Polymerase Chain Reaction

The PCR technique was used for amplification of hOAT3 cDNA. Polymerase Chain Reaction is the enzymatic method of making multiple copies of pre-selected segment of DNA. The basic components of the reaction are: the cDNA template, containing the sequence of interest; two synthetic oligonucleotide primers, designed with homology to the 5’ and 3’ ends of the target sequence; four deoxyribonucleoside triphoshpates (dNTPs); thermostable DNA polymerase and the buffer, usually containing Mg²⁺. The reaction mixture are undergoes the temperature cycling. Cycling begins with heating to 95°C, with is required for the denaturation of the DNA template. Then it is rapidly cooled to the temperature necessary for the annealing of the oligonucleotide primers to their target on the single-stranded template. The primers annealing temperature (Tm) depends on their length and nucleotide base composition. After annealing, the temperature goes to 72°C, optimal for the functioning of DNA polymerase, which extends the primers at their 3’-ends. Since the 3’-ends of the primers directed towards each other, repeated cycles of heating and cooling lead to the chain reaction, an exponential synthesis of many copies of the specific segment of interest. After cycling,

the PRC results usually visualized by an agarose gel electrophoresis, purified with the help of adequate PCR-purification kits and analyzed by sequencing.

3.1.3.1 Standard RT-PCR

For amplification of hOAT3 cDNA fragments, primers were designed on the basis of the published human sequence (nucleotides 1 to 2179, GenBank accession number AB042505). For the primer design, the regions were chosen that indicated both greatest homologies between OAT3s from different species whilst at the same time maximal divergence from the OAT1s.

Reagents used

Template 1 µl of cDNA, reverse transcribed with Oligo dT Primer

Forward Primer 20 pmol

Reverse Primer 20 pmol

5x PCR Buffer 2 µl (50 mM KCl, 10 mM Tris, pH 9.0, 1.5 mM MgCl₂, 0.1% Tritone-X-100)

dNTPs 200 µM of each

DNA Polymerase Takara Tag, 1U

Nuclease-free H₂0 To a final volume of 20 µl

The thermo-cycling parameters were: 94°C for 1 minute followed by 15 cycles with comprised: 94°C for 30 seconds, 61°C (-0.4°C per cycle) for 45 seconds, and 72°C for 1 minute; followed by 15 cycles of: 94°C for 30 seconds, 55°C for 45 seconds, and 72°C for 1 minute; and finally 72°C for 7 minutes. The PCR fragments obtained were purified and cloned into pCR^TMII and sequenced.

3.1.3.2 Rapid amplification of cDNA ends (RACE)

The RACE (rapid amplification of cDNA ends) technique was used to amplify 5’ and 3’-ends of hOAT3 cDNA. The basic principles of this method are following. From the already known internal stretch of sequence gene-specific primers are chosen, that are oriented in the direction of the missing sequence. Extension of the partial cDNAs from the unknown end of the message back to the known region is achieved using primers that anneal to the preexisting poly(A) tail (3’-end) or an appended homopolymer tail (5’-end).

3’ RACE

To generate “3’ end” partial cDNA clone, mRNA was reverse transcribed using the universal RACE “anchor” primer (QoQi-dT) that consists of 17 nucleotides of oligo(dT)

followed by a unique 35-base oligonucleotide sequence (QoQi; table 2.2). Amplification was than performed using a primer containing part of the “anchor” sequence (Qo) and a primer derived from the gene of interest gene specific primer 1 (GSP1). To quench the amplification of nonspecific products, a second set of amplification cycles was then carried out using “nested” primers Qi and GSP2.

Reagents used

First amplification set Nested amplification set Template cDNA, reverse transcribed

with QoQi-dT primer

1 µl initial PCR reaction

Primer Q_O, 200 ng Qi, 200 ng

Gene-Specific Primer GSP1, 200 ng GSP2, 200 ng 5x PCR Buffer

dNTPs 200 µM 200 µM

DNA Polymerase Takara Tag, 1U Takara Tag, 1U

For amplification of 3’-end of hOAT3, in the first nest reaction as primers were used universal RACE primer Qo and GSP1 (3’RACE-hOAT3(1240)F primer, specific for hOAT3). Thermo-cycling conditions for first stage PCR were: 35 cycles of 94°C for 20 seconds, 65°C for 30 seconds and 72°C for 1.5 minute. For the second nested PCR were used universal “nested” RACE primer Qi and GSP2 (3’RACE-hOAT3(1519)F primer, specific for hOAT3). 1 µl of initial PCR reaction was used as a template for the

“nested” reaction. The cycling conditions were the same as used for the initial stage.

The product of reaction was cloned into pCR^TMII vector and sequenced.

5’ RACE Reagents used

Synthesis of poly(A) tail

Template 50ng cDNA, reverse-transcribed with random hexanucleotides

dATP 200 µM

Terminal Transferase 15 U

RNase-free Water To 20 µl of total volume

First amplification set Nested amplification set Template Tailed first-strand cDNA, 1 µl initial PCR reaction Primer QoQi-dT, 20 ng and Q_O, 200 ng Qi, 200 ng

Gene-Specific Primer So, 200 ng Si, 200 ng 5x PCR Buffer

dNTPs 200 µM 200 µM

DNA Polymerase Takara Tag, 1U Takara Tag, 1U

To isolate the 5’ end of a cDNA, reverse transcription was carried out using random hexanucleotides (Invitrogen^TM life technologies) to generate first-strand product. Then, a poly(A) tail was appended using terminal deoxynucleotidyltranserase (TdT) and dATP. Amplification was then achieved using the hybrid “anchor” primer (QoQi-dT) to form second strand of cDNA, the Qo primer and the gene specific primer oriented in the 5’-end direction (So). Finally, a second set of PCR cycles was carried out using nested primers (Qi and Si) to increase specificity. The conditions for the tailing reaction were: 10 minutes at 37°C and then 15 minutes at 65°C, necessary for inactivation of enzyme. In the first stage reaction the primers used were: “anchor” primer QoQi-dT, universal RACE primer Qo and gene-specific primer So (5’RACE-hOAT3(383)R, specific for hOAT3). Thermo-cycling conditions for first stage PCR were: 3 cycles of 94°C for 20 seconds, 42°C for 2 minutes and 72°C for 3 minutes; followed by 35 cycles of 94°C for 20 seconds, 65°C for 20 seconds and 72°C for 1.5 minutes. The second

“nested” PCR was carried out with 1 µl of initial PCR reaction, universal “nested” RACE primer Qi and sequence-specific primer Si (3’RACE-hOAT3(511)R, specific for hOAT3). The cycling conditions were: 35 cycles of 94°C for 20 seconds, 65°C for 20 seconds and 72°C for 1.5 minutes. The product of reaction was cloned into pCR^TMII vector and sequenced.

3.1.3.3 Amplification of full-length clone

Based on the RT-PCR data, sequence-specific primers (with restriction sites incorporated) were designed for PCR- amplification of the full-length hOAT3 clone from human kidney cDNA. The amplification reaction composition was as for usual PCR.

The template was cDNA synthesized using the “anchor” (QoQi-dT) primer. The thermocycling parameters were: 94°C for 2 minutes; 10 cycles of 94°C for 20 seconds, 58°C for 30 seconds and 72°C for 2 minutes; followed by 20 cycles of 94°C for 20 seconds, 58°C for 30 seconds, and 72°C for 2 minutes + 5 seconds per each next cycle; followed by a final extension period of 10 minutes at 72°C. The required extension time was empirically calculated from the expected product length. The full-length cDNA was cloned into pCR^TMII vector and sequenced.