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THE FUTURE OF PCR

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RT PCR

Overview of Assay

RNA oligonucleotides from cells and viruses can be amplified for detection using the methods of Reverse Transcription and Polymerase Chain Reaction (RT-PCR).  In a first step, a complementary strand of DNA (cDNA) is synthesized using a primer and reverse transcriptase.  The cDNA is then amplified using standard PCR techniques.  This can be accomplished by taking an aliquot from the reverse transcription reaction and adding a DNA-dependent DNA polymerase (e.g. Taq) and other reagents and then doing PCR.  That method is commonly called "Two-step RT-PCR", or "Two-tube RT-PCR".  Alternatively, all reagents, including both enzymes and gene-specific primers can be included in a single tube in order to do both reverse trancription and PCR without the need to open the tube between those reactions.  That method is commonly called "One-Step RT-PCR" or "One-Tube RT-PCR".  Both methods can include primers for a single target or for multiple targets (i.e. multiplex RT-PCR).

 

Common Problems

The majority of PCR is now done using a "hot-start" method to keep Taq inactive until it reaches reaction temperatures.  In contrast, the vast majority of reverse transcrption to date has been done without hot start even though reverse transcriptases have significant activity at room temperature and even below.  Due to this activity at low temperature and the high stability of DNA-RNA hybrids, primers intended to be gene-specific may hybridize with undesired targets that are partially complementary, leading to non-specific amplification.  This problem is increased substantially when multiple primers are included for multiplex RT-PCR.

How ThermaStop-RT Overcomes Common Problems

ThermaStop-RT is a simple-to-use reagent for One-Step or Two-Step RT-PCR initiated with gene-specific primers.  ThermaStop-RT interacts with reverse transcriptase at low temperatures to inhibit enzyme activity that can lead to non-specific products.  By eliminating these errors, ThermaStop-RT significantly increases the yield of the desired specific products and increases assay sensitivity.  Reverse transcriptase activity increases at temperatures above 45oC, enabling transcription of the desired RNA sequence.

Documents

Documents Manuals and Protocols

 

Material safety data sheets (MSDS)

 

Single-target-sequence (monoplex) RT-PCR

 

Approximately 1,000 copies of an RNA sequence from Hepatitis C Virus (HCV) Genotype 2b Armored RNA® [link: http://asuragen.com/wp-content/uploads/2016/04/Armored-RNA-Catalog-Rev-08.16_Final.pdf ] in either the presence or absence of ThermaStop-RT was reverse transcribed using SuperScript® III (Invitrogen) and amplified in a non-symmetric PCR using Platinum™ Tfi polymerase (Invitrogen).  Both one-step and two-step RT-PCR protocols were tested.  Figure 1 shows the end-point fluorescent signals from a hybridization probe.  The results indicate that for either one-step or two-step RT-PCR, over 3 times as much of the HCV amplification product was generated in samples containing ThermaStop-RT.

 

A large decrease in the amount of non-specific PCR product is also observed in samples containing ThermaStop-RT  Figure 2 shows the SYBR Green fluorescence derivative from the post-PCR melt of the two-step RT-PCR experiment above.  Samples containing ThermaStop-RT (purple lines) have a single melt peak at 85oC detecting the melting of the HCV-specific amplification product.  In contrast, samples without ThermaStop-RT (blue lines) have a much smaller melting peak at 85oC, consistent with reduced specific amplification, and have additional, non-specific product peaks.

 

Figure 1.  ThermaStop-RT (TS-RT) greatly increases probe signals for detecting Hepatitis C Virus (HCV) RNA sequence using either two-step or one-step RT-PCR.

Figure 2.  Melting analysis following two-step RT-PCR of an HCV RNA sequence detects a single, specific product in samples with ThermaStop-RT (purple lines). Non-specific products are detected in samples without ThermaStop-RT (blue lines).

Lane 1; 100 bp ladder, lanes 2-6; samples without ThermaStop-RT, lanes 7 -11; samples with 1 unit of ThermaStop-RT.

Details:

Lanes 2 and 6 have plant RNA (10-2 dilution from stock) for nad5 transcript amplification, but no viroid RNA.

Lanes 3 and 7 have viroid synthetic RNA (10-9 dilution), but no plant RNA

Lanes 4 and 8 have plant RNA (10-2) and viroid RNA (10-8)

Lanes 5 and 9 have plant RNA (10-2) and viroid RNA (10-9)

Lanes 6 and 11 have plant RNA (10-2) and viroid RNA (10-10)

Note that the plant RNA (control) is at the same concentration in all samples except viroid only samples; the Viroid synthetic RNA was tested at dilutions from 10-6 to 10-11, viroid amplification was observed in all samples at concentrations at or above the 10-10 dilution.