PCR and the preparation of monodisperse DNA samples . 35

With the PCR high quality both ends modified double-stranded DNA (dsDNA) can be synthesized. At the starting point of the PCR procedure the dsDNA is briefly heated so that it melts and separates in to two single-stranded DNA molecules (ssDNA). After the strands have been separated the solution is cooled down to a temperature where a short ssDNA sequence called a primer⁷, which are in large excess in the solution, hybridizes to the matching complementary base-pair combination at the long ssDNA (annealing). A protein called Taq polymerase, which is also in large excess in the solution with the four types of

5Cy3 has its absorbtion maximum at 552 nm and emission maximum at 565 nm.

6GenePix Personal 4100A, Molecular Devices.

7Primers are typically some 20−30 base-pairs long.

Laser

CPU PMT

Objective and scanner Filter

(a) (b)

Figure 3.2: a) The principle of the microarray reader: A laser excites the fluo-rophores found at the sample and the emitted fluorescence is collected, filtered and directed to the PMT. By scanning the sample slide a full picture of the sam-ple can be made. b) Typical sample of ours: a plastic tape was glued on top of a glass slide as a mask and then on the spots Cy3-modified DNA and free oligo mixture was left to dry.

deoxyribonucleosides, starts from the location where the primer is hybridized at the ssDNA and builds the complementary DNA strand out of the four deoxyri-bonucleoside triphosphates. The first cycle is ready when the polymerase has finished its work and the cycle starts once again from the start and the newly synthesized fragments serve as templates for the next round. In practice in the PCR program must be enough time reserved for the Taq polymerase to finish the synthesis (extension). This parameter depends naturally strongly of the length of the desired product. In each cycle the amount of dsDNA is doubled and typically some 20−30 cycles are done [14]. The principle of the PCR is also presented in the Fig. 3.3.

The primers are commercially available with the same modifications as oligos (biotin, amino- and thiol-group) but with the advantage that when a product is successfully gained it has in both of its ends a primer with modification which needs not be the case when modifying λ- DNA with oligos. In other words when a primer does not bind then there is no starting point for the polymerase and therefore there is no product. Another question is of course if every primer really carries the modification.

The work with PCR was started by producing first short 2 kbp pieces of DNA modified only from one end⁸. As a first step a suitable template and primers⁹

8The 2 kbp DNA sample is used in chapter 6.

9End-modified primer: biotin thiol- or amino-group-5⁰-gct gcg cgt aac cac cac acc-3⁰ and unmodified primer: 5⁰-ctg cgg cca act tac ttc tga caa-3⁰.

3`

DNA synthesis is ready and the cycle can start again

Figure 3.3: The principle of PCR: First the dsDNA is melted by a brief heat treatment. In the subsequent step the temperature is lowered allowing primers, which are in large excess in the solution, to hybridize to complementary sequences on the melted ssDNA strands. After that Tag polymerase (P) synthesizes the complementary strand from the four deoxyribonucleoside triphosphates (also in large excess in the solution). After the polymerase has finished its work the cycle can start again by heating up the solution and melting DNA. As in the following cycles the newly synthesized DNA strands (primer limited) will serve as a template, the DNA which is between the primers will only be copied. This leads to the fact that after several cycles essentially all the DNA have a unique length limited by primers. The end-modifications M₁ and M₂ are introduced to DNA by using end-modified primers. The both ends modified DNA is finally created when the single stranded end-modified DNA molecules are hybridized together (this step is not shown). The letters3⁰ and 5⁰ show the direction of the backbone.

Table 3.1: The composition of a single vial in order to produce 2 kbp DNA with PCR. This DNA was modified only from one end and therefore the other primer is unmodified. We used either biotin, thiol- or amino-group as a end-modification depending on the experimental need.

must be chosen. For 2 kbp pieces the template was a plasmid called Bluescript 2KS which is 2961 base-pairs long. The protocol itself started by placing plastic vial for the PCR solution in a cool metal rack. We pipetted first a stock solution into the cooled vial which was later on divided into smaller vials. The content of each vial is presented in Table 3.1. The Taq polymerase was pipetted into the vials just before putting them in to the ”PCR machine”. In the ”PCR machine” repeated steps of cooling and heating can be performed after a user defined program. We used a program where in the first step the PCR solution was left for 5 minutes at 94^◦C in order to activate the Taq polymerase followed by 35 cycles of repeated heating and cooling where one cycle consisted of 15 seconds at 94^◦C, 30 seconds in 56^◦C and 1 minute at 72^◦C respectively. After the 35 cycles the solution was kept for 7 minutes at 72^◦C and after that the program was finished and the product was kept at 4^◦C.

As with 2 kbp pieces of DNA also with 20 kbp pieces a suitable template and primers¹⁰ must be chosen. For 20 kbp pieces we use λ - DNA as a template.

10Direct primer: biotin-5⁰-ctg atg agt tcg tgt ccg tac aac tgg cgt aat c-3⁰ and reverse primer:

thiol-5⁰-gtg cac cat gca aca tga ata aca gtg ggt tat c 3⁰. For the unmodified 20 kbp DNA the sequence is the same but without end modifications.

Table 3.2: The composition of a single vial in order to produce biotin- and thiol-modified20 kbp DNA with PCR.

Reagent Quantity Stock

The protocol for synthesizing 20 kbp biotin- and thiol-modified DNA is shown in the Table 3.2. Here, again the polymerase was pipetted into the vials just before putting them in to the ”PCR machine”. With 20 kbp we used a program where in the first step the PCR solution was left for 2 minutes at 94^◦C in order to activate the polymerase. After the activation followed 10 cycles of repeated heating and cooling where one cycle consists of 20 seconds at 94^◦C and 14 minutes at 68^◦C.

After the first 10 cycles came additionally another 15 cycles which were otherwise the same but here to each cycle of the 14 minutes at 68^◦C were 10 seconds added.

Finally in the end of the program the solution was kept for 10 minutes at 72^◦C and after the program was finished the product was kept at 4^◦C.

The PCR products were proofed with gel electrophoresis, as described in context with FIGE with the difference that here we did not need pulsed field but constant field was already enough to separate the product. The length of the PCR product was checked with a marker, which length distribution is known, by letting it to run at a neighboring lane. After the gel electrophoresis the agarose gel with DNA was loaded with ethidium bromide for 10 minutes, washed and

looked under UV light. The 2 kbp long DNA was further processed by cutting it out of the gel and purifying it with Qiagen MinElute PCR purification kit according to manufacturers instructions. The purification with the kit is based on the binding of nucleic acids on a silica gel membrane at high salt buffer and elution at low salt buffer and so all salts, agarose, ethidium bromide, and other impurities from the DNA sample can be removed.

The biotin- and thiol-modified 20 kbp DNA could also be purified by cutting it out of the gel and so to ensure that the only DNA the sample contains is really the 20 kbp DNA. The yield of the DNA when purified out of gel is, however, low and therefore we purified DNA directly out of the ready PCR solution. This was done with the Qiagen QIAquick^r PCR purification kit according to manufacturers instructions. The purification with this kit is also based to the binding of nucleic acids on the silica gel membrane at high salt buffer and elution at low salt buffer.

3.3 Results and discussion