, 51% GC), and high-GC (P. aeruginosa, 68% GC) content was amplified using EquiPhi29 and Phi29 DNA polymerases as well as a DNA polymerase from another supplier. For each genome, the GC content of the reference genome, in 100 bp windows indicated in gray, was plotted versus the coverage normalized to the unamplified genome mix, indicated in green. In the absence of sequencing bias, all windows should be equally distributed close to the normalized coverage of 1, indicated in light blue. The normalized coverage obtained after amplification using different polymerases is shown. EquiPhi29 DNA Polymerase amplifies DNA with the lowest GC bias across all GC contents when compared to other DNA polymerases (EquiPhi29 DNA Polymerase is indicated in yellow).

Thermo Scientific Bsm DNA Polymerase is an equivalent to Bst DNA polymerase and provides with strong displacement activity. This enzyme is suitable for isothermal DNA amplification methods such as LAMP, WGA, and other molecular assays.

Loop-mediated isothermal amplification (LAMP) setup using Bsm DNA polymerase, DNA sample, and 4 to 6 primers

Figure 12. LAMP reaction setup using Bsm DNA Polymerase. Loop-mediated isothermal amplification is a single tube technique for the amplification of DNA. It uses 4-6 primers, which form loop structures to facilitate subsequent rounds of amplification. Bsm has strong strand displacement activity and an optimum temperature of 60°C. Amplification is very efficient with DNA being copied a billion-fold in as little as 15 minutes. The enzyme is highly resistant to inhibitors in complex samples, so plant tissue, blood, urine, or saliva can be assayed with minimal processing.

Thermo Scientific EquiPhi29 DNA Polymerase is a proprietary phi29 DNA Polymerase mutant developed through in vitro protein evolution. This enzyme is significantly improved over phi29 DNA Polymerase in protein thermostability, reaction speed, product yield, and amplification bias. This enzyme is useful in unbiased whole genome amplification (WGA), providing high yields from minimal template amount and highly accurate DNA synthesis in a shorter amount of time.

EquiPhi29 DNA polymerase demonstrates low GC bias when amplifying 3 whole genomes from bacteria

Figure 11. EquiPhi29 DNA Polymerase demonstrated low GC bias when amplifying 3 bacterial genomes. A mixture of bacterial genomes with low-GC (S. aureus, 33% GC), moderate-GC (E. coli, 51% GC), and high-GC (P. aeruginosa, 68% GC) content was amplified using EquiPhi29 and Phi29 DNA polymerases as well as a DNA polymerase from another supplier. For each genome, the GC content of the reference genome, in 100 bp windows indicated in gray, was plotted versus the coverage normalized to the unamplified genome mix, indicated in green. In the absence of sequencing bias, all windows should be equally distributed close to the normalized coverage of 1, indicated in light blue. The normalized coverage obtained after amplification using different polymerases is shown. EquiPhi29 DNA Polymerase amplifies DNA with the lowest GC bias across all GC contents when compared to other DNA polymerases (EquiPhi29 DNA Polymerase is indicated in yellow).

Thermo Scientific Bsm DNA Polymerase is an equivalent to Bst DNA polymerase and provides with strong displacement activity. This enzyme is suitable for isothermal DNA amplification methods such as LAMP, WGA, and other molecular assays.

Loop-mediated isothermal amplification (LAMP) setup using Bsm DNA polymerase, DNA sample, and 4 to 6 primers

Figure 12. LAMP reaction setup using Bsm DNA Polymerase. Loop-mediated isothermal amplification is a single tube technique for the amplification of DNA. It uses 4-6 primers, which form loop structures to facilitate subsequent rounds of amplification. Bsm has strong strand displacement activity and an optimum temperature of 60°C. Amplification is very efficient with DNA being copied a billion-fold in as little as 15 minutes. The enzyme is highly resistant to inhibitors in complex samples, so plant tissue, blood, urine, or saliva can be assayed with minimal processing.

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