Chapter 2. Materials and Methods
2.1 General techniques
2.1.2 Methods
2.1.2.2 Polymerase chain reaction (PCR)
Various PCR strategies were used to synthesize and clone the different target genes (Figure 2.1). The Expand High Fidelity PCR System (Roche) was used to perform PCR reactions.
This system contains the thermostable Taq DNA Polymerase and the thermostable Tgo DNA polymerase, which provides proofreading activity (Expand High fidelity enzyme mix). The corresponding buffer system (10 x Expand buffer) contains MgCl2 at a final concentration of 15 mM. Resulting final PCR products were purified using gel electrophoresis (see section 2.1.2.10).
Strategy A:
DNA fragments encoding the various target genes were amplified from either plasmid DNA or human cDNA library using PCR. Specific restriction sites used for further cloning procedures were incorporated in both PCR oligonucleotides at the 5’ ends (Figure 2.1A). 5-10 cycles with a partial annealing temperature (Tpa) were performed to allow initial binding of the primers to the template. A subsequent step of 15-20 cycles using the full annealing temperature (Tfa) was carried out for full binding of the primers to the template. The following conditions were used for a 50 μl reaction:
Reaction:
Reagent Final concentration Volume
Template DNA 0.1–200 ng Variable
10 x Expand buffer with 15 mM MgCl2 1 x 5 μl 10 mM dNTP mix 200 μM of each dNTP 4 μl
Top strand primer (10 pmol/μl) 200 nM 1 μl Bottom strand primer (10 pmol/μl) 200 nM 1 μl
Expand High fidelity enzyme mix 1.3 U/reaction 0.375 μl Sterile dH2O Add up to 50 μl Settings:
Temperature Time Cycles
Initial Denaturation 95˚C 5 min 1 x Denaturation
Partial annealing Elongation
95˚C 45-60˚C
72˚C
30 s 30 s 1 min
5–10 x Denaturation
Full annealing Elongation
95˚C 55-70˚C
72˚C
30 s 30 s 1 min
15–25 x Final Elongation 72˚C 5 min 1 x
Cooling 4˚C Unlimited time 1 x
Strategy B:
For de novo synthesis of the template, various overlapping oligonucleotides (P1, P2, P3, P4, P5 and P6) were annealed using the following conditions for a 50 μl reaction (Figure 2.1B):
Reaction:
Reagent Final concentration Volume
10 x Expand buffer with 15 mM MgCl2 1 x 5 μl 10 mM dNTP mix 200 μM of each dNTP 4 μl
Primer P1–P6 (10 pmol/μl) 200 nM of each primer 1 μl of each primer Expand High fidelity enzyme mix 1.3 U/reaction 0.375 μl
Sterile dH2O Add up to 50 μl Settings:
Temperature Time Cycles
Initial Denaturation 95˚C 5 min 1 x Denaturation
Primer annealing Elongation
95˚C 45-55˚C
72˚C
30 s 30 s 1 min
10 x Final Elongation 72˚C 5 min 1 x
Cooling 4˚C Unlimited time 1 x
The annealed oligonucleotides were purified using a standard QIAgen PCR purification kit and eluted with 50 μl of 0.1 x elution buffer (EB buffer). The resulting product was then amplified with a pair of external oligonucleotides (P1 and P6) using the following conditions (Figure 2.1B):
Reaction:
Reagent Final concentration Volume
Template DNA variable 5 μl
10 x Expand buffer with 15 mM MgCl2 1 x 5 μl 10 mM dNTP mix 200 μM of each dNTP 4 μl
Top strand primer (10 pmol/μl) 200 nM 1 μl Bottom strand primer (10 pmol/μl) 200 nM 1 μl
Expand High fidelity enzyme mix 1.3 U/reaction 0.375 μl Sterile dH2O Add up to 50 μl
Settings:
Temperature Time Cycles Initial Denaturation 95˚C 5 min 1 x
Denaturation Full annealing
Elongation
95˚C 55-70˚C
72˚C
30 s 30 s 1 min
20 x Final Elongation 72˚C 5 min 1 x
Cooling 4˚C Unlimited time 1 x
Strategy C:
A three step PCR approach was applied to create target genes consisting of two different fragments that are fused together. In the first step (step 1), the 2 fragments of interest were individually amplified using standard PCR conditions as described below. Primer P2 and P3 carried a common region at the 5’ end (red box in Figure 2.1C) which was used for the subsequent fusion PCR step (Step 2). In a final step (Step 3), the resulting PCR fusion product was amplified with a pair of external oligonucleotides (P1 and P4).
Step 1 was performed as described in Strategy A. Both amplified fragments were then purified using a standard QIAgen PCR clean up kit and eluted with 50 μl of 0.1 x EB buffer.
To anneal the resulting products together the following conditions for a 50 μl reaction were used (Step 2):
Reaction:
Reagent Final concentration Volume
PCR Fragment 1 Variable 5 μl PCR Fragment 2 Variable 5 μl
10 x Expand buffer with 15 mM MgCl2 1 x 5 μl 10 mM dNTP mix 200 μM of each dNTP 4 μl
Expand High fidelity enzyme mix 1.3 U/reaction 0.375 μl Sterile dH2O Add up to 50 μl
Settings:
Temperature Time Cycles Initial Denaturation 95˚C 5 min 1 x
Denaturation Full annealing
Elongation
95˚C 55-70˚C
72˚C
30 s 1 min 1 min
4 x Final Elongation 72˚C 5 min 1 x Cooling 4˚C Unlimited time 1 x
The resulting PCR fusion product was purified using a standard QIAgen PCR purification kit, eluted with 50 μl of 0.1 x EB buffer and amplified with a pair of external oligonucleotides (P1 and P4) as described in Strategy B (Step 3).
Strategy D:
To introduce site directed mutations into the target sequence, a three step PCR approach was applied similar as described in Strategy C (Figure 2.1D). In the first step, the left and right cassette of the target gene was individually amplified using standard PCR conditions.
Overlapping sequence regions containing the desired point mutation were used as internal primers (P2 and P3) for the subsequent fusion PCR step. In a final step, the resulting PCR fusion product was amplified with a pair of external oligonucleotides (P1 and P4). All 3 steps were performed as described in Strategy C. Note that all constructs harboring single point mutations were generated using this strategy if not otherwise stated.
RS I
Figure 2.1 Scheme of PCR strategies used to synthesize and amplify target genes. (A) DNA fragments encoding the target gene were amplified from available template DNA. Specific restriction sites (RS I and RS II) used for further cloning procedures were incorporated in both PCR oligonucleotides (P1 and P2) at their 5’
ends. (B) For de novo synthesis of the target gene, various overlapping oligonucleotides (P1, P2, P3, P4, P5 and P6, overlapping region depicted as red lines) were annealed together and amplified with a pair of external oligonucleotides (P1 and P6). (C) A target gene composed of two different fragments was generated in 3 steps.
The individual fragments (left and rightcassette) were amplified using standard PCR (as described in A). Primer P2 and P3 carried a common region at the 5’ end (red box) which was used to fuse the two fragments together.
The resulting fusion product was amplified with a pair of external oligonucleotides (P1 and P4). (D) To introduce site directed point mutations into the target sequence, a left and a right cassette of the target gene were individually amplified. Overlapping sequence regions containing the desired mutation (indicated by red X) were incorporated in the internal primers (P2 and P3) to subsequently fuse the two cassettes together. In a final step, the resulting PCR fusion product was amplified with a pair of external oligonucleotides (P1 and P4).