Simplified oligonucleotide gene mixing assay
Simplified oligonucleotide gene mixing assay
Improvements in the biochemical properties of proteases can be achieved through misdirected mutagenesis and DNA hybridization of the enzyme genes. The hybridization technique can be used for a set of mutants of the same gene or for new combinations of fragments of related family genes to produce chimeric mutant gene products. The source of this experiment is "A Laboratory Guide to Modern Protein Engineering" [German] K.M. Arndt, K.M. Miller, eds.
Operation method
short oligonucleotide gene mix
Materials and Instruments
pBSII KS Plasmid E. coli DH5α Move We take the DNA recombinant D. thermophilum Rt46B.1 xylanase gene (xynB) and its five related xylanase genes as an example [7] to illustrate the general process of DOGS. Figure 11.1 shows the framework diagram of the whole experiment. It includes the design of primers, primer extension to amplify gene fragments, then integration of these fragments, and finally amplification of full-length chimeric genes (Note 1). For more product details, please visit Aladdin Scientific website.
Pfx polymerase Alkaline phosphatase Universal buffer Cover solution Congo red solution Decolorizing solution Birch xylan substrate solution PAHBAH Storage solution
3.1 Complementary primer pairs allow for efficient amplification of gene fragments and easy joining of overlapping neighboring fragments.
The most common method of isolating distantly related genes is to design parsimonious primers based on highly conserved sequences in genes and then isolate these genes by PCR. The difficulty with this method is that as primer parsimony increases to fit more genes, the number of correct primers to guide synthesis of the PCR reaction decreases, and these primers may be used up in the first few cycles of the reaction. At this point nonspecific amplification can occur in the presence of a large number of primers that are not involved in the amplification of the target gene, especially if less stringent annealing temperatures are used during the mismatch template process.
In order to overcome these problems with parsimonious primers in parsimonious oligonucleotide gene recombination, Rose et al. [13] invented a method called Consensus-Only Parsimonious Oligonucleotide Hybridization (CODEHOP).CODEHOP primers consist of a relatively short parsimonious Y-terminus and a universal unparsimonious 5' terminus.Minimizing the length of the 3' terminus reduces the number of primers in the entire parsimonious primer library. Binding of the parsimonious 3' end to the template can be stabilized by the non-parsimonious 5' end, allowing for higher annealing temperatures without the need to increase library parsimony. Although potential mismatches between the 5' end and the target sequence can occur during the initial PCR cycle, they are relatively far from the 3' end extension site, and therefore the mismatch rarely affects the initiation of polymerase extension. Further amplification of the gene product is significantly enhanced by the similarity of primer sequences in the primer pool; this also suggests that more primers are available.The DOGS method allows for more efficient amplification of gene fragments in the overlap region by improving CODEHOP. The overlapping extension of neighboring gene fragments from different genes can lead to the generation of chimeric genes. Suitable primer sequence design is the most important aspect of the DOGS method.
Improvements to the CODEHOP method require well-designed complementary primer pairs. Each primer has a non-compact core flanked by the compact 5' and 3' ends, which are referred to here as complementary compact end (CDE) primers. Like the primers in CODEHOP, the condensed 3' end allows the CDE primer to bind specifically to the template, while the non-condensed region stabilizes the PCR cycle that follows. The condensed 5' end does not contribute to the binding of the CDE primer to the template during the PCR process, but is mainly used to efficiently bind and overlap the PCR products (gene fragments) that are separated from each other and amplified by the forward or reverse CDE primers, respectively.
The core region of each CDE primer that is not parsimonious is usually based on the coding sequence on the corresponding gene, which is often the parental gene used for hybridization. This ensures that the chimeric gene retains the sequence of the parental gene at the site of recombination.
3.1.1 Design and use of gene-specific nested end primers
( 1 ) Design and synthesize suitable forward and reverse primers for amplification of each gene to be chimerized. Each primer should contain 17-20 nucleotides at the gene-specific 3' end and 17-20 nucleotides at the universal 5' end. We introduce cleavage sites into the universal primers for targeted ligation of PCR products into the pBSII KS vector.
( 2 ) Two nested primers are synthesized with sequences relative to the universal end of the gene-specific forward and reverse nested primers. This nested primer will be used in combination with the CDE primer to amplify the first and last parts of each gene.
( 3 ) Amplify each gene with the designed gene-specific primers, typically from the genome or from other clones that have the gene.
3.1.2 Summary of CDE Primer Characteristics
CDE primers can efficiently amplify fragments from genes that are less homologous. the 5' condensed end of the CDE primer ensures that fragments amplified by forward and reverse complementary CDE primers will anneal efficiently in the subsequent overlapping extension PCR step. Furthermore, consecutive gene fragments generated by multiple CDE primers carry complementary ends suitable for overlap extension and PCR, allowing for the generation of recombinant fragments. Figure 11.2 gives an example of CDE primers designed to amplify gene fragments from related genes and to generate chimeras in subsequent overlapping extensions.
CDE primers can also be used in combination with complementary condensed primers that do not contain a non-simplex core region to obtain recombinant gene fragments by end complementation as well as overlapping extension PCR. Mixing these fragments from related genes followed by overlap extension and PCR can efficiently produce chimeric gene fragments. The non-simplex region of a CDE primer can (but does not have to) be designed based on a parental gene. Finally, fragments from different genes can be mixed in unequal amounts, allowing control of the new genes generated by the integration of the different fragments (see Note 2). Figure 11.3 summarizes the process of amplifying and overlapping extended gene fragments with CDE primers. 
3.1.3 Designing CDE primers to ensure efficient amplification of gene fragments and ligation of neighboring fragments
( 1 ) Perform sequence comparison of related proteins using suitable sequence comparison software, such as ChistalX.
( 2 ) Determine the conserved amino acid motifs. In our example, the genes of the xylanase family can be divided into 8 parts according to their conserved regions.
( 3 ) Compare the nucleotide sequences of the genes with a suitable sequence comparison software, such as Tranalign [ 15 ].
( 4 ) Based on the conserved DNA sequences, design CDE forward and reverse primers to amplify the conserved DNA. The gene fragments were then amplified with the appropriate addition of universal nested primers at the 5' and 3' ends (Figure 11.2).
( 5 ) Each fragment of each gene was amplified using adjacent CDE primers and universal 5' end and 3' end nested primers. In this example, the conditions of the PCR reaction were: 95°C for 1 min, 1 cycle; then 95°C for 30 s, 35°C for 20 s, and 72°C for 40 s, for a total of 35 cycles; and finally 72°C for 5 min, 1 cycle. The archaeal DNA polymerase, Platinum Pfx, is used in the PCR reaction (see Notes 3 and 4).
( 6 ) Each PCR product was recovered on a gel. 
3.1.4 Overlap extension of gene fragments
( 1 ) The fragments of each gene should be mixed in a suitable ratio to produce chimerism. For example, six candidate genes, G1 to G6, were used, where G1 is the D . ZiermojiiZmn Rt46B.1 gene. When it is decided to use G1 as the primary parental gene in the recombination, the PCR fragments for each gene are then mixed at a ratio of 8.75 times the G1 gene relative to an equal number of other genes so that, on average, 5/8 of the chimeric genes are derived from the Rt46B.1xynB (Note 5 gives a simple formula for this).
( 2 ) Next, a mixture of 50-100 ng of fragments was used as a template for overlap extension [16], applying the following PCR reaction system: 95°C for 1 min, 1 cycle; then 95°C for 30 s, 35°C for 20 s, 72°C for 40 s, for a total of 35 cycles; and finally 72°C for 5 min, 1 cycle. The archaeal DNA polymerase, Platinum Pfx, was used in the PCR reaction (see Note 3).
3.1.5 Amplification of full-length chimeric genes
The full-length chimeric recombinant gene is amplified by PCR using the overlapping extension product (50-100 ng, see 11.3.1.4) as a template with universal 5' and 3' nested primers under the following PCR conditions: 95°C for 1 min, 1 cycle, then 95°C for 30 s, 50°C for 20 s, 72°C for 40 s, for a total of 35 cycles, and finally 72°C for 5 min, 1 cycle. The PCR reaction conditions were as follows: 95°C for 1 min, then 95°C for 30s, 50°C for 20s, 72°C for 40s for a total of 35 cycles, and finally 72°C for 5 min and 1 cycle. Platinum Pfx polymerase was used in the PCR reaction.
3.1.6 Cloning of recombinant genes
( 1 ) The PCR product of DOGS was digested with Bam HI and Hind III enzymes.
(2) The pBSII KS-vector was digested with Bam HI and Hind III and then treated with shrimp alkaline phosphatase.
( 3 ) The PCR product was spliced to the pBSII KS-vector.
( 4 ) The conjugated vector (with ampicillin resistance) was transferred to the DH5α strain and applied to an LB plate containing 100 μg/ml ampicillin resistance and 5 mmol/L IPTG.
( 5 ) The monoclones were picked and backed up on a new LB plate with 100 μg/ml ampicillin resistance and 5 mmol/L IPTG, and then assayed for xylanase expression and activity according to the Congo Red Overlay method [17] described in section 11.3.1.7.
3.1.7 Congo Red Screening
( 1 ) Add 4 ml of overlay solution cooled to approximately 50°C to a plate with clones. The plate should be pre-warmed at 37°C to ensure uniform coverage.
( 2 ) When the coverage layer is formed, invert the plate into a sealed pocket. Leave sealed at 70°C for 3 h.
( 3 ) The flat plate is then removed, the bag removed and left to cool at room temperature.
( 4 ) Add 5 ml of Congo red solution to each plate so that it can fully cover the covering layer. Then leave for 5~10 min.
( 5 ) Pour out the remaining Congo red solution, and then dip the plate into the decolorizing solution to decolorize.
( 6 ) Clones that are not colored are xylanase gene positive clones.
Figure 11. 4 shows an example of recombinant xylanase production from the genes for six xylanases using the DOGS method (see Note 6). 
3.1.8 Xylanase activity assay (PAHBAH method)
Recombinant xylanase is extracted in whole cells for enzyme activity analysis using the protein extraction reagent BPER II. Xylanase activity analysis was determined by the Lever method using birch xylan as substrate. A standard reaction mixture with a final volume of 0.03 ml consisted of 120 mmol/L universal buffer (pH 6.5) [19], 0.5% ( m/V) xylan, and reductase. The reaction mixture was placed at 60°C for 20 min.
1 ) Cell lysis and enzyme extraction
( 1 ) Identified positive transformants were cultured overnight in 2 ml of LB containing 100 μg/ml ampicillin and IPTG.
( 2 ) Remove 1.5 ml of the overnight cultured LB, centrifuge at 11000 g for 30 s, and remove the supernatant.
( 3 ) Suspend the above cells by shaking, then add 150 μl of BPER II solution.
( 4 ) Lysed the cells by shaking for 1 min and centrifuged at 11000 g for 1 min to remove the cell debris.
( 5 ) Transfer the supernatant to a new tube and store at 4℃.
2 ) Xylanase activity assay
( 1 ) In a 0.2 ml PCR tube, add 5 μl of appropriately diluted enzyme extract to birch xylan substrate solution to a final volume of 50 μl.
( 2 ) Place the PCR tube in a PCR instrument with a heated top and incubate for 10~30 min at a suitable temperature.
( 3 ) Place the PCR tubes in ice water, add 100 μl of PAHBAH reservoir solution to terminate the enzyme reaction, and mix well.
( 4 ) Heat the above mixture in the PCR instrument at 99°C for 5 min for color change reaction. Ensure that the heatable lid is in the closed position. Then cool immediately to 4°C to stop the color change.
( 5 ) Mix the solution in the tube and transfer 100 μl to a flat-bottomed microtiter plate. Detect the absorption at A420 nm using a spectrometer with a suitable readable plate.