Difference between revisions of "Team:Bielefeld-CeBiTec/Results/unnatural base pair/development of new methods"

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<h2> Übergeordneter Titel der gesamten Seite </h2>
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<h3> Mutation Analysis Xplorer – Results  </h3>
 
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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 <i>Eci</i>I, <i>Bsa</i>I, <i>Sap</i>I and <i>Mnl</i>I. We postulate that non of these restriction enzymes will recognize their respective recognition sequences if the basepair between isoG and isoC<sup>m</sup> is present at this position.  
 
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 <i>Eci</i>I, <i>Bsa</i>I, <i>Sap</i>I and <i>Mnl</i>I. We postulate that non of these restriction enzymes will recognize their respective recognition sequences if the basepair between isoG and isoC<sup>m</sup> is present at this position.  
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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 OD<sub>260</sub> value, while an  OD<sub>260</sub> of 1 equals 33&nbspµg ml<sup>-1</sup> <a href=" https://nebiocalculator.neb.com/#!/od260"> (NEB calculator, September 2017) </a> and the molecular mass of each oligo.   
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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 OD<sub>260</sub> value, while an  OD<sub>260</sub> of 1 equals 33 µg ml<sup>-1</sup> <a href=" https://nebiocalculator.neb.com/#!/od260"> (NEB calculator, September 2017) </a> and the molecular mass of each oligo.   
 
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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.  
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<br>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.</br>
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Subsequently, we tested different amounts of annealed DNA, varying from 50&nbspµmol&nbsp L<sup>-1</sup> to 0.25&nbspµmol&nbspL<sup>-1</sup>, which still shows visible bands on the gel.  
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<br>Subsequently, we tested different amounts of annealed DNA, varying from 50 µmol  L<sup>-1</sup> to 0.25 µmol L<sup>-1</sup>, which still shows visible bands on the gel.  
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After first results, a final annealing concentration of 0.5&nbspµmol&nbspL<sup>-1</sup> seems to be a good choice in terms of visibility and low DNA quantity for complete digestion. For the following annealing reactions, 1&nbspµmol&nbspL<sup>-1</sup> of ssDNA was used to get 0.5&nbspµL&nbsp L<sup>-1</sup> of annealed dsDNA.  
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<br>After first results, a final annealing concentration of 0.5 µmol L<sup>-1</sup> 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<sup>-1</sup> of ssDNA was used to get 0.5 µL  L<sup>-1</sup> of annealed dsDNA.  
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<h4>Restriction digest</h4>
 
<h4>Restriction digest</h4>
 
 
 
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<br>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.  
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<br>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. </br>
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To ensure the digestion is complete, we calculated the amount of DNA which is digested per unit of enzyme in 1&nbsphour at 37&nbsp°C. 1 unit is defined as the amount of restriction enzyme needed to digest 1&nbspµg of lambda DNA. The lambda DNA consists of 48,502&nbspbp <a href=" https://www.neb.com/products/n3011-lambda-dna#Product%20Information"> (NEB) </a>, which equals 1.99&nbsp∙&nbsp10<sup>10</sup> molecules per µL. Depending on the sum of recognition sites of each enzyme, we calculated the cuts per hour of each enzyme.
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<br>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 <a href=" https://www.neb.com/products/n3011-lambda-dna#Product%20Information"> (NEB) </a>, which equals 1.99 ∙ 10<sup>10</sup> molecules per µL. Depending on the sum of recognition sites of each enzyme, we calculated the cuts per hour of each enzyme.
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<br>In an annealing reaction with 0.5&nbspµmol&nbspL<sup>-1</sup> DNA in a total reaction volume of 50&nbspµL, we have 3.011&nbsp∙&nbsp10<sup>8</sup> 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.  
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<br>In an annealing reaction with 0.5 µmol L<sup>-1</sup> DNA in a total reaction volume of 50 µL, we have 3.011 ∙ 10<sup>8</sup> 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.  
 
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Revision as of 16:02, 31 October 2017

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 isoCm 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.