Tth DNA Polymerase is a thermostable enzyme of approximately 94kDa isolated from eubacterium Thermus thermophilus strain HB8. This enzyme replicates DNA at 74°C, reveals RNA-dependent DNA-polymerase activity in the presence of Mn²⁺ ions. Meanwhile, the concentration of RNA template for effective reverse transcription with Tth DNA polymerase should be higher if to compare with reverse transcription directed by Reverse Transcriptases (M-MuLV, AMV).

Tth DNA Polymerase is a thermostable DNA polymerase with intrinsic reverse transcription (RT), but no RNAse H activity(6.). The error rate of Tth DNA polymerase increase in the present of Mn²⁺ ions (5., 8., 12 and 13.).Tth DNA polymerase can reverse transcript and amplify fragments up to 2-3 kb. However, the fragment should be ideally smaller 1 kb. The error rate is 3.0 x 10^-5 in PCR reaction(8.). Although Tth DNA polymerase adds 3´dA overhangs, it is not recommended for PCR product cloning because the error rate is similar to Taq polymerase.Tth DNA polymerase accepts modified dNTPs and can therefore be used to label DNA fragments with modified dNTPs labeled with digoxigenin, biotin or fluorescein. Several buffer systems were used for 1-tube or step (6.) and 2-tubes/steps RT-PCR (5.) with Tth-polymerase. One step means intrinsic reverse transcription and amplification is mediated by one enzyme in one tube.Two steps means that the first step is a RTPCR reaction (first strand synthesis of the cDNA) with the reverse primer and a reverse transcription buffer which is including Mn²⁺ which is followed by a second PCR reaction (including the forward primer, second strand synthesis of cDNA) with a PCR buffer (Mg²⁺).

Tth DNA polymerase displayed the unique property of maintaining both DNA- and RNA-dependent DNA polymerase activities in the presence of 2%-5% (vol/vol) of phenol-saturated PBS buffer. Tth DNA polymerase mediated reverse transcriptase activity was unaffected by phenol-saturated phosphate-buffered saline concentrations as high as 15% (vol/vol). By contrast, Taq DNA Polymerase was inactive under these conditions. The ability to function in the presence of phenol can greatly simplify reverse transcriptase, PCR and reverse transcription-PCR protocols since the phenol-saturated aqueous phase of a phenol partition can be added directly to the reaction mixtures (9.).

Successful RT – PCR depends on the quality of the RNA. Use highest purity of RNA (A₂₆₀/A₂₈₀ ratio of 1.7 or higher). RNA should be DNA free. Total RNA, messenger RNA or viral RNA can be used. The quality of template RNA can be assessed using a positive control primer pair for a housekeeping gene (ß-actin, GAPDH).The 16S rRNA gene from various bacterial cultures was amplified by the polymerase chain reaction without DNA purification, and sequenced directly by using a laser fluorescent DNA sequencer and Tth polymerase with a cycle sequencing protocol. The described procedures provide almost complete 16S rDNA sequence data within a couple of days and facilitate systematic studies (10.).

The use of Tth-DNA polymerase which, is also a reverse transcriptase, is active in the presence of manganese and accepts both RNA and DNA as matrix, enables the whole reaction to be performed as a “one-step” RT-PCR analysis (6). This experimental approach include the amplification of fragments only to a maximum of 1 kb and a relatively high error rate for DNA polymerase as a result of the manganese ion concentration (7.).

Reverse transcription could be performed at 60°C minimizing problems from RNA secondary structure and high G/C content.

The “one enzyme/one tube” Tth DNA polymerase assay uses bicine buffers containing Mn²⁺ ions that are compatible with both RT and subsequent PCR (6., 11.)

For RT-PCR amplification (reverse transcription and amplification in one step), the concentration of Mn²⁺ needs to be determined by testing Mn²⁺ -concentrations from 1- 4 mM for each reaction.

One step reaction eliminates the risk of cross contaminations associated with two step RT-PCR.

Number of cycles ranges from 20 – 50 in the literature. If the template is limited, increasing the number of cycles may result in nonspecific product yield.

*depends on your primers, 70 - 75°C is the optimal reaction temperature for Tth DNA polymerase. Tth DNA polymerase is resistant to prolonged incubations (20 min Half - Life time at 95°C ) at high temperatures (94°C) and can therefore be used for PCR.

Two steps means that the first step is a RT-PCR reaction (first strand synthesis of the cDNA) with the reverse primer and a reverse transcription buffer which is including Mn²⁺ which is followed by a second PCR reaction (including the forward primer, second strand synthesis of cDNA) with a PCR buffer (Mg²⁺).
The error rate of Tth DNA polymerase is increased in present of Mn²⁺ – ions (5). Therefore a Two step RT – PCR is recommended for PCr products which are cloned and used for subsequent further investigations.

** For a 0.5 M EGTA stock: dissolve 19.2 g EGTA in 70 ml deionized water, adjust pH 8.0 with NaOH (10 M). Add deionized water to 100 ml final volume. Filter sterilize with 0.22 μm and store at room temperature).