Team:Bielefeld-CeBiTec/Results/unnatural base pair/development of new methods

Development of New Methods

Mutation Analysis Xplorer – Results

Primer annealing

To test the best annealing efficiency, we applied three different annealing methods.

We designed five ssDNA pairs with different bases at position 40. Each of the natural bases A, T, G, and C will lead to a recognition sequence of one of the restriction enzymes EciI, BsaI, SapI and MnlI. We postulate that non of these restriction enzymes will recognize their respective recognition sequences if the basepair between isoG and isoC^m is present at this position. For good annealing efficiency, it is necessary to add the two oligo strands together in equal molar amounts. The concentration can be calculated by the OD₂₆₀ value, while an OD₂₆₀ of 1 equals 33 µg ml^-1 (NEB calculator, September 2017) and the molecular mass of each oligo.

Figure 1:Sequenzes of M.A.X targets MutA, mutT, mutG ad mutC with relevant restriction sites as well as the sequence of UBP_target.

All reactions showed a nearly complete alignment. We prosecuted further experiments with the aqua annealing in order to avoid affecting subsequent digestion reactions by influencing the buffer conditions.

Subsequently, we tested different amounts of annealed DNA, varying from 50 µmol L^-1 to 0.25 µmol L^-1, which still shows visible bands on the gel.

After first results, a final annealing concentration of 0.5 µmol L^-1 seems to be a good choice in terms of visibility and low DNA quantity for complete digestion. For the following annealing reactions, 1 µmol L^-1 of ssDNA was used to get 0.5 µL L^-1 of annealed dsDNA.

Restriction digest

The DNA strands were designed such that the partial restriction sites of four different restriction enzymes are located at the same position. In case of a mutation, we can validate to which base the unnatural base mutated without sequencing it. To test the practicability and quality of the restriction system, we performed several test restriction digests.

To ensure the digestion is complete, we calculated the amount of DNA which is digested per unit of enzyme in 1 hour at 37 °C. 1 unit is defined as the amount of restriction enzyme needed to digest 1 µg of lambda DNA. The lambda DNA consists of 48,502 bp (NEB) , which equals 1.99 ∙ 10¹⁰ molecules per µL. Depending on the sum of recognition sites of each enzyme, we calculated the cuts per hour of each enzyme.

Table 1: Calculation of restrictions per hour (1 unit) of the M.A.X enzymes.

	Enzyme	unit per µL	restriction sites in lambda DNA	restriction per hour (1 unit)
MutG	SapI	10	10	1.99∙10¹²
MutC	MnlI	5	262	2.35∙10¹³
MutT	BsaI	10	2	3.6∙10¹¹
MutA	EciI	2	29	1.44∙10¹²

In an annealing reaction with 0.5 µmol L^-1 DNA in a total reaction volume of 50 µL, we have 3.011 ∙ 10⁸ molecules per µL, each contain one or two restriction site. Theoretically, more than 1 mL of the annealing DNA should be digested by 1 unit of restriction enzyme per hour.

Figure 2: Native DNA PAGE of annealed mutA oligos. Samples (FLTR): UBP_target ssDNA, UBP_target annealing, restricted UBP_target (EciI), restricted mutA (EciI), 17jf ssDNA primer, annealed mutA.

Figure 3: Native DNA PAGE of annealed oligos. Samples (FLTR): annealed mutT, 17jj ssDNA primer, restricted mutT (BsaI), restricted UBP_target (SapI), UBP_target annealing, UBP_target ssDNA.

Figure 4: Native DNA PAGE of annealed mutG oligos. Samples (FLTR): UBP_target ssDNA, UBP_target annealing, restricted UBP_target (SapI) restricted mutG (SapI), 17jh ssDNA primer, annealed mutG.

Figure 5: Native DNA PAGE of annealed mutC oligos. Samples (FLTR): UBP_target ssDNA, UBP_target annealing, restricted UBP_target (MnlI) restricted mutC (MnlI), 17jl ssDNA primer, annealed mutC.

All Figures show the expected band pattern. The digest of the M.A.X targets is not complete. Later experiments revealed that a longer incubation time is necessary. The UBP_target annealings are not digested, indicating that the UBP prevents sequence recognition of the tested restriction enzymes. This proofs that the M.A.X restriction system is a good detection system for UBP retention and mutation event analysis in selected DNA sequences.

PCR with UBPs

Based on Sismour&nbsp et al. (Sismour and Benner, 2005) and Johnson et al. (Johnson et al., 2004), we designed a novel protocol for PCR with the unnatural base pair isoG and isoC^m. We first started to reproduce positive results with Titanium Taq (TiTaq) polymerase. While Johnson et al. presented an efficiency of 96&nbsp%&nbsp±&nbsp3&nbsp%, Sismour&nbspet al. showed a reduced fidelity using the klenow fragment of TiTaq polymerase. Without thymidine analogues, the fidelity per round PCR decreases rapidly to less than 60&nbsp% after 20 rounds of PCR.

For endpoint determination, we performed PCR reactions with 30 rounds to find out if there is any polymerase activity with template DNA containing the unnatural bases isoG and isoC^m.

The PCR templates were prepared by ligating each of the annealed 80 bp M.A.X targets mutA, mutT, mutG and mutC into pSB1C3_RuBisCo . For this purpose, the plasmid backbone was linearized by digestion with BmtI and XbaI. For complementary sticky ends, the annealed oligos were digested with BmtI and SpeI. After ligation, subsequent digestion with XbaI, lambda exonuclease and exonuclease I was performed to reduce the amount of unintended DNA template.

Figure 6: Plasmidcard of pSB1C3_Rubisco which is used as backbone for M.A.X targets and UBP_target during PCR.

To increase the possibility of the insertion of the unnatural bases, we used 100&nbspµM dNTPs and 200&nbspµM isoG and 200&nbspµM isoC^m for each reaction. After variations of template concentrations from 1&nbspng&nbspµL^-1 to 50&nbspng&nbspµL^-1, the best concentrations to acquire high-quality bands were 1&nbspng&nbspµL^-1 for the M.A.X targets and 25&nbspng&nbspµL^-1 for the UBP_target template.

Figure 7: PCR with Titanium Taq polymerase of pSB1C3_RuBisCo with the inserts mutA, mutT, mutG, mutC (5 ng µL^-1) and UBP_target (25 ng µL^-1) (FLTR). The expected fragment is 351 bp long.

To quantify the efficiency of the incorporation of isoG and isoC^m, all PCR products were tested/restricted with the M.A.X system. In order to achieve complete digestion, different incubation times from 1&nbsph to 15&nbsph were tested. The best results with BsaI and MnlI were achieved with an incubation of 15&nbsph overnight. For the less stable enzymes EciI and SapI, a 2&nbsph digestion with an addition of further enzyme after 1&nbsph turned out to be optimal. Nevertheless, EciI and SapI could not digest the complete sample even if the concentrations are lowered. Therefore we expected undigested bands in the M.A.X targets mutA and mutG for the whole experiment.

After the first successful PCR, we tested if the presence of isoG and isoC^m has any influence on the efficiency of the polymerase. So we added both unnatural bases to every PCR reaction with the M.A.X targets as template to see if the intensity of the bands decreases.

Figure 8: Digested PCR with Titanium Taq polymerase of pSB1C3_RuBisCo with all M.A.X targets and the UBP_target insert with isoG and isoCm in each reaction of the M.A.X targets and no UBPs in the reactions with the UBP_target insert. There seems to be no efficiency difference to reactions without the unnatural base pairs. All samples are digested to the same fragment size so no UBPs are incorporated.

In comparison to fig.7, the intensity is the same. In contrast, we did not add UBPs to the reaction with the UBP_target fragment as template. The restriction digest shows the same bands for every sample so the presence of UBPs does not influence the polymerase activity.

The next step was to test if different polymerases can incorporate the unnatural bases. Therefore we tested 7 other polymerases from different manufacturers.
Titanium Taq Polymerase (Clontech) lacks 5’-exonuclease activity of wild-type DNA.

Figure 9: PCR with Titanium Taq of pSB1C3_RuBisCo with of pSB1C3_RuBisCo with the inserts mutA, mutT, mutG, mutC (5 ng µL^-1) and UBP_target (25 ng µL^-1) after the restriction digest with EciI (mutA) and SapI (mutG) for 2 h and BsaI (muttT) and MnlI (mutC) for 15 h.

GoTaq G2 polymerase (Promega) with 5’-3’ exonuclease activity.

Figure 10: PCR with Go Taq G2 of pSB1C3_RuBisCo with of pSB1C3_RuBisCo with the inserts mutA, mutT, mutG, mutC (5 ng µL^-1) and UBP_target (25 ng µL^-1) after the restriction digest with EciI (mutA) and SapI (mutG) for 2 h and BsaI (mutT) and MnlI (mutC) for 15 h.

Allin HiFi DNA Polymerase (highQu) is derived from Pfu polymerase with several mutations and proof reading function.

Figure 11:PCR with Allin HiFi DNA Polymerase of pSB1C3_RuBisCo with of pSB1C3_RuBisCo with the inserts mutA, mutT, mutG, mutC (5 ng µL^-1) and UBP_target (25&ngbspng µL^-1) after the restriction digest with EciI (mutA) and SapI (mutG) for 2 h and BsaI (muttT) and MnlI (mutC) for 15 h

innuDRY polymerase is a specific hot-start Taq DNA polymerase.

Figure 12: PCR with innuDRY polymerase of pSB1C3_RuBisCo with of pSB1C3_RuBisCo with the inserts mutA, mutT, mutG, mutC (5 ng µL^-1) and UBP_target (25 ng µL^-1) after the restriction digest with EciI (mutA) and SapI (mutG) for 2 h and BsaI (muttT) and MnlI (mutC) for 15 h.

BioMaster-HS Taq PCR polymerase (Biolabmix) is also a hot-start Taq DNA polymerase.

Figure 13: PCR with BioMaster-HS Taq PCR polymerase of pSB1C3_RuBisCo with of pSB1C3_RuBisCo with the inserts mutA, mutT, mutG, mutC (5 ng µL^-1) and UBP_target (25 ng µL^-1) after the restriction digest with EciI (mutA) and SapI (mutG) for 2 h and BsaI (muttT) and MnlI (mutC) for 15 h.

FirePol DNA polymerase (Solis Biodyne) had genetic modifications, such that it is stable at room temperature for 1&nbspmonth. It has a 5’-3’ polymerization-dependent exonuclease replacement, but lacks 3’-5’ exonuclease activity.

Figure 14: PCR with FirePol DNA polymerase of pSB1C3_RuBisCo with of pSB1C3_RuBisCo with the inserts mutA, mutT, mutG, mutC (5 ng µL^-1) and UBP_target (25 ng µL^-1) after the restriction digest with EciI (mutA) and SapI (mutG) for 2 h and BsaI (muttT) and MnlI (mutC) for 15 h.

The Phusion DNA polymerase (NEB) is a derived Pyrococcus enzyme fused with a processivity-enhancing domain. It possesses 5’-3’ polymerase activity and 3’-5’ exonuclease activity so blunt-ended products are generated.

Figure 15: PCR with Phusion DNA polymerase of pSB1C3_RuBisCo with of pSB1C3_RuBisCo with the inserts mutA, mutT, mutG, mutC (5 ng µL^-1) and UBP_target (25 ng µL^-1) after the restriction digest with EciI (mutA) and SapI (mutG) for 2 h and BsaI (muttT) and MnlI (mutC) for 15 h.

The Q5 DNA polymerase (NEB) is a polymerase that is fused to the processivity-enhancing Sso7d DNA binding domain with an error rate ~280-fold lower than of Taq DNA polymerase.

Figure 16: PCR with Q5 DNA polymerase of pSB1C3_RuBisCo with of pSB1C3_RuBisCo with the inserts mutA, mutT, mutG, mutC (5 ng µL^-1) and UBP_target (25 ng µL^-1) after the restriction digest with EciI (mutA) and SapI (mutG) for 2 h and BsaI (mutT) and MnlI (mutC) for 15 h.

We always used the same template concentration and the same primers to ensure the comparability between the DNA polymerases. As can be seen in the figures above, all Taq based polymerases are able to incorporate the unnatural base pair in the DNA. The best results were apparently achieved with the Go Taq G2 DNA polymerase. All lanes with the UBPs show clear bands and no mutations to T or A. The M.A.X restriction digest showed there are some mutations to C. The PCR product of the UBP_target fragment was digested because of the second MnlI restriction site, but one can see a difference between the M.A.X target mutC and the UBP_target PCR product. The digested PCR products of the BioMaster-HS Taq PCR polymerase and the Allin HiFi DNA Polymerase show more mutations to A than the other polymerases. But also the TiTaq polymerase seems to miss incorporate the A instead of isoG. Moreover, the Phusion DNA polymerase proceeds to miss the incorporated G while the Q5 DNA polymerase does not show any bands containing the UBPs. Both polymerases have a proofreading function in contrast to the other polymerases.

In the final analysis, the faster and the stronger the poof reading function of a polymerase is, the worse is the incorporation of the UBPs.

The M.A.X system seems to be a good method for the first review of the efficiency of the polymerases. One can see if there is any incorporation of UBPs, so that sequencing is worthwhile. Minor deviations are not detectable by gel electrophoresis. It is also difficult to make a clear statement about the proportion of correctly or incorrectly incorporated unnatural bases, because the digested fragments seem to be less intensive than intact sequences.