Nowadays, PCR has become a powerful research tool in the field of molecular biology and is used in all aspects of modern molecular biology. A variety of PCR methods have been invented to clone genes, and thus PCR has been widely used in gene cloning and vector construction, and has become an essential complement to conventional recombinant gene technology. Conventional cloning and recombinant DNA techniques require digestion of DNA fragments with restriction enzymes and joining of two fragments with ligase. However, sometimes the gene to be cloned does not have a suitable restriction site and cannot be cloned by conventional recombinant DNA technology. To overcome this difficulty, PCR cloning methods that do not require a ligation reaction have been developed, and thus ligation-independent cloning PCR (LIC-PCR) has emerged. Two different ligation-independent PCR cloning methods have been invented, both of which produce single-stranded ends of about 10-12 bases at the ends of the amplified DNA fragments, which anneal specifically to the complementary sequence of the vector and can then be used to transform the susceptible bacterium without the need for a ligation reaction. One was invented by Aslanidis and deJongW, who designed their PCR primers so that only three nucleic acids were present in the 12 or more bases at the open end of the primer - the resulting PCR product was then digested with T4 DNA polymerase and the digest was spiked with nucleic acids that were not present at the 3' end of the product. Another method of creating protruding ends on both ends of a PCR product is to use uracil DNA glycosylase (UDG).
Principle
The basic principle of ligation-independent cloning PCR experiments is the use of uracil DNA glycosylase (UDG), an enzyme involved in the biosynthesis of TTP that hydrolyzes the N-glycosylation bond between the deoxyribose moiety and uracil. This enzymatic reaction produces a dU residue that is dealkylated, thereby disrupting the base pairing of the DNA. The method involves adding a deoxyuracil-doped tail to the 5' end of the primer and selectively removing the protruding deoxyuracil from the 5' end of the PCR product with UDG. These products can then be annealed to a suitable vector containing complementary sequences so that the vector and the inserted exogenous fragment form a loop. This recombinant plasmid can then be efficiently transformed into E. coli (Figures 10-15). It is important in this method that the 3' protruding end of the product contains a sufficient number of nucleic acid residues to allow stable and efficient annealing of the PCR-generated DNA fragments inserted into the vector. It is also important to insert dUMP at the junction of the PCR template and the complementary sequence of the vector during primer design, as well as to add a few dUMP residues at the 5' end of the oligonucleotide primer, which, after UDG cleavage, causes the other bases in the oligonucleotide to lose interaction with the complementary strand, resulting in the protruding end of the exiting single-stranded DNA fragment.
Appliance
Common applications of PCR experiments for ligation-independent cloning are as follows: 1. Site-specific mutagenesis using PCR and UDG cloning The use of deoxyuridine-containing oligonucleotides as primers in the PCR process is a convenient way to generate overhanging mutations at the 3' end of the nucleotide (5). In the case of plasmid point mutations, two overlapping primers containing the target nucleic acid mutation were synthesized, and all the 5' thymidine residues were replaced with deoxyuridine. The 5' end of the PCR product was sensitive to UDG, and the dU residue was excised from the PCR product by treatment with UDG, resulting in the creation of a 3' mucosal terminus, which was annealed, cyclized, and repaired in vivo after transformation into Escherichia coli, resulting in the generation of a new plasmid molecule that was not only the desired plasmid molecule, but also the new plasmid molecule, which was also the first to be transformed into a plasmid. The new plasmid molecule is identical to the wild-type parent plasmid except for the desired mutation. Sometimes the gene of interest is not a plasmid, or the plasmid is too large for complete amplification. Then the gene can be amplified twice and cloned using UDG. In this process, two mutant primers are designed that contain dUs and can overlap, and each mutant primer is then subjected to independent PCR with a suitable 5' or 3' primer, both of which contain the dU sequence, so that the UDG can be cloned into a suitable vector. The obtained amplification products were mixed with the vector and UDG in a test tube to produce chimeric molecules containing the target mutation, and the obtained circular chimeric plasmids were directly used for transformation of receptive E. coli cells, and the clones carrying the chimeric plasmids contained the target mutation. 2. Generation of new genes Rashtchian et al. (6) used LIC-PCR to clone the gene encoding the human Ciliary Neurotropism Factor (CNTF), because CNTF is a gene that encodes the human Ciliary neuroproliferative factor. Rashtchian et al. (6) used LIC-PCR to clone the gene encoding the human Ciliary Neurotrophic Transforming Factor (CNTF), which consists of an intron and two exons separated by an intron, and needed to be cloned from the genome by PCR. In the PCR primer design, deoxyuridine was used instead of thymidine to produce 3 single strands (as shown in Figure 10-16). In this way, the two exons were assembled into the full-length sequence of CNTF, and the assembled sequence was sequenced and analyzed, and it was identical to the wild-type cDNA sequence. In the same way, the precursor sequence of nerve growth factor was cloned. There are countless precedents in molecular biology that the exon sequences of genes were amplified from genomic DNA by PCR, and then assembled into the full sequence of genes by splicing. RACE has been successfully used to obtain cDNA when mRNA is scarce, and UDG cloning has been used successfully in 3" RACE and 5" RACE [E]. Other applications include the design of chimeric toxins, chimeric antibodies, structural domain changes in proteins, and other mutations. PCR is now applied to a diverse family of antibodies. This may be mainly due to the fact that both the variable and constant regions of antibodies have conserved sequences at the 5' and 3' ends. Direct amplification of the conserved sequences of an antibody allows the construction of an antibody cD- NA library containing a large number of different light and heavy chains. In this library, a single vector can express different fragments of each antibody. Of course, these techniques can only be applied in some laboratories, and the application of UDG in antibody engineering also includes humanized antibodies, etc. 3. Future Applications PCR has been widely used in molecular biology, and with the emergence of new PCR techniques, its application will be even more extensive. Recently, significant progress has been made in the amplification of DNA fragments by PCR. Barnes (1) mixed DNA polymerase and a DNA polymerase with 3'-end exonuclease activity, and successfully amplified a 35 kb long fragment of DNA.O Because long fragments of DNA may contain a large number of restriction sites, it may be impractical to use restriction enzyme cloning methods. The TA cloning method is also impractical because a polymerase with 3' end exonuclease activity removes the 3' end of the overhang in long fragment PCR, creating a flat end. In this case, the ligation-free PCR method is particularly suitable because it does not require restriction enzyme digestion and removal of the extra nucleic acid at the 3' end. PCR without ligation is used in gene synthesis, where oligonucleotides with overlapping fragments can be linked together by PCR annealing without ligation. Although most researchers are constantly looking for high-fidelity methods to clone genes, high-fidelity PCR is certainly useful. However, Cadwell and JoyceW make full use of low-fidelity PCR reactions to generate a range of mutants, and Arnold also uses this method to generate mutant proteins. The random mutations produced by this method are used to screen for mutants that increase catalytic activity. Combined with DNA shuffling, it can be used to rapidly study the evolution of proteins in vivo, and thus to discover new active pairs of proteins.7 Of course, for these methods to be successful, a large number of different genes have to be cloned efficiently and screened effectively. PCR cloning methods that do not require a ligation reaction, with their high ligation efficiencies and ease of use in DNA libraries, will allow for their development in these areas. The use of UDG to clone DNA fragments from a range of genes has proved to be an extremely efficient method. In addition, it is a common method for joining DNA fragments at any point in the DNA sequence without utilizing restriction sites. This feature allows for the rapid discovery of new genes and DNA structures, giving molecular biologists and protein engineers more options.
Operation method
Cloning PCR experiments that do not depend on the ligation reaction
Principle
The basic principle of ligation-independent cloning PCR experiments is the use of uracil DNA glycosylase (UDG), an enzyme involved in the biosynthesis of TTP that hydrolyzes the N-glycosylation bond between the deoxyribose moiety and uracil. This enzymatic reaction produces a dU residue that is dealkylated, thereby disrupting the base pairing of the DNA. The method involves adding a deoxyuracil-doped tail to the 5' end of the primer and selectively removing the protruding deoxyuracil from the 5' end of the PCR product with UDG. These products can then be annealed to a suitable vector containing complementary sequences so that the vector and the inserted exogenous fragment form a loop. This recombinant plasmid can then be efficiently transformed into E. coli (Figures 10-15). It is important in this method that the 3' protruding end of the product contains a sufficient number of nucleic acid residues to allow stable and efficient annealing of the PCR-generated DNA fragments inserted into the vector. It is also important to insert dUMP at the junction of the PCR template and the complementary sequence of the vector during primer design, as well as to add a few dUMP residues at the 5' end of the oligonucleotide primers, which, after UDG cleavage, cause the other bases in the oligonucleotide to lose interaction with the complementary strand, resulting in the protruding end of the exiting single-stranded DNA fragment.
Materials and Instruments
Equipment: Move The basic process of cloning PCR experiments that do not depend on linkage reactions can be divided into the following steps:
PCR instrument, electrophoresis apparatus.
Reagents:
① Upstream primer P1 (CUACUACUACUACUA at the 5' end) and downstream primer P2 (CAU- CAUCAUCAUCAU at the 5' end) of the gene to be amplified.
②Template (mRNA or cDNA)
(iii) Heat-stabilized polymerase and 10X buffer.
④10 mmol/L dNTP
⑤ pAMPl vector (25 ng/μL)
⑥Annealing buffer
⑦ Uracil DNA glycosylase (1 U/μL)
(i) Primer design
A The upstream primer R for LIC-PCR is CUACUACUACUACUA at the 5' end, plus the complementary sequence of the gene to be amplified; the downstream primer P2 is CAUCAUCAUCAUCAU at the 5' end, plus the complementary sequence of the gene to be amplified.
(ii) PCR reaction
A Follow the standard PCR reaction system, add the required reagents and carry out the standard PCR reaction.
(iii) Annealing reaction
A Add the following reagents to a 0.5 mL centrifuge tube (on ice)
B Mix well and incubate at 37 °C for 30 min on ice.
(iv) Conversion
A Take 1~5 mL of the annealed reaction solution and transform the DH5a receptor bacteria.
Caveat
1 A 44 bp positive control DNA is provided in the CLONEAMP kit and is annealed with Knock 1 (approximately 25 ng).2 The diversity of recombinants is directly related to the diversity of PCR products, i.e., if a PCR product contains many different amplicons, it is likely that each PCR product will contain a primer-specific linker fragment of deoxyribonucleic acid at the end. This is an ideal way to build PCR libraries so that you can select the ideal clones for your needs.3 Some amplification reactions may produce off-purpose products such as primer dimers, which require primer redesign. dTMP can be used instead of dUMP so that the problem of primer dimerization caused by dU does not arise.
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