The genome of
Polymerase chain reaction (PCR) based cloning of gene of interest with high GC content is a long recognized problem. PCR is a most sensitive tool and various factors have to be optimized for amplification of gene of interest. Primer is one of the precise control elements in this process. Designing of primers directly influences the result of standardized cloning procedures. High GC content of the gene generates complication during primer designing like mismatch and high annealing temperature, self-dimer formation, and secondary structure. Sometimes, amplification of gene is not routinely achieved by normal PCR techniques. The most prominent problem associated is hairpin loop, which directly interferes during annealing of primers on difficult DNA template that leads to no amplification. Different strategies have been proposed to sort out this problem. Use of DMSO and glycerol was reported to reduce the annealing temperature and denaturation temperature, increase the chances of breakage of secondary structure, and increase the efficiency of amplification [
In an attempt to clone GC rich genes (
Degeneracy of codon is normally used to overcome the existing problem including change of base at wobble position specific for coding sequence of
Comparison of standard (normal) and modified primer sequences and its secondary structure for amplification of GC rich sequences.
Gene | Primers (5′-3′) | GC content | Secondary structure |
|
Gene annealed at |
---|---|---|---|---|---|
16s DNA |
|
60 | — | — | 58°C |
|
60 | ||||
|
|||||
|
|
60 |
|
−0.92 | 63.5°C |
|
55.6 |
|
−1.29 | ||
|
|||||
|
|
64 |
|
−3.67 | — |
|
55.6 |
|
−1.87 | ||
|
60 |
|
−0.31 | 64.5°C | |
|
55.6 |
|
−1.25 | ||
|
|||||
|
|
60 |
|
−0.97 | — |
|
46.4 |
|
−1.45 | ||
|
57.7 |
|
−0.97 | 62°C | |
|
53.3 |
|
−1.19 |
NF: normal forward; NR: normal reverse; MF: modified forward; MR: modified reverse.
Data evaluation by oligo-analyzer tools on IDT: integrative DNA technology.
The primers were designed based on the genomic sequence deposited in GenBank under accession number NC_000962.3 and sequence of
Strategies for the cloning of complicated DNA sequences are of the most significance and it has to be optimised through simple procedures. For PCR based cloning of genes, primer is one of the crucial factors for successful amplification of the genes to be cloned. Length of primer and annealed matches increase the specificity of the reaction but it may not always be an authentic reason to get desired amplicons. With the development of sequencing technology, many tools have been developed to design primers. But the noticeable points with these databases were the variability of primers properties like annealing temperature, prediction of secondary structure, and so forth. Successful amplifications were performed even with primer pairs that were generated through integrative oligoanlyser tools, at annealing temperature close to predicted value (
Prediction of secondary structure. (a) Presence of secondary structures in the primers designed by standard primer designing. Secondary structure halts the progression of polymerase. (b) Modification of bases at wobble position distorts secondary structure and allows moving of polymerase. Base modifications are shown in light red circle and the possible hydrogen bond is shown in square box.
Amplification pattern of 16S rDNA,
On analysis of terminal regions of the
A major benefit of this protocol was to resolve the problem of Tm mismatch as well as existence of secondary structure in the primer pairs of high GC rich sequences. By using this approach we could adjust the Tm mismatch without increasing the length of primers which may be responsible for dimerization. The high Tm of primers, which become a setback in carrying out PCR, was easily solved by this method (Figure
Schematic presentation of the annealing primer mismatch and use of wobble hypothesis approach to sort out this problem.
By using the wobble hypothesis approach in primer designing, we were able to tackle the ongoing amplification problem associated with complicated gene of
The authors declare that there is no conflict of interests regarding the publication of this paper.
Authors duly acknowledge the Department of Biotechnology, Council of Scientific and Industrial Research (CSIR), Indian Council of Medical Research (ICMR), India, for providing financial assistance to Jagdeep Kaur and SRF to Arbind Kumar.