Id |
Subject |
Object |
Predicate |
Lexical cue |
T90 |
0-4 |
Sentence |
denotes |
1.3. |
T91 |
5-40 |
Sentence |
denotes |
Multiple Displacement Amplification |
T92 |
41-194 |
Sentence |
denotes |
The proof-of-principle amplification of choice is a MDA reaction, which is a non-specific isothermal method of amplification performed around 30 °C [50]. |
T93 |
195-285 |
Sentence |
denotes |
MDA is a method of whole genome amplification (WGA), as it amplifies all present DNA [52]. |
T94 |
286-356 |
Sentence |
denotes |
It is commonly used when the initial amount of DNA sample is very low. |
T95 |
357-492 |
Sentence |
denotes |
After the WGA is performed, a sequence specific amplification can be done since the quality the amplified DNA by MDA is very high [53]. |
T96 |
493-629 |
Sentence |
denotes |
The amplification reaction is illustrated below in Figure 1 (the contour of the amplified sequence is highlighted in black for clarity). |
T97 |
630-846 |
Sentence |
denotes |
Starting with a double stranded DNA (dsDNA) molecule, a denaturation step at 95 °C is required, giving the random hexamer-primers and the ϕ29 DNA polymerase access to the bases of single stranded DNA (ssDNA) strands. |
T98 |
847-916 |
Sentence |
denotes |
The hexamers anneal themself to aleatory parts of the ssDNA sequence. |
T99 |
917-985 |
Sentence |
denotes |
These hexamers work as initiation sites for the ϕ29 DNA polymerases. |
T100 |
986-1100 |
Sentence |
denotes |
After denaturation at 95 °C, the mixture is cooled down to ice temperature and the rest of the reagents are added. |
T101 |
1101-1301 |
Sentence |
denotes |
The mixture is heated up to ~30 °C so the polymerase starts to complete the complementary ssDNA sequence, creating again a dsDNA strand, eventually it encounters a hexamer from another annealing site. |
T102 |
1302-1439 |
Sentence |
denotes |
Once this happens the polymerase will lift up that hexamer and starts to separate the amplified sequence formed from that annealing site. |
T103 |
1440-1537 |
Sentence |
denotes |
As the polymerase displaces the formed strand ahead of it, it continues to complete the sequence. |
T104 |
1538-1757 |
Sentence |
denotes |
The displaced strand becomes a new ssDNA strand and therefore, it gives new sites for more primers to attach and initiation sites for the polymerase, continuing the amplification, and thus creating a web of DNA strands. |
T105 |
1758-1844 |
Sentence |
denotes |
Finally, the inactivation of the polymerase is done by heating up the system to 65 °C. |
T106 |
1845-2089 |
Sentence |
denotes |
Even though MDA is considered an isothermal process, prior to the reaction and to the addition of most reactants, the dsDNA and a buffer are heated up to 95 °C to denature the dsDNA to ssDNA and to give hexamers the initial access to the ssDNA. |
T107 |
2090-2170 |
Sentence |
denotes |
After the amplification reaction, the polymerase has to be inactivated at 65 °C. |
T108 |
2171-2314 |
Sentence |
denotes |
However, this does not require fast temperature changes, as would be the case with, for example, the temperature cycling in PCR amplifications. |
T109 |
2315-2531 |
Sentence |
denotes |
This, together with the robustness of the amplification (it is a self-limiting reaction that amplifies all present DNA [50]) makes MDA perfectly suitable as proof-of-principle amplification reaction for such devices. |