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Jul 2017

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High-throughput Site-directed Scanning Mutagenesis Using a Two-fragment PCR Approach
利用双片段PCR法进行高通量定点扫描突变   

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Abstract

Site-directed scanning mutagenesis is a useful tool applied in studying protein function and designing proteins with new properties, such as increased stability or enzymatic activity. Creating a systematic library of hundreds of site-directed mutants is still a demanding and expensive task. The established protocols for making such libraries include PCR amplification of the recombinant DNA using a pair of primers carrying a target mutation in the same PCR. Unfortunately, this approach is very often coupled with PCR artifacts which compromise overall efficiency of site-directed mutagenesis. To reduce the failure rate due to the PCR artifacts, we have set up a high-throughput mutagenesis protocol based on a two-fragment PCR approach. To this end, each mutation is introduced in two separate PCRs resulting in two linear fragments of the mutated plasmid. In the next steps, the PCR template is digested and the two matching plasmid fragments are joined together using Gibson assembly. Separating the corresponding mutagenic primers into two different PCRs decreases a number of artifacts and thus increases overall cloning efficiency. Furthermore, free software that we developed facilitates both high-throughput primer design and analysis of sequencing results. Overall, this protocol enabled us to efficiently produce several alanine-scanning libraries of 400 single-point mutations with complete coverage of the protein sequence.

Keywords: Scanning mutagenesis (扫描诱变), Site-directed (定点), High throughput mutagenesis (高通量突变), Two-fragment PCR (双片段PCR), Gibson assembly (Gibson 组装)

Background

High-throughput cloning techniques are widely used for research involving expression and purification of proteins. Site-directed scanning mutagenesis techniques in particular are often used to generate mutants for functional studies or to generate stabilized proteins for biophysics and structural biology. Libraries necessary for these purposes may contain several hundred mutants and their generation can be expensive and time consuming. Many single-point mutant libraries have been created using single vector PCRs with overlapping primers, e.g. QuikChange (Agilent). Although this and similar mutagenesis techniques are well established and widely used (for a recent review see Ortega et al., 2019), they may lead to a number of different errors in the final vector (Liu and Naismith, 2008; Edelheit et al., 2009; Sun et al., 2013; Heydenreich et al., 2017) caused by inherent sequence overlap of primers. Such errors include additional mutations and primer repeats, which necessitates sequencing of multiple colonies or additional rounds of mutagenesis. The protocol presented here is based on a two-fragment PCR mutagenesis, where the primers used in each PCR reaction have no sequence complementarity. The two halves of a plasmid to be cloned are amplified in two separate PCRs, followed by in vitro circularisation using Gibson assembly reaction (Figure 1). As a result, the number of successful mutagenesis reactions after sequencing only one clone per mutant is increased, allowing us to generate multiple alanine-scanning libraries of complete proteins of ca. 400 amino acids in length within approx. 6 weeks each (Heydenreich et al., 2017). In addition, our protocol can also be used to insert multiple point mutations in one round of cloning and for sequence modification (e.g., addition of purification tags, small insertions and deletions) with increased efficiency.


Figure 1. Overview of the mutagenesis technique. AAscan software (Sun et al., 2013) is used to design mutagenic primers for the high-throughput site-directed scanning mutagenesis. Two PCR reactions are done per mutant, in each of them approximately half of the vector is amplified. Two fragments containing one mutation are combined, followed by DpnI digestion at 37 °C overnight. Reaction clean-up is performed to purify DNA fragments, which are then assembled by Gibson assembly reaction. Bacteria are transformed with the resulting circular plasmid and plated on selective LB agar plates. One clone per mutant is sent for sequencing either on a selective LB agar 96-well plate or as purified DNA. All steps excluding the plating of the bacteria are done in 96-well plates. Sequencing results are analyzed in a high-throughput manner using MutantChecker program (Sun et al., 2013).

Materials and Reagents

Note: All reagents must be of analytical grade or higher and stored according to recommendations from the manufacturer. All plasticware must be DNase-free.

  1. LB-agar 96-well plates with the appropriate antibiotic(s) (can be ordered from a sequencing company; if not, see Recipes)
  2. Pipette tips, 10 μl, 200 μl and 1,250 μl (VWR, European catalog numbers: 613-1093, 613-1092 and 613-1087)
  3. (Optional) Sterile toothpicks
  4. Polypropylene conical tubes 15 ml and 50 ml (Falcon, catalog numbers: 352096 and 352070)
  5. Microcentrifuge tubes 1.5 ml (Simport, catalog number: T330-7G)
  6. Pipetting reservoir (Diversified Biotech, catalog number: REBP-3000)
  7. 96-well PCR plates, 0.2 ml (VWR, European catalog number: 211-0297)
  8. Microseal ’B’ PCR plate seals (Bio-Rad, catalog number: MSB1001)
  9. Deep-well plates 96/2,000 μl (Eppendorf, catalog number: 651033405)
  10. Adhesive gas-permeable film for culture plates (VWR, European catalog number: 391-1261)
  11. Sterile Petri dishes (e.g., ThermoScientific NuclonTM Delta Surface, catalog number 150350)
  12. Disposible sterile spreaders (e.g., VWR, catalog number: 612-5497) or autoclaved glass beads 2 mm (e.g., Supelco, catalog number: 1.04014)
  13. ZR-96 DNA Clean & Concentrator (Zymo Research, catalog number: D4024)
  14. Chemically competent Escherichia coli XL-1 Blue cells (Agilent, catalog number: 200249) or some other cell strain which is used for cloning (Mach1, DH5α, TOP10 etc.)
  15. Cloning primers
    ColE1A: 5′-GGAGCGAACGACCTACACCGAACTGAGATACCTACAGCG-3′
    ColE1B: 5′-CGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCC-3′
    Note: This pair of primers can be used for all plasmids containing the ColE1 origin of replication.
  16. Plasmid to be used as a template in PCR
  17. Nicotinamide adenine dinucleotide, NAD+, 50 mM (New England Biolabs, catalog number: B9007L)
  18. Deoxynucleotide (dNTPs) solution mix, 10 mM each dNTP (New England Biolabs, catalog number: N0447S)
  19. T5 exonuclease, 10 U/μl (New England Biolabs, catalog number: M0363S)
  20. Phusion HF DNA polymerase, 2 U/μl (New England Biolabs, catalog number: M0530L)
  21. Taq DNA ligase, 40 U/μl (New England Biolabs, catalog number: M0208L)
  22. DpnI, 20 U/μl (New England Biolabs, catalog number: R0176L)
  23. Nuclease-free water (Cell Signaling Technology, catalog number: 12931S)
  24. Phusion High-Fidelity PCR Master Mix with GC Buffer (New England Biolabs, catalog number: M0532L) or some other appropriate DNA polymerase master mix (see Procedure B. PCR)
  25. PEG-8000 (Sigma-Aldrich, catalog number: P5413)
  26. Tris-HCl buffer, 1 M, pH 7.5 (Thermo Fischer Scientific, catalog number: 15567027)
  27. MgCl2, 1 M (Sigma-Aldrich, catalog number: M1028)
  28. DL-dithiothreitol (DTT), 1 M (Sigma-Aldrich, catalog number: 43816)
  29. SOC outgrowth medium (New England Biolabs, catalog number: B9020S)
  30. Antibiotic stock solution(s) (depending on an antibiotic resistance gene(s) carried by the plasmid of interest)
  31. Lysogeny broth (LB) broth with agar (Sigma-Aldrich, catalog number: L2897)
  32. Ultrapure deionized water with electrical resistivity of 18 MΩ cm at 25 °C
  33. 5x isothermal reaction (IT) buffer (see Recipes)
  34. 1.33x Gibson assembly mixture (see Recipes)
  35. LB-agar plates with the appropriate antibiotic(s)-Petri dishes (see Recipes)

Equipment

  1. Diverse pipettes including multi-channel pipettes and repeat pipettors
  2. Thermocycler that can accommodate 96-well PCR plates (e.g., EppendorfTM MastercyclerTM Nexus Thermal Cycler, catalog number: E6332000029)
  3. Centrifuge for deepwell plates (e.g., Eppendorf 5810 R with swing-bucket rotor A-4-62, catalog number: 5811000320)
  4. Laboratory incubator set at 37 °C
  5. Laboratory incubator/shaker set at 37 °C

Software

  1. AAscan (Sun et al., 2013; https://github.com/dmitryveprintsev/AAScan/)
  2. MutantChecker (Sun et al., 2013; https://github.com/dmitryveprintsev/AAScan/)

Procedure

  1. Primer design and ordering
    1. Download and open AAscan software (Sun et al., 2013) to design mutagenic primers for the site-directed scanning mutagenesis. A screenshot of the AAscan software interface is shown in Figure 2.
    2. Replace the default DNA sequence given in AAscan with a DNA sequence which is to be mutated, including the flanking regions (40-50 nucleotides) at the 5′ and 3′ ends (DNA seq. incl.40-50 nt flanking regions).
    3. Define the position of the first nucleotide in the first triplet codon (Nt position of 1st triplet). This relates the given DNA sequence with the amino-acid numbering. In the example given in Figure 2, the 50 nucleotides from the vector upstream of the protein coding sequence are included as a flanking region. Therefore, the nucleotide A from the ATG codon, which corresponds to amino-acid residue 1, is at the position 51. Whenever possible, we recommend using a standard numbering scheme for the protein of interest. This does not have to match the amino-acid numbering in the expressed protein construct (which might include a signal peptide, purification tags, protease cleavage sites, linkers or fusion protein partners).
    4. Define up to two codons coding for an amino acid (Xxx) by which you want to replace other amino acids in the scanning mutagenesis, e.g., ‘GCG’ and ‘GCA’ for Ala (mut codon 1 and 2). If only one particular codon is to be used, include it into both mut codon 1 and 2 text fields. We recommend choosing the codons based on their frequency in the expression host and the GC content of the gene of interest. Select those codons which are most frequent in the expression host and, if possible, avoid those with high or low GC content if your gene of interest has a very high (> 60%) or very low (< 40%) GC content, respectively. For several constructs with GC content in a range 50-55 % which were to be expressed in human cell culture (HEK 293S), we used the most frequent codons for this host: GCC/GCT for Ala, GGC/GGA for Gly, and GTG/GTC for Val. On the other hand, if the gene of interest has a high GC content, it might be better to use codons with lower GC content, i.e., GCT/GCA for Ala, GGA/GGT for Gly and GTT/GTA for Val. Such codon choice minimizes the number of designed mutagenic primers with relatively high annealing temperatures.
    5. If a particular amino acid in the scanning mutagenesis region is already Ala, replace it with another amino acid Yyy by defining up to two corresponding codons, e.g., in alanine scanning mutagenesis replacing an existing alanine with a glycine (if Ala, use Gly). In choosing the codons, use the recommendations given in the previous step.
    6. Enter an amino acid mutagenesis range (text fields From and To) by using the amino-acid numbering scheme defined in Step A3.
    7. Define the primer design parameters. Based on our experience (Sun et al., 2013; Heydenreich et al., 2017), we recommend using the following parameters: minimal primer length (min length)–18 nucleotides (nt); maximal primer length (max length)–60 nt; minimal GC clamp (MinGCclamp)–2; optimized GC clamp (OptimisedGCclamp); minimal primer melting temperature (MinTm)–60 °C; maximal primer melting temperature (MaxTm)–70 °C; maximal difference between melting temperatures of forward and reverse primer (MaxDeltaTm)–5 °C; minimal distance from the mutation codon to the 3′ end of the primer (MinAnnealLen)–15 nt; length of the overlap sequence between the ends of the resulting PCR fragment (MinOverlap and MaxOverlap)–between 15 and 27 nt; the number of primer pairs designed for the mutation of every specified amino acid position (MaxSuggestions)–1.
    8. Define the output format. Use separate result columns for forward and reverse primers (Separate F and R). User can choose among the more (Long1 and Long2) or less (Short and mFASTA) detailed outputs.
    9. Click or touch Batch button. This gives two separate lists of the designed forward (‘n_F’) and reverse (‘n_R’) primers including their names and sequences (and other primer properties if output format Long1 or Long2 was chosen). ‘n’ in a primer name corresponds to an amino acid number which is to be mutated by using that primer.
    10. Order the designed primers from a company that provides synthesis of DNA oligonucleotides. Such company usually makes available an order sheet template file into which the AAscan output (obtained in Step A10) can easily be copied. When ordering, use a 96-well plate format with forward and reverse primers separated into different 96-well plates, but in such a way that a position of each forward primer corresponds to exactly the same position of the related reverse primer in the other 96-well plate (e.g., primers 103_F and 103_R at the position A6 in each 96-well plate). Order desalted DNA oligonucleotides already dissolved at concentration 100 μM either in nuclease-free water or in some standard buffer provided by the company (usually 10 mM Tris-HCl, 0.1 mM EDTA, pH 7.5); see Note 1.
    11. Purchase a pair of the desalted reverse-complementary primers which anneal approximately on the opposite side of the plasmid. It is advisable to choose such primers that bind either to the origin of replication or the antibiotic resistance gene. In this case, only the properly assembled plasmid DNA will allow growing the transformed bacteria on a plate with selection antibiotic. We use origin-of-replication ColE1 primers (see Materials and Reagents).


      Figure 2. AAscan software interface. In the example shown, the coding sequence is written in capital letters and the flanking regions are given in small letters (DNA seq incl. 40-50 nt flanking regions). The first nucleotide in the first triplet codon (ATG) is at the position 51 of the specified DNA sequence (Nt position of 1st triplet). The mutagenic primers designed for amino-acid residues 2-371 are separated into two output windows: the forward primers are listed in the left one and the reverse primers in the right one.

  2. PCR
    Each mutation is generated in two separate PCRs (denoted A and B) resulting in two PCR fragments that are combined in subsequent Gibson assembly. Before performing PCR for all of the target mutations, we recommend testing PCR conditions on a small number (20-30) of randomly chosen mutations and modifying the protocol accordingly, as detailed in the original article by Heydenreich et al. (2017). We recommend testing different polymerases and buffer condition as well as DMSO addition. To identify efficient PCR conditions, we analyze each PCR product from the test mutation set in agarose gel electrophoresis. Nevertheless, we do not include this or any other control in the large-scale mutagenesis.
    1. Before doing the experiment, check the orientation of the gene of interest in its plasmid. This is important to determine how to combine in PCR mutagenic primers with the origin-of-replication ColE1 primers. A mutagenic primer and the appropriate ColE1 primer should anneal to complementary DNA strands; e.g., if the forward mutagenic primers are combined with ColE1B, the reverse mutagenic primers should be combined with ColE1A (and vice versa).
    2. Dilute the plasmid DNA template with nuclease-free water to concentration 1 ng/μl.
    3. Dilute the primers ColE1A and ColE1B with nuclease-free water to concentration 6 μM.
    4. Dilute the mutagenic primers in a 96-well plate with nuclease-free water to 6 μM.
    5. Two sets of PCRs are necessary. For each set of x mutagenesis PCR reactions, prepare a PCR master mix as defined in Table 1.

      Table 1. PCR master mixes. Each mixture is enough for x mutagenesis PCRs in 20 μl reaction volume.


    6. To prepare PCR mixtures A, pipette using a multichannel pipette 19 μl PCR Master Mix A into each used well of a 96-well 200-μl PCR plate on ice and add to it 1 μl mutagenic primer (6 μM) from the appropriate primer set (forward or reverse, as discussed in Step B1). Close the 96-well PCR plate with a PCR seal.
    7. Prepare a PCR program as in Table 2 (see Note 2).

      Table 2. PCR program for DNA multiplication using Phusion polymerase

      *Size of the longest DNA fragment in kilobase pairs (kbp) to be amplified in PCR; the extension time is 30 s per 1 kbp.

    8. Preheat a thermocycler: a block to 98 °C (denaturation temperature) and a lid to 103 °C (see Note 3).
    9. Transfer the sealed 96-well PCR plate with the reaction mixtures directly from ice to the preheated thermocycler, close the lid and start the PCR program defined in Step B7.
    10. Keep the 96-well PCR plate with completed PCR on ice until further processing.
    11. To prepare PCR mixtures B, repeat the Steps B6-B9 by using PCR Master Mix B and the corresponding mutagenic primers (the second mutagenic primer set, as discussed in Step B1).

  3. DNA template digestion and reaction clean-up
    1. After completion of both PCRs A and B, transfer 20 μl PCR mixture A to the corresponding 20 μl PCR mixture B in a 96-well PCR plate using a multichannel pipette. In this step, we combine two fragment PCR products possessing the same mutation.
    2. Add 0.5 μl DpnI (20 U·μl-1) to each mixture of fragments, seal the 96-well plates and incubate them at 37 °C to digest methylated DNA template. DpnI digestion for 1 h is already enough, but the mixtures can be incubated for up to 18 h if this is more convenient in terms of time management.
    3. In order to purify the PCR fragments from the reaction mixture after the DpnI digestion, use ZR-96 DNA Clean & Concentrator-5 KitTM (Zymo Research) according to the manufacturer’s protocol, but use 10 μl elution buffer in the last step. This small volume should result in higher DNA concentrations for the subsequent Gibson assembly.

  4. Gibson assembly
    1. In a 96-well PCR plate on ice, mix 1 μl each mixture of cleaned PCR fragments with 3 μl 1.33x Gibson assembly mix (Recipe 2) by pipetting gently up and down ten times using a multichannel pipette set at volume 3 µl to avoid foaming (see Note 4).
    2. Transfer the sealed 96-well plate with Gibson assembly reaction mixtures into a preheated thermocycler (a block at 50 °C and a lid at 53 °C) and incubate it for 10 min at 50 °C followed by 1 h at 37 °C and a hold at 10 °C. Alternatively and especially for longer nucleotide overlaps within a pair of mutagenic primers (e.g., 21 or more nt; see Step A8), the Gibson assembly reaction can be conducted for 1 h at 50 °C followed by a hold at 10 °C. Which Gibson assembly conditions are better suited can be checked first on a small number (20-30) of randomly chosen mutations, as detailed by Heydenreich et al., 2017 (see Note 5).

  5. Cell transformation
    1. Thaw chemically competent Escherichia coli XL-1 Blue cell suspension on ice (see Note 6).
    2. Onto a 96-well PCR plate on ice, distribute 20 μl cell suspension per each well and mutation using a repeat pipette.
    3. Add 2 μl Gibson assembly mixture to each 20-μl cell aliquot, seal the plate with a PCR plate seal and incubate on ice for 10-20 min.
    4. Transfer the 96-well plate with cells-DNA mixtures into a preheated thermocycler (a block at 42 °C and a lid at 42 °C) and incubate it for 60 s at 42 °C.
    5. Immediately put the cells-DNA mixtures on ice and incubate for 2-5 min. If the plasmid carries the ampicillin resistance gene, you can proceed immediately to Step E8 (although Steps E6 and E7 increase cloning efficiency in this case as well).
    6. Using a multichannel pipette, transfer the transformed cells to a 96-deep-well plate filled with 300 μl SOC media (without any antibiotic), close the plate with a gas-permeable seal and incubate for 1 h at 37 °C while shaking at 160 rpm.
    7. Spin down the cells (3,000 x g, 5 min) and carefully remove 220 μl supernatant using a multichannel pipette.
    8. Resuspend a cell pellet in the remaining supernatant (ca. 100 μl) by pipetting up and down using a multichannel pipette set at 80 µl (to avoid foaming) until all cell pellets are resuspended without visible clumps during pipetting. Plate the cell suspension onto an LB-agar plate with the appropriate antibiotic. To save some storage space and material, you can plate four different mutants per LB-agar plate (one mutant onto each plate quadrant) or 24 mutants on a 24-well LB-agar plate (one mutant per well) by using an expanding multi-channel pipette.
    9. Allow the plates to dry for ca. 10 min and then incubate them upside-down at 37 °C overnight. Next day, you should find bacterial colonies on the LB-agar plates.

  6. Plasmid DNA preparation and sequencing
    For high-throughput projects, sequencing companies offer plasmid preparation and sequencing directly from a bacterial colony on 96-well plates without extra costs in comparison to sending already extracted plasmid samples. We strongly recommend this option.
    1. Label a position of a selected colony on an LB-agar plate, pick it by a sterile pipette tip or a toothpick and transfer it to LB-agar with the appropriate antibiotic in a 96-well plate. Such plates are usually offered by a sequencing company; if not, you can easily prepare them by your own (Recipe 3).
    2. Order the sequencing with the appropriate sequencing primer (forward or reverse). For reliable results, the sequencing primer should anneal at least 30-40 nucleotides upstream from the expected mutation. Depending on a sequencing service, one can usually expect reliable sequence read lengths of at least ca. 600 nucleotides (which corresponds to ca. 200 amino acids). For longer sequences, one should use additional sequencing primer(s) to cover the whole region of interest. Send the 96-well LB-agar plates with one colony per mutant to the sequencing company and wait for the sequencing results. Sending one mutant per sequencing round reduces sequencing costs.
    3. Analyze the sequencing results in MutantChecker software (Sun et al., 2013), as detailed in the Data analysis section.
    4. If the mutation is not confirmed in a sequencing round, send a new colony in the next sequencing round (see Note 8).
    5. You can obtain plasmid solutions in a 96-well plate from the sequencing company. Alternatively or in addition, you can prepare plasmids by yourself from the colonies labeled on LB-agar plates which were positive in sequencing.

Data analysis

Analysis of sequencing data

  1. Save the sequencing data as separate .fasta, .seq or .txt files in a folder on the computer.
  2. Open MutantChecker (Figure 3) and paste the reference non-mutated DNA sequence into the indicated field (Reference sequence). The sequence should start with the first codon as defined in Procedure, Step A3. Analyze mutant DNAs sequenced with forward primers separately from those sequenced with reverse primers. For more detailed information see Sun et al. (2013).
  3. For DNA sequenced with forward primers, change the default values provided in Report mut from # and To in the respective fields, using amino acid numbers. Generally, a forward sequence should cover about 200 to 250 amino acids.
  4. Once you have checked the program settings, click on “File” → “Load File List”. Select one or multiple files to be checked. We recommend checking files one by one initially. If several files are analyzed at once, an alignment will only be displayed for the last file selected.
  5. The largest window will now display the alignment of the reference sequence and the sequencing result (Figure 3). The graph on the right indicates how many possible alignments have been found. Ideally, you should see a single sharp peak. The window on the lower right displays sequence names followed by any mutation found within the limits entered in step 3 (see Note 7).
  6. For DNA sequenced with reverse primers, indicate the range of amino acids where a mutation should be reported using the fields RC From and RC To. RC stands for reverse complement. Tick the ReverseComplement box when checking DNA sequenced with reverse primers.
  7. Repeat steps 4-5 to analyze the remaining sequencing data.


    Figure 3. MutantChecker software interface. Sequencing data analysis for mutation R104A is shown as an example. Graph on the right side depicts the alignment score function vs. offset between the sequencing result and the reference sequence (Sun et al., 2013). In this case, there is a single peak which indicates that one possible alignment was found. Multiple peaks would have implied several alternative alignments due to repeated sequences (usually an indication of a cloning artifact). Mutations identified in the specified amino acid range (1-200) are listed in the lower right output window. ‘E198@’ denotes that sequencing trace starts to be unclear (“n” in the sequencing result) from amino acid E198.

Notes

  1. No primer modification (like phosphorylation) is necessary for Gibson assembly and the minimum synthesis scale of DNA oligonucleotides is usually more than sufficient. Store the frozen primers at -20 °C for up to 2 years. Several freeze-thaw cycles have no considerable influence on the stability of DNA oligonucleotides.
  2. Different PCR protocols might be needed for DNA polymerases other than Phusion.
  3. This step minimizes mis-priming during initial denaturation. It may not be necessary if using hot-start polymerases.
  4. Instead of the homemade Gibson assembly mix, one can use a commercial Gibson assembly master mix or a similar DNA assembly product.
  5. More recently, we successfully used incubation for 1 h at 45 °C for which it was shown to increase the efficiency of Gibson assembly (Gibson et al., 2010). This may be especially beneficial if the primers were designed with relatively short overlap to improve their specificity (e.g., 15 bp).
  6. The cells should be prepared by the Inoue method (Sambrook and Russell, 2006) and have a transformation efficiency of at least 107 colonies per 1 μg pBR322 (or a similar plasmid). Alternatively, one can use some other E. coli strain that is customary for routine cloning, such as Mach1, DH5α or TOP10.
  7. Depending on the company which sequenced the DNA, the annotation of the traces may vary. Some companies decide not to display an “N” when the trace is unclear, but instead opt for the highest peak. In both cases, a mismatch will be reported, but it may be perceived as a real mutation when the trace does not contain Ns. We recommend that you check the trace in cases like that even if the reported mutation occurs at the very beginning or end of a sequencing result. Especially towards the end of a sequence, a repeat of four or more bases of the same type (all Gs, all Cs, all As or all Ts) may lead to difficult-to-interpret traces, which can lead to n bases being read as n-1 or n+1, resulting in a frameshift. This is usually a sequencing problem rather than a true frameshift. These cases, or sequences trailed by many Ns may lead to additional peaks close to the main peak.
  8. For troubleshooting, please refer to the original publication (Heydenreich et al., 2017).

Recipes

  1. 5x Isothermal reaction (IT) buffer (6 ml)
    1.5 g PEG 8000
    3 ml Tris-HCl (1 M, pH 7.5)
    300 μl MgCl2 (1 M)
    300 μl DTT (1 M)
    600 μl NAD+ (50 mM)
    600 μl dNTPs solution mix (10 mM each dNTP)
    Prepare 500-μl aliquots of 5x IT buffer and store them at -80 °C
  2. 1.33x Gibson assembly mixture (1.8 ml)
    480 μl 5x IT buffer
    0.96 μl T5 exonuclease (10 U/μl)
    30 µl Phusion HF DNA polymerase (2 U/μl)
    240 µl Taq DNA ligase (40 U/μl)
    1,049 μl nuclease-free water
    Make 50-μl aliquots and store at -20 °C (or -80 °C for long-term storage)
    Note: Gibson assembly mixture can be stored for more than a year at -20 °C and the enzymes are still active after more than 10 freeze-thaw cycles (Gibson et al., 2009).
  3. LB-agar plates with antibiotic
    1. Add 35 g LB broth with agar in 1 L ultrapure deionized water
    2. Autoclave at 121 °C for 15 min to dissolve the powder and sterilize LB-agar
    3. Let it cool down to hand-hot temperature (ca. 45 °C), add stock solution of the appropriate antibiotic to achieve the target antibiotic concentration and swirl vigorously to mix
    4. Pour ca. 15 ml melted LB-agar with antibiotic into each 10-cm-diameter Petri dish and let it solidify
    5. For 96-well LB-agar plates, use a multi-channel pipette to transfer 150 μl LB-agar with antibiotic per well into a 96-well plate with flat bottom and let the agar solidify
    6. The plates should be stored upside-down in sealed plastic bags at 4 °C until use

Acknowledgments

This protocol was derived from our previous publication (Heydenreich et al., 2017). This work was supported by Swiss National Science Foundation Doc.Mobility fellowship 165219 to FMH, Erasmus, EMBO Short Term Fellowship ASTF 420–2016 and Swiss National Science Foundation Doctoral Mobility Grant 31003A_159748/2 travel fellowships, and NanoMem ITN network funding to TM, NRW Strategy Project BioSC BOOST FUND “RIPE” to the Institute of Biochemical Plant Physiology at the Heinrich Heine University, Marie Curie (274497) and UBS Promedica Foundation post-doctoral fellowships to DM, and Swiss National Science Foundation grants 141898, 159748 to DBV and COST Action CM1207 GLISTEN.

Competing interests

The authors declare no competing financial or non-financial interests.

References

  1. Edelheit, O., Hanukoglu, A. and Hanukoglu, I. (2009). Simple and efficient site-directed mutagenesis using two single-primer reactions in parallel to generate mutants for protein structure-function studies. BMC Biotechnol 9: 61.
  2. Gibson, D. G., Smith, H. O., Hutchison, C. A., 3rd, Venter, J. C. and Merryman, C. (2010). Chemical synthesis of the mouse mitochondrial genome. Nat Methods 7(11): 901-903.
  3. Gibson, D. G., Young, L., Chuang, R. Y., Venter, J. C., Hutchison, C. A., 3rd and Smith, H. O. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6(5): 343-345.
  4. Heydenreich, F. M., Miljuš, T., Jaussi, R., Benoit, R., Milić, D. and Veprintsev, D. B. (2017). High-throughput mutagenesis using a two-fragment PCR approach. Sci Rep 7(1): 6787.
  5. Liu, H. and Naismith, J. H. (2008). An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol. BMC Biotechnol 8: 91.
  6. Ortega, C., Abreu, C., Oppezzo, P. and Correa, A. (2019). Overview of high-throughput cloning methods for the post-genomic era. Methods Mol Biol 2025: 3-32.
  7. Sambrook, J. and Russell, D. W. (2006). The inoue method for preparation and transformation of competent E. coli: "ultra-competent" cells. CSH Protoc 2006(1).
  8. Sun, D., Ostermaier, M. K., Heydenreich, F. M., Mayer, D., Jaussi, R., Standfuss, J. and Veprintsev, D. B. (2013). AAscan, PCRdesign and MutantChecker: a suite of programs for primer design and sequence analysis for high-throughput scanning mutagenesis. PLoS One 8(10): e78878.

简介

定点扫描诱变是一种有用的工具,可用于研究蛋白质功能和设计具有新特性(例如增加的稳定性或酶活性)的蛋白质。创建包含数百个定点突变体的系统库仍然是一项艰巨而昂贵的任务。建立此类文库的已建立方案包括在同一PCR中使用携带靶突变的一对引物对重组DNA进行PCR扩增。不幸的是,这种方法经常与PCR伪像结合使用,从而影响了定点诱变的整体效率。为了降低由于PCR伪影而导致的故障率,我们基于两片段PCR方法建立了高通量诱变方案。为此,将每种突变引入两个单独的PCR中,从而产生突变质粒的两个线性片段。在下一步中,将PCR模板消化,并使用Gibson组装将两个匹配的质粒片段连接在一起。将相应的诱变引物分成两个不同的PCR可以减少许多假象,从而提高总体克隆效率。此外,我们开发的免费软件可促进高通量引物设计和测序结果分析。总体而言,该协议使我们能够有效地产生具有400个单点突变且完全覆盖蛋白质序列的多个丙氨酸扫描文库。
【背景】高通量克隆技术被广泛用于涉及蛋白质表达和纯化的研究。特别是定点扫描诱变技术通常用于生成功能研究的突变体或用于生物物理学和结构生物学的稳定蛋白。这些目的所需的库可能包含数百个突变体,它们的生成可能既昂贵又耗时。使用带有重叠引物 eg QuikChange(Agilent)的单载体PCR创建了许多单点突变体文库。尽管这种诱变技术和类似的诱变技术已经很好地建立并广泛使用(有关最近的综述,请参见Ortega等,2019),但它们可能会导致最终载体产生许多不同的错误(Liu和Naismith ,2008年; Edelheit等人,2009年; Sun等人,2013年; Heydenreich等人,2017年),这是由内在序列重叠引起的底漆。此类错误包括其他突变和引物重复,这需要对多个菌落进行测序或进行另外几轮诱变。此处介绍的协议基于两片段PCR诱变,其中每个PCR反应中使用的引物均无序列互补性。将要克隆的质粒的两半分别在两个单独的PCR中扩增,然后使用Gibson组装反应进行体外环化(图1)。结果,每个突变体仅测序一个克隆后成功诱变反应的次数增加了,这使我们能够生成完整的 ca 蛋白质的丙氨酸扫描文库,其长度约为400个氨基酸。每个6周(Heydenreich等人,2017)。此外,我们的协议还可以用于在一轮克隆中插入多个点突变,并以更高的效率用于序列修饰(例如,添加纯化标签,小的插入和缺失)。

“”
图1.诱变技术概述。使用Asscan软件(Sun et al。,2013)设计用于高通量定点扫描诱变的诱变引物。每个突变体进行两次PCR反应,在每个突变体中,大约扩增了一半的载体。将包含一个突变的两个片段合并,然后在37°C下DpnI消化过夜。进行反应净化以纯化DNA片段,然后通过Gibson组装反应将其组装。用所得的环状质粒转化细菌,并将其铺在选择性的LB琼脂平板上。将每个突变体一个克隆发送到选择性LB琼脂96孔板上或以纯化的DNA进行测序。除细菌接种外,所有步骤均在96孔板上完成。使用MutantChecker程序以高通量的方式分析测序结果(Sun et al。,2013)。

关键字:扫描诱变, 定点, 高通量突变, 双片段PCR, Gibson 组装

材料和试剂

注意:所有试剂必须为分析纯或更高,并根据制造商的建议进行存储。所有塑料制品都必须不含DNase。

  1. 具有适当抗生素的LB-琼脂96孔板(可以从测序公司订购;如果没有,请参见食谱)
  2. 移液器吸头,10μl,200μl和1,250μl(VWR,欧洲目录号:613-1093、613-1092和613-1087)
  3. (可选)无菌牙签
  4. 15毫升和50毫升聚丙烯锥形管(Falcon,目录号:352096和352070)
  5. 1.5 ml微量离心管(进口,目录号:T330-7G)
  6. 移液容器(Diversified Biotech,目录号:REBP-3000)
  7. 96孔PCR板,0.2毫升(VWR,欧洲目录号:211-0297)
  8. Microseal'B'PCR板密封件(Bio-Rad,目录号:MSB1001)
  9. 深孔板96 / 2,000μl(Eppendorf,目录号:651033405)
  10. 用于培养板的透气性薄膜(VWR,欧洲目录号:391-1261)
  11. 无菌培养皿(例如,ThermoScientific Nuclon TM Delta Surface,目录号150350)
  12. 一次性无菌涂抹器(例如,VWR,目录号:612-5497)或2 mm高压灭菌的玻璃珠(例如,Supelco,目录号:1.04014)
  13. ZR-96 DNA清洁剂 集中器(Zymo Research,目录号:D4024)
  14. 具有化学活性的大肠杆菌 XL-1蓝色细胞(安捷伦,目录号:200249)或用于克隆的其他一些细胞株(Mach1,DH5α,TOP10 等。)
  15. 克隆引物
    ColE1A:5'-GGAGCGAACGACCTACACCGAACTGAGATACCTACAGCG-3'
    ColE1B:5'-CGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCC-3'
    注意:这对引物可用于所有含有ColE1复制起点的质粒。
  16. 用作PCR模板的质粒
  17. 烟酰胺腺嘌呤二核苷酸,NAD + ,50 mM(New England Biolabs,目录号:B9007L)
  18. 脱氧核苷酸(dNTP)溶液混合物,每个dNTP均为10 mM(New England Biolabs,目录号:N0447S)
  19. T5核酸外切酶,10 U /μl(New England Biolabs,目录号:M0363S)
  20. Phusion HF DNA聚合酶,2 U /μl(New England Biolabs,目录号:M0530L)
  21. Taq DNA连接酶,40 U /μl(New England Biolabs,目录号:M0208L)
  22. DpnI,20 U /μl(New England Biolabs,目录号:R0176L)
  23. 无核酸酶的水(细胞信号技术,目录号:12931S)
  24. 具有GC缓冲液的Phusion高保真PCR预混液(New England Biolabs,目录号:M0532L)或其他一些合适的DNA聚合酶预混液(请参阅步骤B. PCR)
  25. PEG-8000(Sigma-Aldrich,目录号:P5413)
  26. Tris-HCl缓冲液,1 M,pH 7.5(Thermo Fischer Scientific,目录号:15567027)
  27. MgCl 2 ,1 M(Sigma-Aldrich,目录号:M1028)
  28. DL-二硫苏糖醇(DTT),1 M(Sigma-Aldrich,目录号:43816)
  29. SOC增长培养基(新英格兰生物实验室,目录号:B9020S)
  30. 抗生素原液(取决于目标质粒携带的抗生素抗性基因)
  31. 带有琼脂的溶源性肉汤(LB)肉汤(Sigma-Aldrich,目录号:L2897)
  32. 25°C时电阻率为18MΩcm的超纯去离子水
  33. 5个等温反应(IT)缓冲液(请参阅食谱)
  34. 1.33x吉布森装配混合物(请参阅食谱)
  35. 具有适当抗生素的LB-琼脂平板-培养皿(请参见食谱)

设备

  1. 多种移液器,包括多通道移液器和重复移液器
  2. 可容纳96孔PCR板的热循环仪(例如,Eppendorf TM Mastercycler TM Nexus热循环仪,目录号:E6332000029)
  3. 用于深孔板的离心机(例如,带有摆斗式转子A-4-62的Eppendorf 5810 R,目录号:5811000320)
  4. 实验室培养箱设置为37°C
  5. 实验室培养箱/振荡器设置在37°C

软件

  1. AAscan(Sun等,2013年; https://github.com/dmitryveprintsev/ AAScan / )
  2. MutantChecker(Sun et al。,2013; https://github.com/dmitryveprintsev/ AAScan / )

程序

  1. 底漆设计和订购
    1. 下载并打开Aascan软件(Sun et al。,2013),以设计用于定点扫描诱变的诱变引物。Aascan软件界面的屏幕截图如图2所示。
    2. 用要突变的DNA序列替换Asscan中提供的默认DNA序列,包括5'和3'末端的侧翼区域(40-50个核苷酸)( DNA序列,包括40-50 nt侧翼)地区)。
    3. 定义第一个三联体密码子中第一个核苷酸的位置(1 st 三联体的 Nt位置)。这使给定的DNA序列与氨基酸编号相关。在图2给出的例子中,来自蛋白质编码序列上游载体的50个核苷酸被包括为侧翼区。因此,来自ATG密码子的核苷酸A(对应于氨基酸残基1)位于51位置。只要有可能,我们建议对目标蛋白质使用标准编号方案。这不必与表达的蛋白质构建体(可能包括信号肽,纯化标签,蛋白酶切割位点,接头或融合蛋白伴侣)中的氨基酸编号相匹配。
    4. 定义最多两个编码氨基酸( Xxx )的密码子,在扫描诱变中您要通过其替换其他氨基酸,例如 eg ,“ GCG”和“ GCA”表示Ala( mut密码1和2)。如果只使用一个特定的密码子,请将其同时包含在 mut密码子1和2 文本字段中。我们建议根据密码子在表达宿主中的频率和感兴趣基因的GC含量来选择密码子。选择表达宿主中最常见的密码子,如果可能,请避免如果您感兴趣的基因的GC含量非常高(> 60%)或非常低(<40%),则不要选择GC含量高或低的密码子。 , 分别。对于在人类细胞培养中表达的几种GC含量在50-55%之间的构建体(HEK 293S),我们对该宿主使用了最常见的密码子:Ala的GCC / GCT,Gly的GGC / GGA和GTG / GTC值 另一方面,如果感兴趣的基因具有较高的GC含量,则最好使用GC含量较低的密码子,例如 ie ,Ala的GCT / GCA,Gly的GGA / GGT和GTT / GTA Val。这样的密码子选择使具有相对高的退火温度的诱变引物的设计数量最小化。
    5. 如果扫描诱变区域中的特定氨基酸已经是Ala,则通过在丙氨酸扫描诱变替换中最多定义两个对应的密码子( eg ),将其替换为另一个氨基酸 Yyy 现有的带有丙氨酸的丙氨酸(如果是Ala,则使用Gly )。在选择密码子时,请使用上一步中给出的建议。
    6. 使用步骤A3中定义的氨基酸编号方案,输入氨基酸诱变范围(文本字段 From 和 To )。
    7. 定义底漆设计参数。根据我们的经验(Sun等,2013年; Heydenreich等,2017年),我们建议使用以下参数:最小引物长度(最小长度) )– 18个核苷酸(nt);最大引物长度(最大长度)– 60 nt;最小GC钳位( MinGCclamp )– 2;优化的GC钳位( OptimizedGCclamp ); 最低引物熔化温度( MinTm )– 60°C;引物最高熔化温度( MaxTm )– 70°C;正向和反向引物的熔化温度之间的最大差异( MaxDeltaTm )– 5°C;从突变密码子到引物( MinAnnealLen )– 15 nt 3'端的最小距离;所得PCR片段末端之间的重叠序列长度( MinOverlap和MaxOverlap )– 15到27 nt之间;每个特定氨基酸位置( MaxSuggestions )– 1突变设计的引物对的数量。
    8. 定义输出格式。正向和反向引物使用单独的结果列(分别的F和R )。用户可以选择更多( Long1 和 Long2 )或更少( Short 和 mFASTA )详细输出。
    9. 点击或触摸批处理按钮。这给出了设计的正向(' n _F')和反向(' n _R')引物的两个单独列表,包括它们的名称和序列(以及输出的其他引物特性)格式为 Long1 或 Long2 )。引物名称中的“ n ”对应于要通过使用该引物进行突变的氨基酸编号。
    10. 向提供DNA寡核苷酸合成的公司订购设计的引物。该公司通常会提供一个订单模板文件,可以轻松地将Aascan输出(在步骤A10中获得)复制到该模板文件中。订购时,请使用96孔板形式,正向和反向引物分为不同的96孔板,但以这种方式,每个正向引物的位置均与其他96孔中相关反向引物的位置完全相同孔板( eg ,每个96孔板中A6位置的引物103_F和103_R)。订购已脱盐的DNA寡核苷酸,它们已以100μM的浓度溶解在无核酸酶的水或公司提供的某些标准缓冲液中(通常为10 mM Tris-HCl,0.1 mM EDTA,pH 7.5); 见注1。
    11. 购买一对脱盐的反向互补引物,它们大约在质粒的相对侧上退火。建议选择与复制起点或抗生素抗性基因结合的引物。在这种情况下,只有正确组装的质粒DNA才能使转化的细菌在带有选择抗生素的平板上生长。我们使用复制起点的ColE1引物(请参阅材料和试剂)。


      图2. AAscan软件界面。在所示的示例中,编码序列以大写字母写出,侧翼区域以小写字母给出( DNA seq,包括40-50 nt侧翼区域)。第一个三联体密码子(ATG)中的第一个核苷酸位于指定DNA序列的51位(1 st 三联体的 Nt位置)。为氨基酸残基2–371设计的诱变引物分为两个输出窗口:正向引物列在左侧,反向引物列于右侧。

  2. PCR
    每个突变均在两个单独的PCR(分别表示为A和B)中生成,从而导致两个PCR片段在随后的Gibson组装中组合在一起。在对所有目标突变进行PCR之前,我们建议先对少量(20-30)随机选择的突变进行PCR条件测试,并相应地修改方案,如Heydenreich 等人在原始文章中所详述。 (2017)。我们建议测试不同的聚合酶和缓冲液条件以及添加DMSO。为了确定有效的PCR条件,我们从琼脂糖凝胶电泳中的测试突变集分析了每种PCR产物。尽管如此,我们并未在大规模诱变中包括此控件或任何其他控件。
    1. 在进行实验之前,请检查其质粒中目标基因的方向。这对于确定如何在PCR诱变引物中与复制起点ColE1引物结合非常重要。诱变引物和适当的ColE1引物应与互补DNA链退火; eg ,如果将正向诱变引物与ColE1B结合,则应将反向诱变引物与ColE1A结合(反之亦然)。
    2. 用无核酸酶的水稀释质粒DNA模板至浓度为1 ng /μl。
    3. 用无核酸酶的水稀释引物ColE1A和ColE1B到浓度为6μM。
    4. 用无核酸酶的水在96孔板中稀释诱变引物至6μM。
    5. 需要两组PCR。对于每组 x 诱变PCR反应,准备表1中定义的PCR预混液。

      表1. PCR主混合物。每个混合物足以进行20 µl反应体积的 x 诱变PCR。


    6. 要制备PCR混合物A,请使用多通道移液器将19μlPCR Master Mix A移至冰上96孔200μlPCR板的每个已用孔中,并向其中添加来自适当引物组的1μl诱变引物(6μM) (正向或反向,如步骤B1所述)。用PCR密封垫封闭96孔PCR板。
    7. 准备表2中所示的PCR程序(请参阅注释2)。

      表2。使用Phusion聚合酶进行DNA扩增的PCR程序

      *要在PCR中扩增的最长DNA片段的大小,以千碱基对(kbp)为单位;扩展时间为每1 kbp 30 s。

    8. 将热循环仪预热:将加热块加热至98°C(变性温度),将盖子加热至103°C(请参阅注3)。
    9. 将带有反应混合物的密封96孔PCR板直接从冰上转移到预热的热循环仪中,盖上盖子并启动步骤B7中定义的PCR程序。
    10. 将96孔PCR板和完整的PCR放在冰上,直至进一步处理。
    11. 要制备PCR混合物B,请使用PCR Master Mix B和相应的诱变引物(第二个诱变引物组,如步骤B1中所述)重复步骤B6-B9。

  3. DNA模板消化和反应净化
    1. 完成PCR A和PCR B后,使用多道移液器将20μlPCR混合物A转移到96孔PCR板中的相应20μlPCR混合物B中。在此步骤中,我们将具有相同突变的两个片段PCR产物组合在一起。
    2. 向每种片段混合物中加入0.5μlDpnI(20 U·μl -1 ),密封96孔板,在37°C下孵育以消化甲基化的DNA模板。DpnI消化1小时已经足够,但是如果在时间管理方面更方便的话,混合物可以孵育18小时。
    3. 为了在DpnI消化后从反应混合物中纯化PCR片段,请使用ZR-96 DNA Clean&amp; Concentrator-5 Kit TM (Zymo Research)根据制造商的规程,但在最后一步中使用10μl洗脱缓冲液。这个小体积将导致随后的Gibson组装中更高的DNA浓度。

  4. 吉布森大会
    1. 在冰上的96孔PCR板中,将每份干净的PCR片段混合物与3μl1.33x Gibson装配混合物(配方2)混合1μl,使用设置为3μl的多通道移液器轻轻上下移液十次,以避免起泡(见注4)。
    2. 将带有Gibson组装反应混合物的密封96孔板转移到预热的热循环仪中(在50°C下封闭,在53°C上放置盖子),在50°C下孵育10分钟,然后在37°C下孵育1 h,保持在10°C。另外,对于一对诱变引物(例如,21个或更多核苷酸;请参见步骤A8)中较长的核苷酸重叠,可以在50°C下进行Gibson组装反应1 h,然后进行保持在10°C。首先可以通过少量(20-30)随机选择的突变来检查哪种Gibson装配条件更合适,如Heydenreich et al。,2017年所详述(请参见注释5)。

  5. 细胞转化
    1. 在冰上解冻具有化学感受态的大肠杆菌 XL-1蓝细胞悬液(请参见注释6)。
    2. 在冰上的96孔PCR板上,每孔分配20μl细胞悬液,并使用重复移液器进行突变。
    3. 将2μlGibson装配混合物添加到每个20μl细胞等分试样中,用PCR板密封垫密封板,并在冰上孵育10-20分钟。
    4. 将带有细胞-DNA混合物的96孔板转移到预热的热循环仪中(在42°C时封闭,在42°C时放置盖子),在42°C孵育60 s。
    5. 立即将细胞-DNA混合物放在冰上,孵育2-5分钟。如果质粒带有氨苄青霉素抗性基因,则可以立即进入步骤E8(尽管在这种情况下,步骤E6和E7也会提高克隆效率)。
    6. 使用多通道移液器,将转化的细胞转移至装有300μlSOC培养基(不含任何抗生素)的96深孔板中,用透气密封垫封闭板并在37°C下孵育1 h,同时在160转/分。
    7. 旋转细胞(3,000 x g ,5分钟),并使用多通道移液器小心除去220μl上清液。
    8. 通过使用设置为80 µl的多通道移液器上下吸液,将细胞沉淀重悬在剩余的上清液中( ca。 100μl),直到所有细胞沉淀都重新悬浮而在移液过程中没有可见的团块为止。将细胞悬液接种到含有适当抗生素的LB琼脂平板上。为了节省一些存储空间和材料,您可以使用一个扩展的多孔板将每个LB-琼脂平板上四个不同的突变体(每个突变体上一个突变体)接种到24孔LB-琼脂板上的24个突变体(每个孔一个突变体)上。通道移液器。
    9. 让板干燥10分钟,然后在37°C下颠倒孵育过夜。第二天,您应该在LB-琼脂平板上发现细菌菌落。

  6. 质粒DNA的制备和测序
    对于高通量项目,测序公司可以直接从96孔板上的细菌菌落中进行质粒制备和测序,与发送已提取的质粒样品相比,无需额外费用。我们强烈建议您使用此选项。
    1. 在LB-琼脂平板上标记一个选定菌落的位置,用无菌移液器尖端或牙签将其挑选出来,然后将其与带有适当抗生素的96孔平板一起转移到LB-琼脂上。这种板通常由测序公司提供;如果没有,您可以轻松地自己准备它们(食谱3)。
    2. 使用适当的测序引物(正向或反向)订购测序。为了获得可靠的结果,测序引物应退火预期突变上游至少30-40个核苷酸。根据测序服务的不同,通常可以期望可靠的序列读取长度至少为 ca。 600个核苷酸(对应于 ca。 200个氨基酸)。对于更长的序列,应使用其他测序引物覆盖整个目标区域。将每个突变体带有一个菌落的96孔LB琼脂平板发送到测序公司,等待测序结果。每测序循环发送一个突变体可降低测序成本。
    3. 如“数据分析”部分所述,在MutantChecker软件中分析测序结果(Sun et al。,2013)。
    4. 如果未在测序轮次中确认突变,则在下一轮测序轮次中发送一个新的菌落(请参见注释8)。
    5. 您可以从测序公司的96孔板中获得质粒溶液。另外,您也可以自己从LB-琼脂板上标记的菌落制备质粒,这些菌落在测序中呈阳性。

数据分析

测序数据分析

  1. 将测序数据另存为计算机中的文件夹中的.fasta,.seq或.txt文件。
  2. 打开MutantChecker(图3),然后将参考的未突变DNA序列粘贴到指示的字段(参考序列)中。序列应以“程序”步骤A3中定义的第一个密码子开始。分别分析使用正向引物测序的突变DNA和使用反向引物测序的突变DNA。有关更多详细信息,请参见Sun et al。(2013)。
  3. 对于使用正向引物测序的DNA,请使用氨基酸编号更改相应字段中 Report mut from #和 To 中提供的默认值。通常,正向序列应覆盖约200至250个氨基酸。
  4. 检查程序设置后,单击“文件”→“加载文件列表”。选择一个或多个要检查的文件。我们建议最初一开始检查文件。如果一次分析多个文件,则只会显示最后选择的文件的对齐方式。
  5. 现在,最大的窗口将显示参考序列和测序结果的比对(图3)。右边的图表指示已找到多少可能的路线。理想情况下,您应该会看到一个尖峰。右下角的窗口显示序列名称,后跟在步骤3输入的限制内发现的任何突变(请参见注释7)。
  6. 对于使用反向引物测序的DNA,请使用 RC From 和 RC To 字段指示应报告突变的氨基酸范围。RC代表反向补码。检查使用反向引物测序的DNA时,勾选 ReverseComplement 框。
  7. 重复步骤4-5分析剩余的测序数据。


    图3. MutantChecker软件界面。以突变R104A的测序数据分析为例。右侧的图显示了比对得分函数 vs。与测序结果和参考序列之间的偏差(Sun et al。,2013)。在这种情况下,只有一个峰,表明找到了一个可能的比对。由于重复的序列(通常表示克隆伪像),多个峰可能暗示了几种替代比对。右下方的输出窗口中列出了在指定的氨基酸范围(1-200)中识别的突变。“ E198 @”表示从氨基酸E198开始不清楚测序迹线(测序结果中的“ n”)。

笔记

  1. Gibson组装不需要引物修饰(如磷酸化),DNA寡核苷酸的最小合成规模通常绰绰有余。将冷冻的引物在-20°C下存储最多2年。几个冻融循环对DNA寡核苷酸的稳定性没有重大影响。
  2. 除Phusion外,DNA聚合酶可能需要不同的PCR方案。
  3. 此步骤可将初始变性过程中的底涂最小化。如果使用热启动聚合酶,则可能没有必要。
  4. 可以使用商业Gibson装配预混液或类似的DNA装配产品来代替自制的Gibson装配合剂。
  5. 最近,我们成功地在45°C下孵育了1 h,事实证明它可以提高Gibson组装的效率(Gibson et al。,2010)。如果将引物设计成相对较短的重叠以提高其特异性(例如,15 bp),则这可能特别有益。
  6. 细胞应采用井上法制备(Sambrook和Russell,2006),每1μgpBR322(或类似质粒)的转化效率至少为10 7 个菌落。或者,可以使用其他一些 E。常规克隆常用的大肠杆菌菌株,例如Mach1,DH5α或TOP10。
  7. 根据对DNA进行测序的公司,痕迹的注释可能会有所不同。一些公司决定在迹线不清楚时不显示“ N”,而是选择最高峰。在这两种情况下,都将报告不匹配,但是当迹线不包含Ns时,可以将其视为真正的突变。我们建议您在类似情况下检查跟踪,即使报告的突变发生在测序结果的开头或结尾。尤其是在序列末尾,四个或多个相同类型碱基(所有G,所有C,所有As或所有T)的重复可能导致难以解释的痕迹,从而导致 n 基被读取为 n -1或 n +1,从而导致移码。通常这是一个排序问题,而不是真正的移码。这些情况或许多Ns拖尾的序列可能导致接近主峰的其他峰。
  8. 有关故障排除,请参阅原始出版物(Heydenreich 等人,2017年)。

菜谱

  1. 5个等温反应(IT)缓冲液(6毫升)
    1.5克PEG 8000
    3毫升Tris-HCl(1 M,pH 7.5)
    300μlMgCl 2 (1 M)
    300μlDTT(1 M)
    600μlNAD + (50 mM)
    600μldNTPs溶液混合物(每个dNTP 10 mM)
    准备5倍IT缓冲液的500μl等分试样并将其储存在-80°C
  2. 1.33x吉布森装配混合物(1.8毫升)
    480μl5x IT缓冲液
    0.96μlT5核酸外切酶(10 U /μl)
    30 µl Phusion HF DNA聚合酶(2 U /μl)
    240μlTaq DNA连接酶(40 U /μl)
    1,049μl无核酸酶水
    制成50微升等分试样,并在-20°C(或-80°C进行长期存储)中存储
    注意:Gibson装配混合物可以在-20°C下保存一年以上,并且经过10个以上的冻融循环后,酶仍然具有活性(Gibson等,2009)。
  3. 具有抗生素的LB-琼脂平板
    1. 在1升超纯去离子水中添加35克LB肉汤和琼脂
    2. 在121°C下高压灭菌15分钟,以溶解粉末并对LB-琼脂灭菌
    3. 让其冷却至手热温度( ca。 45°C),添加适当抗生素的储备溶液以达到目标抗生素浓度,并剧烈旋转以使其混合
    4. 向每个直径10厘米的培养皿中倒入 ca。 15毫升融化的带有抗生素的LB琼脂,使其固化
    5. 对于96孔LB琼脂板,请使用多通道移液器将每孔150μl含抗生素的LB琼脂转移到平底96孔板中,并使琼脂凝固
    6. 板应倒置保存在4°C的密封塑料袋中,直到使用

致谢

该协议源自我们先前的出版物(Heydenreich et al。,2017)。这项工作得到了FMH,Erasmus,EMBO短期研究金ASTF 420–2016的瑞士国家科学基金会移动研究金165219的支持以及瑞士国家科学基金会博士机动性补助金31003A_159748 / 2旅行研究金以及NanoMem ITN网络对TM,NRW的资助战略项目BioSC BOOST资金“ RIPE”授予海因里希海涅大学生物化学植物生理学研究所,居里夫人(274497)和瑞银Promedica基金会为DM提供博士后研究金,瑞士国家科学基金会为DBV和COST授予141898、159748行动CM1207闪亮。

利益争夺

作者声明没有任何竞争性的金融或非金融利益。

参考文献

  1. Edelheit,O.,Hanukoglu,A.和Hanukoglu,I.(2009)。简单高效的定点诱变,同时使用两个单引物反应来生成蛋白质结构突变体功能研究。 BMC Biotechnol 9:61。
  2. 吉布森(Gibson)DG,史密斯(Smith),何(HO),哈奇森(Hutchison),加利福尼亚,第3,温特(JC)和迈里曼(C. 小鼠线粒体基因组的化学合成。 自然方法 7(11):901-903。
  3. Gibson,DG,Young,L.,Chuang,RY,Venter,JC,Hutchison,CA,3rd and Smith,HO(2009)。酶促组装高达数百千碱基的DNA分子。 自然方法< 6(5):343-345。
  4. Heydenreich,FM,Miljuš,T.,Jaussi,R.中,Benoit,R.,米利ć ,D。和Veprintsev,DB(2017)。使用两片段PCR方法进行高通量诱变。 科学代表 7(1):6787。
  5. Liu H.和Naismith,JH(2008)。一种有效的一步定点定点缺失,插入,单和多位点质粒诱变方案。 BMC Biotechnol 8:91。
  6. 奥尔特加角,阿布鲁乌角,奥佩佐,P。和科雷亚角(2019)。 后基因组时代的高通量克隆方法概述。 方法分子生物学2025:3-32。
  7. Sambrook,J。和Russell,DW(2006)。制备和转化胜任的 E的井上方法。大肠杆菌:“超能力”细胞。 CSH协议 2006(1)。
  8. Sun,D.,Ostermaier,MK,Heydenreich,FM,Mayer,D.,Jaussi,R.,Standfuss,J.和Veprintsev,DB(2013)。 AAscan,PCRdesign和MutantChecker:一套用于高通量扫描的引物设计和序列分析程序诱变。 PLoS One 8(10):e78878。
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引用:Heydenreich, F. M., Miljuš, T., Milić, D. and Veprintsev, D. B. (2020). High-throughput Site-directed Scanning Mutagenesis Using a Two-fragment PCR Approach. Bio-protocol 10(1): e3484. DOI: 10.21769/BioProtoc.3484.
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