DNA amount
II.4. W ORKING WITH DNA AND RNA
II.4.5. A PPLICATION OF POLYMERASE CHAIN REACTION (PCR)
Polymerase chain reactions (PCRs) are a potent tool to detect, analyze, amplify, and modify specific DNA sequences in vitro by amplification reactions using DNA polymerases. Within this thesis, PCRs were used for different purposes, such as cloning, mutagenesis, transcript analysis, and sequencing. All cycling reactions, covering denaturation, annealing, and elongation steps, were conducted with a RoboCycler 96 (Stratagene) PCR machine.
AM P L I F I C A T I O N O F DNA SE QU E N C E S F O R C L O N I N G
The amplification of DNA sequences necessary for cloning was performed in 50 μl reactions using the Phusion High‐Fidelity DNA Polymerase (NEB). Due to its 3’5’‐exonuclease activity, the Phusion DNA polymerase possesses proof‐reading activity and enables high accuracy within the DNA amplification. The reactions contained either 20‐300 ng of plasmid DNA or 50‐70 ng of cDNA as template, dNTPs in a final concentration of 200 μM each, the respective forward (FW, for) and reverse (RV, rev) primers in a final concentration of 200 nM each, and 1 U of the Phusion High‐Fidelity DNA Polymerase (NEB) in 1x Phusion HF Buffer (NEB). The DNA was amplified in 38 cycles running the PCR program listed in Table II‐5.
Table II‐5: PCR program using the Phusion High‐Fidelity DNA Polymerase.
98 °C 30‐60 sec 98 °C 20‐30 sec
38 cycles TM+3 °C 30‐45 sec
72 °C 30 sec/kb 72 °C 5 min
TM, highest salt adjusted melting temperature of primers (given by Metabion).
The PCR products were either immediately employed for downstream processes (analysis, purification, cloning) or stored at ‐20 °C.
SI T E‐S PE C I F I C M U T A G E N E S I S OF P L A S M I D DNA
Two different set‐ups were used for site‐specific single nucleotide exchanges in the coding sequences in plasmids. In both cases, the mutation was generated by the amplification of complete plasmids via PCR. For this, complementary primer pairs carrying the mutation in the middle of the sequence were used.
To insert a mutation in the Ensembl AKR1B15‐201 (ENST00000423958.1) transcript sequence encoding for AKR1B15.1 which results in the AKR1B15.1 S8R mutant, a protocol derived from Sawano & Miyawaki [217] was followed. The 50 μl reactions contained 50 ng of the wild type plasmid DNA template, dNTPs in a final concentration of 200 μM each, the 5’‐phosphorylated mutagenesis primer pair # 2680 and # 2681 in a final concentration of 300 nM each, as well as 1 U of the Phusion High‐Fidelity DNA Polymerase (NEB), necessary for the amplification of the plasmid DNA strand after primer annealing, and 20 U of the Taq DNA Ligase (NEB), necessary for the ring closure of plasmid DNA. In order to provide optimal buffer conditions, the reactions were carried out in a mixture of 0.5x Phusion HF Buffer (NEB) and 0.5x Taq DNA Ligase Buffer (NEB). The PCR program applied for the amplification of mutated plasmids is shown in Table II‐6.
Table II‐6: PCR program for site‐specific mutagenesis of AKR1B15.1 according to Sawano & Miyawaki [217].
65 °C 5 min 98 °C 2 min 98 °C 30 sec
17 cycles 62 °C 30 sec
65 °C 5.5 min 72 °C 7 min
Subsequently, 20 U of DpnI (NEB) were added to the mutagenesis reactions and the reactions were incubated at 37 °C for 90 min. In doing so, DpnI digested the methylated wild type plasmid DNA template which was originally purified from in E. coli.
After the DpnI treatment, potentially arisen strand breaks in the mutated plasmids were repaired by two PCR cycles [Table II‐7].
Table II‐7: PCR program for strand break repair after mutagenesis.
98 °C 2 min 98 °C 30 sec
2 cycles 60 °C 1 min
72 °C 7 min
Finally, 10 μl of the mutagenesis reactions were transformed into chemically competent E. coli DH5alpha (see II.1.2).
For the mutagenesis of the N‐terminal sequence in AKR1B10 fused to AcGFP the QuikChange Lightning Site‐Directed Mutagenesis Kit (Agilent) was used. According to the manufacturer’s instructions, 50 ng of the wild type pAcGFP‐AKR1B10 (Met1‐Ala38) plasmid DNA template, the respective mutagenesis primer pair (# 2919 + # 2920 or # 2921 + # 2922) in a final concentration of 200 nM each, 1 μl of the provided dNTP mix, as well as 1.5 μl of the QuikSolution reagent were mixed in 50 μl 1x reaction buffer before 1 μl of the QuikChange Lightning Enzyme was added. The mutagenesis was carried out using the slightly modified
Colony screen PCRs served for the identification of potentially “positive E. coli clones”
resulting from cloning reactions and were performed in 22 μl reactions. 20 μl of a master mix, containing dNTPs in a final concentration of 200 μM each, the respective forward and reverse primer pair in a final concentration of 250 nM each, and 0.5 μl of a lab‐made Taq polymerase in 1.1x Standard Taq buffer, were added to 2 μl of the colony screen cultures. The amplification was conducted with 30 cycles using the PCR program listed in Table II‐9.
Afterwards, PCR products were analyzed via agarose gel electrophoresis [II.4.6].
DE T E CT I O N O F AKR1B15 T R A N S C R I P T S IN CDNA S A M P L E S
In order to determine the abundance of AKR1B15 splice variants (Ensembl transcripts AKR1B15‐001 [ENST00000457545.2] and AKR1B15‐201 [ENST00000423958.1]; referred to as AKR1B15.2 and AKR1B15.1, respectively) in comparison to that of the AKR1B10 transcript in different human tissues as well as cell lines, semi‐quantitative end‐point RT‐PCR was carried out. The 20 μl reactions included 50 ng of the respective cDNA sample, dNTPs in a final concentration of 200 μM each, the transcript specific primer pairs [Table II‐10, Figure III‐1, VI.4] in a final concentration of 250 nM each, and 1.25 U of the commercial available DreamTaq DNA Polymerase (Thermo Scientific) in 1x DreamTaq Green Buffer (Thermo Scientific). The amplification of human GAPDH (hGAPDH) served as control [Table II‐10].
Table II‐10: Transcript specific primer pairs used for semi‐quantitative RT‐PCR.
transcript primer pair TM product size AKR1B15.2 AKR1B15.2‐TK‐FW (# 2715)
The PCR programs applied for the amplification of the human AKR1B15 and AKR1B10 transcripts (hAKR1B’s) or hGAPDH are listed in Table II‐11.
The PCR products were analyzed by gel electrophoresis using 2.5 % agarose gels. In addition, the specificity of AKR1B15 primer pairs was verified by Sanger sequencing of the respective PCR products.
SE Q U E N C I N G O F DNA
The sequencing of plasmid DNA was conducted in‐house. To enable the in‐house DNA sequence analysis according to Sanger [218, 219], sequencing PCR reactions were set up with the BigDye3.1 Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). For this, 1 μl of plasmid DNA (≥ 150 ng/μl) or 2 μl of plasmid DNA (≤ 150 ng/μl) were mixed with 1 μl of the respective primer (10 μM), 1 μl of 5x Buffer (Applied Biosystems), 1 μl of BigDye Terminator v3.1 Ready Reaction Mix (Applied Biosystems), and MilliQ‐H2O to a reach final volume of 5 μl. Afterwards, the PCR program listed in Table II‐12 was applied resulting in single‐
stranded fluorophore‐labeled DNA amplicons.
The PCR products were purified via the Montage SEQ96 Sequencing Reaction Cleanup Kit (Merck Millipore) according to the manufacturer’s instructions and, finally, analyzed by an ABI 3730 DNA Analyzer (Applied Biosystems).
II.4.6. A
NALYSIS OR PURIFICATION OFDNA
VIA AGAROSE GEL ELECTROPHORESISPCR products and restriction digestions of DNA were analyzed or separated for downstream purification by agarose gel electrophoresis. For this, the DNA samples were mixed with an appropriate volume of 6x loading‐dye and loaded on agarose gels containing either 0.025 ‰ (v/v) Midori Green Advance DNA stain (Nippon Genetics) or 0.1 ‰ (v/v) ethidium bromide solution (500 μg/ml, Sigma‐Aldrich). Depending on the size of DNA fragments and purpose, 1‐2.5 % (w/v) agarose in 1x TBE buffer gels were applied [Table II‐13].
Table II‐13: Percentages, application, and run time of agarose gels.
agarose gel fragment / purpose run time
1 %
The gels were run in a (Mini‐)Sub‐Cell GT (Bio‐Rad) horizontal electrophoresis chamber filled with 1x TBE buffer at 100 mA for 20‐60 min. Afterwards, the separated DNA was visualized upon UV illumination using a Bio‐Vision Gel Documentation system (PeqLab).
When DNA was purified via agarose gels, only 70 % UV‐light intensity were used for the visualization of DNA bands. Appropriate bands were cut from the gel and the gel slices subjected to the “Purification of linear DNA” [II.4.7] using the Wizard SV Gel and PCR Clean‐Up System (Promega).