4.3 General molecular biological methods
4.3.4 Polymerase chain reaction (PCR)
The polymerase chain reaction (PCR) allows in vitro synthesis of large amounts of DNA by sequence-specific polymerization of nucleotide triphosphates catalyzed by DNA polymerase (Mullis et al., 1986). The polymerization reaction is “primed” with small oligonucleotides that anneal to the template DNA strand through base pairing, giving the reaction its specificity by defining the borders of the segment to be amplified. Standard applications of PCR reactions are explained in the following and are used unless otherwise mentioned. More specialized applications are explained in more detail within the specific method.
4.3.4.1 Primer design
Unless otherwise mentioned sequences for generating primers were extracted using the UCSC Genome Browser. In general primers were designed using PerlPrimer Software and controlled using PCR and BLAT functions of the UCSC Genome Browser and GeneRunner Software. Following settings were used to design primers:
Primer Tm: 65-68°C
Primer length: 18-28 bp
Amplicon size: 80-150 bp
4.3.4.2 Standard PCR for cloning or sequencing of gDNA
PCRs were generally performed in “thick” PCR tubes with a reaction volume of 20-100 µl in a MJ research PTC 200 thermocycler (Biozym). The "calculated temperature" feature was used to decrease temperature hold times and additionally the lid was heated to 105°C to prevent vaporisation. The nucleotide sequences of the utilized primers are given in section 3.6. The primer annealing temperatures varied between 57 and 65°C. For a typical reaction the PhusionTM Hot Start High-Fidelity DNA Polymerase (Finnzymes; Espoo, Finnland) was used with the following basic reaction conditions:
Component Volume Final concentration
H2O Add 50 µl
5×Phusion HF buffer 10 µl 1×
10 mM dNTPs 1 µl 200 µM each
Primer S 1 µl 0.2 µM
Primer AS 1 µl 0.2 µM
Template DNA X µl
Phusion Polymerase (2 U/ml) 0.5 µl 0.02 U/µl
General parameter settings for analytical PCR are summarized in Table 4-5 Reaction parameter for analytical PCR.
Table 4-5 Reaction parameter for analytical PCR
Quantitative real-time PCR (qPCR) enables both detection and quantification (as absolute number of copies or relative amount when normalized to DNA input or additional normalizing genes) of a specific sequence in a DNA sample. The procedure follows the general principle of polymerase chain reaction; its key feature is that the amplified DNA is quantified as it accumulates in the reaction in real-time after each amplification cycle. On the one hand, this method was used to quantify cDNA after reverse transcription (see section 4.3.5.3), on the other hand to quantitatively analyze genomic DNA after fragmentation by methyl-CpG immunoprecipitation (MCIp, see section 4.4.4) or after chromatin immunoprecipitation. PCR reactions were performed using the Quantifast SYBR Green Kit from Qiagen either in glass capillaries using the LightCycler system from Roche (total volume: 20 µl) or in 96-well format adapted to the Eppendorf Realplex Mastercycler EpGradient S (Eppendorf, Hamburg, Germany). The relative amount of amplified DNA is measured through the emission of light by the SYBR green dye when it is intercalated in double-stranded DNA.
Reaction setup: 5 µl SYBR Green mix (2×)
To calculate amplification efficiency, a standard curve was generated for each primer pair by amplifying four dilutions (1:10, 1:50, 1:100, 1:1000). Realplex software calculated automatically DNA amounts based on the generated slope and intercept. Specific amplification was controlled by melting-curve analysis and data was imported and processed in Microsoft Excel 2007. All samples were measured in duplicates and normalized to the ß-actin or the HPRT housekeeper when analyzing mRNA expression. Duplicates of ChIP samples were normalized to the input or a not affected upstream control region.
4.3.4.4 MassARRAY quantitative gene expression (QGE) analysis
4.3.4.4.1 Principle
The MassARRAY QGE method combines real-competitive PCR (rcPCR) with the iPLEX primer extension reaction, followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). cDNA or MCIp-enriched DNA is spiked with a competitor, which matches the sequence of the targeted cDNA region in all positions except a single base and serves as an internal standard. DNA and competitor are PCR-amplified and then SAP (shrimp alkaline phosphatase) -treated to dephosphorylate remaining nucleotides. After SAP inactivation, a primer extension cocktail is added. The PCR products from the competitor and the cDNA now serve as templates for the iPLEX reactions.
After primer extension, the products are desalted using clean resin and then dispended on a SpectroCHIP for subsequent MALDI-TOF MS. During mass spectrometric analysis, the peak areas of the distinct mass signals for the competitor and DNA extension products are resolved and peak area ratios are calculated. The QGE Analyzer software plots cDNA frequency versus competitor concentration for each assay and sample. DNA concentrations (expressed as LOGEC50 or EC50) are automatically calculated via non-linear regression analysis and represent the competitor concentration at which the allele frequencies of cDNA and competitor are equal (0.50:0.50). A workflow for conducting MassARRAY QGE experiments is shown in Figure 4-1. Detailed description of the method is given in the MassARRAY QGE-iPLEX Application guide (www.sequenom.com).
Figure 4-1 Schematic outline of the MassARRAY QGE process
cDNA or MCIp-enriched DNA is spiked with a synthetic DNA molecule (competitor), which matches the sequence of the targeted cDNA region in all positions except a single base and serves as internal standard. The cDNA/competitor is PCR-amplified and subjected to a SAP treatment. After inactivation of the SAP, a primer extension cocktail is added. The PCR products from the competitor and the cDNA now serve as templates for the iPLEX reactions. After primer extension, the products are desalted using clean resin and then dispended on a SpectroCHIP for subsequent MALDI-TOF MS (www.sequenom.com).
4.3.4.4.2 Protocol
Primer and competitive template designs were created using the MassARRAY QGE Assay Design software v1.0 (Sequenom, San Diego, CA). Preparing of the competitor plates, PCR, SAP addition, iPLEX reaction, desalting of the iPLEX reaction and MALDI-TOF analysis were performed as described in the Sequenom protocols. Raw data were then processed using the MassARRAY QGE Analyzer software v3.4.
4.3.4.5 Nested PCR for quantitative methylation analysis
Methylation analysis of specific DNA fragments was performed using a nested PCR after bisulfite treatment of genomic DNA (see section 4.4.3). 10 µl of bisulfite-treated DNA were
used for the first nested PCR reaction, generated with an outer primer pair. Afterwards the PCR products are used as a template for a second PCR using a different set of primers inside of the first PCR product (inner primer pair). The reaction was performed as follows:
Components 1st PCR 2nd PCR