The dependence on polymerase chain reaction (PCR) as a fundamental analytical tool for molecular biology tests has increased rapidly. PCR as a synthetic tool can be used for recombining DNA sequences. In this PCR based recombination, the reliance on restriction sites is reduced. In this article, the technique and its uses are discussed briefly. Some related PCR applications are also discussed.
In PCR process, DNA polymerase is used for extension of the primer. A copy of a DNA strand is formed in this reaction step. This strand serves as a template for an extension by a second primer in the opposite orientation. This process is repeated for multiple rounds this leads to exponential accumulation of the sequence of interest. The result of this process is a DNA segment of defined length this is done by incorporating synthetic oligonucleotide primers into its ends. #i end primers should be able to meet certain demands such as it should match the sequence of the template gene and at the 5′ ends is capable of including sequences unrelated to the template gene. The extent of this attribute decides the capability of this primers to act as primers for DNA polymerase. The characteristic of the 5′ end is termed as mispriming it aids in site-directed mutagenesis and in addition of sequences at the end a PCR generated fragment. This technique is limited by the length of an oligonucleotide from the end of the PCR fragment, i.e. the mutation should take place in the primer. This method can make changes at positions close to the restriction sites.
The first use of this method is done by introducing mutations into the center of a PCR fragment. The resultant is a more flexible PCR mutagenesis. The intrinsic error frequency of this method is sufficiently low, making it practically successful in widespread use. A variant of this method made recombination of different segments from two different genes or “spliced” together by overlap extension. This process is termed as gene Splicing by Overlap Extension (SOE) or gene SOEing. These two ends are generated by PCR. The ends of these two fragments are modified by mispriming and they share a region of homology. Then these two fragments are mixed, denatured and reannealed, 3′ end of the top strand anneals with 3′ end of the bottom strand. This overlap can be extended to form a recombinant product. Primers decide the overlap region and they can contain any sequence limited only by the complementary length of the oligomers. Base changes incorporated in these regions leads to site-directed mutagenesis. Also when no new sequences included the overlap can be designed to make a “neat” joint between two fragments. Such a neat joint is described in this article. Construction of a recombinant gene encoding a chimeric protein where class I MHC antigen is replaced with class II MHC antigen is discussed here.
Various modification of PCR method has been used for a different cloning purpose. The methods can be enlisted as
- TA cloning
- Ligation independent cloning (LIC)
- Recombinase-dependent cloning
- PCR-mediated cloning
The choice of any cloning method enlisted above is majorly attributed to the reliability of the particular method. The convenience, price, and efficiency are minor deciding factors. Any method whose monitoring optimization is easy tends to become the most reliable method. The requirement of end modifications that cannot be monitored by gel electrophoresis makes TA cloning and LIC less reliable. Recombinases are generally sold as a preoptimized kit by the manufacturer which makes the optimization unavailable for a user to meet their own demands. Overlap extension PCR is a straightforward, efficient, and reliable.
Mode of working
A linear with plasmid sequences at both ends insert is created by a PCR reaction. The strands of the PCR product formed by these extensions act as a pair of oversized primers on the vector fragment. Following denaturation and annealing, hybridization of the vector and insert strands happen and this extends to form a new double-stranded plasmid. This technique is dependent on the Pusion DNA polymerase as it does not possess strand displacement activity. A double-stranded fusion plasmid consisting of two nicks is formed as the final product. Then these double-stranded plasmid is transferred to competent Escherichia coli cells. DNA repairing enzymes of the host seal the nicks. A thermostable polymerase is recommended to be used in both PCR reaction to avoid the strange behavior of PCR using multiple restriction enzymes, polymerases, glycosylases, recombinases, and ligases.
Factors affecting the efficiency Overlap Extension PCR
To demonstrate the factors affecting the result of this method gfp gene is used for the study. Primers: 5′ ends complementary to the pQE30 plasmid; 3′-end complementary to gfp is used to amplify gfp gene. Five different DNA polymerases in this Overlap extension PCR experiments. The efficiency of the overlap extension is controlled by two factors: High concentrations of the insert and relatively low annealing temperatures in the reaction. The annealing temperature is calculated as 5–10°C below the melting temperature of the primer/plasmid complex. The resultant products are analyzed using agarose gel analysis. Only a part of the PCR products corresponding to the relaxed form of the desirable vector. The reaction with DpnI restriction endonuclease destroyed the original pQE30 vector. A small aliquot of the reaction sample is then transformed into E. coli cells.
The results using different polymerase showed the better suitability of Phusion DNA polymerase for overlap extension PCR cloning than its counterparts. The resultant product of the Phusion DNA polymerase is 10× more processive than the native Pfu polymerase and 46× more colonies as produced. Almost 8% of the colonies transformed with DNA produced by Phusion DNA polymerase were visibly green. This represents a minimal cloning error or carryover of the original vector.
Another parameter to measure the efficiency of the overlap extension PCR cloning is expressed as a function of temperature cycles. The number of recombinant clones showed a geometric increase during the first 15 cycles. The number peaked at 17–18 cycles. While proceeding further with the cycles there was a decrease in the number of clones produced around 30%. An agarose gel analysis showed an accumulation of the high–molecular weight DNA products. Also, the ration of insert/ plasmid had a pronounced effect on the outcome of the reaction. Three different vector: insert ratios (1:5; 1:50 and 1:250) is also studied in overlap extension PCR cloning reaction with Phusion DNA polymerase. Agarose gel electrophoresis and recombinant clone numbers (green number colonies) are used as the judging parameters. The 1:250 ratio produced the most recombinant clones.
This study is further extended to find is suitability with various genes. Genes: GFP (gfp, 1 kb), β-D-glucuronidase (gusA, 1.9 kb), and β-galactosidase (lacZ, 3.2 kb), as well as the entire luxABCDE operon (6 kb), have experimented for this purpose. Protein function and structure of the recombinant vectors is kept as the resultant parameters. Structural analysis is confirmed by restriction analysis. The fraction of colonies that did not exhibit full reporter activity, had <3% of the size of the insert. When there is an increase in insert length the number of error colonies observed on plates after transformation decreased linearly. The upper limit for insert length is 6.7 kb in this technique.
One of the characteristics of the overlap extension PCR cloning reaction is as easy to monitor and optimize as any other long PCR protocol. The reaction conditions of the PCR determines the yield percentage. It should not be too stringent (primers fail to anneal) or too relaxed (nonspecific priming). Both produce empty lanes in the agarose gels, although smears or undesired bands have been seen in the latter condition. Reactant concentrations (primers, template), annealing temperature, buffer ingredients (magnesium, pH, DMSO) or the number of temperature cycles are the controlling parameters of a PCR reaction. The presence of the internal repeated elements does not affect this cloning approach.
Gene Splicing by Overlap Extension or “gene SOEing” described above is discussed in this article with this methodology. It is a PCR-based method of recombining DNA sequences. Non-reliance on restriction sites and the ability to directly generate in vitro mutated DNA fragments are few attributes of this method. By using different sequences that can be incorporated into the 5′-ends of the primers, results in a various pair of polymerase chain reaction products. These pairs share a common sequence at one end. An overlap is formed during PCR reaction. When induced polymerase chain reaction conditions, the common sequence allows strands from two different fragments to hybridize to one another. This forms an overlap. When this overlap is extended by DNA polymerase yields a recombinant molecule.
Materials and methods
Oligonucleotide primers were synthesized DNA synthesizer and desalted on a Sephadex G-50 column.
The class I sequences were derived from a plasmid containing Kb, and the class II sequences were derived from plasmids containing Aαk and Aβk.
The sequences of the eight primers are used. Primers ‘a’ and ‘h’ are the flanking or “outside” primers, which serve to PCR amplify the final recombinant product. They do not contribute to the sequences added at the overlapping ends. Oligomers ‘b’ and ‘c,’ ‘d’ and ‘e,’and ‘f’ and ‘g’ are the SOEing primers. The members of each pair are related because bases have been added to their 5′-ends to make them complementary to one another. In each case, the overlap region between the primers, and the priming region by which each primer recognizes its template was designed to have an estimated Td of approximately 50°C according to the formula Td= 4 (C+G) + 2(A+T) in degrees Celsius. In practice, we have found that simply making these regions 15 to 16 nucleotides long generally works well. We have not made a careful examination of the minimum length of the oligomers.
In the primers all of the complementary bases have been added to one of the two primers (primers ‘b,’ ‘e’ and ‘g’), rather than adding some sequence to each primer. This way, the other primers (‘c,’ ‘d’ and ‘f’) can potentially be used with new SOEing primers (analogous to ‘b,’ ‘d’ and ‘g’) to join these fragments to other genes. Since the two templates share three nucleotides in primers ‘f’ and ‘g,’ these nucleotides contribute both to the overlap and to the priming portion of oligomer ‘g’. The portion of oligomer ‘e’ in parentheses is not related to either template and does not contribute to the overlap. This is an example of insertional mutagenesis being carried out simultaneously with recombination.
As an example of the SOEing process, the complementary regions ‘d’ and ‘e’ containing the sequences which lead to the PCR products AD and EH having overlapping ends.
PCR and SOE reactions were carried out in a thermocycler for 25 cycles, each consisting of 1 min at 94oC, 2 min at 50oC, and 3 min at 72oC. (The reaction probably produces all of the product in fewer than 25 cycles, but we have not examined the minimum number of cycles required.) Taq polymerase was from PerkinElmer Cetus, and the reaction buffer was as recommended by the supplier (50 mM KCl, 10 mM Tris-Cl, pH 8.3, 1.5 mM MgCl2, 0.01% (wt/vol) gelatin). Deoxyribonucleotides were used at a final concentration of 200 μM. The buffer and deoxynucleoside triphosphates were each made as a 10x stock, and 10 μl was used per 100 μl reaction. One-half μl of polymerase (2.5 U) was used per reaction. Reactions were covered with mineral oil before thermal cycling.
Purification of Fragments
PCR and SOEn products which were to be used as templates in further reactions were purified by electrophoresis through agarose ( 1% SeaKem LE agarose + 2% NuSieve GTG agarose) in TAE buffer (0.04 M Tris-acetate, 0.001 M EDTA) with 0.5 μg/ml ethidium bromide in the gel. DNA from the appropriate bands was recovered from the gel fragment by GeneClean. The final recombinant product was similarly gel-purified before cloning.
Cloning of Fragments
The SOEn products were cut with restriction enzymes Sall and Xhol and ligated into the corresponding position of a pUC-derived plasmid which has been designed to act as an expression vector for class I MHC antigen binding regions, as described elsewhere.
Analysis of Products
The cloned product was sequenced from the double-stranded template using a Sequenase kit with a modified protocol. A total of approximately 1700 bases sequenced from the product in the above reaction. One observation of an unplanned mutation is reported for all the cases. This may be caused due to the misincorporation by the polymerase. The error frequency reported, therefore, is compatible for future uses.
This approach can be used for the construction of complicated genes. This methodology has been used to create and express a gene of fusion proteins in which the a helixes are replaced by the corresponding segments of a similar gene. In the above methodology construction of a complicated structure is explained. The methodology constructs a hybrid using four gene segments from three different genes. At least two is used at a time to produce a chimeric product. Simultaneously, a 15-base pair (bp) segment encoding a portion of the class I gene which was in a different exon from the one that was amplified was added at the recombination joint. Thus enabling the use of this method in incorporating complicated structural genes and recombination- insertional mutagenesis is achieved in one step.
Synthetic Uses of PCR
Modifications to the basic SOE concept are technically possible. A modified approach known as “mega-primer” approach is used in several applications. This approach uses fewer primers. Another modification of inserting that fragments directly to a vector, and then recircularized by blunt-end ligation is also demonstrated. The use of restriction enzymes for the construction of recombinant genes in appropriate vectors is entirely avoided in this approach. Different methods of PCR usage as a synthetic tool incorporates a varied approach to this technique. An asymmetric PCR has been used to generate a huge mutagenic oligonucleotide for use. The creation of this huge mutagenic oligonucleotide otherwise uses standard m13- based mutagenesis strategy, which is capable of replacing regions large as an exon.
Limitations of SOE
The major drawback of the gene SOEing technique is that it is necessary to sequence the cloned products even though the frequency of polymerase errors is low, to be certain that you have what you want. This majorly limit the usefulness of gene SOEing to engineering problems while these are answered by more conventional methods. The possibility of random recombinations between related genes present in the same reaction in the PCR reaction, during directed recombination, may also occur. In case of partial elongation of a fragment of one gene, this an act as a primer on a different gene and also this may result in producing a recombinant product. This property may be positive or a hazard based on the application desired.