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 . 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 . 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 [ 15 ] . the genes of m. tuberculosis are being cloned and expressed in e. coli cells in order to identify their possible role in mycobacterium life . the mycobacterium genome has very high gc content ( 66% ) which raised the possibility of hairpin structure in the genomic structure . from genome sequence analysis it was observed that ppe , pe , and pgrs multigene family code for proteins of approximately 11080 amino acids rich in proline and glutamic acid at n - terminal position . proline and glutamic acid residues are mainly coded by triplet of gc bases in mycobacterium genome . most of the genes for membrane proteins of m. tuberculosis were rich in gc content at terminal regions . presence of high gc content increased the annealing temperature beyond the extension temperature ( 72c ) and also repeated stretches generate the hairpin structure . in such cases , effectiveness and reproducibility of pcr amplification depend on detailed analysis of the possible secondary structures of the oligonucleotide primers as well as formation of self - dimers and cross - dimers with other interrelating oligonucleotides . though these problems have been considered by several investigators , no systematic details are available to approach this problem . in an attempt to clone gc rich genes ( rv0519c and rv0774c from m. tuberculosis and ml0314c from m. leprae ) from mycobacterium sp . , we designed primers by using standard method for gene amplification . rv0774c and rv0519c genes demonstrated 100% nucleotide identity in m. tuberculosis h37rv and m. tuberculosis h37ra . therefore , m. tuberculosis h37ra chromosomal dna was used as template for amplification of these two genes . we could amplify rv0774c gene , but rv0519c and ml0314c genes having high gc content at terminal region were not amplified by standard pcr procedures . therefore , an attempt has been made in the present investigation to standardize the conditions and ingredients that favor the amplification of gc rich sequences . taq polymerase and dntps were purchased from fermentas , usa . restriction enzymes were purchased from new england biolabs . kanamycin , middlebrook media , oadc , and tween 80 were purchased from hi - media , india . the strain mycobacterium tuberculosis h37ra was a kind gift from director of national institute of leprosy and other mycobacterial diseases , agra , india . mallika lavania of stanley browne laboratory , the leprosy mission , nandnagri , shahdara , new delhi . m. tuberculosis h37ra strains were routinely cultured for one week on middlebrook , 0.05% tween 80 , enriched with oadc . one week grown m. tuberculosis h37ra cells ( 1.5  ml ) were harvested by centrifugation at 5000  g for 20  min . harvested pellet was resuspended in 400  l tris - edta buffer ( 100  mm tris/10  mm edta , ph 8) . the cells were lysed by putting the sample in boiling water bath for 5  min followed by cooling on ice for 5  min . forty microliters of 20  mg / ml lysozyme and 5  l of proteinase k were added . after 1  h of incubation at 37c , solution a ( 56  l of 10% sds , 64  l of ctab - nacl ) was added to the reaction mixture . after 2  h of incubation at 65c , proteins were removed by 2 - 3 times of washing with phenol  :  chloroform  :  isoamyl alcohol ( 25  :  24  :  1 ) . the genomic dna was precipitated with 0.6 volume of isopropanol at room temperature for 1  h. the precipitated dna was dried and dissolved in sterile water and stored at 20c . degeneracy of codon is normally used to overcome the existing problem including change of base at wobble position specific for coding sequence of mycobacterium genome . designed forward primer of rv0519c contributes about 64% gc content and stretches of gc led to generation of complicated hairpin structure with high value of free energy change g . by carefully examining the hairpin structure , introduction of the small base / pair the incorporation we opted in the primer sequence was as follows : guanine ( g ) base turned into adenosine ( a ) at wobble position of third codon cgg and thymine ( t ) to adenine ( a ) in codon cgt ( table 1 ) . similarly in reverse primer of rv0519c primer sequence , the adenosine ( a ) base was turned into thymine ( t ) at wobble position of last sixth codon cga . the primers were designed based on the genomic sequence deposited in genbank under accession number nc_000962.3 and sequence of ml0314c was retrieved from leprosy database ( http://mycobrowser.epfl.ch/leprosy.html ) . pcr was carried out in 25  l reaction volumes containing 75  ng genomic dna templates , 2.5  mm of dntp mix , 4  mm mgso4 , 1.0  m of each primer sets , 1  u/l taq polymerase , and 1x tris buffer containing kcl ( fermentas , usa ) and 5% dmso ( v / v ) . the sequences of both forward and reverse primers are listed in table 1 . these sets of primers were used to clone rv0774c and rv0519c genes from isolated genomic dna of m. tuberculosis h37ra ( table 1 ) and ml0314c gene from m. leprae genomic dna as follows : denaturation for 4  min at 94c , then 30 cycles consisting of 50  s at 94c , 40  s at 63.3c , and 2  min at 72c and then 7  min at 72c for final extension . pcr product was analyzed on 1.5% agarose gel and purified with rbc column dna extraction kit . the gene sequences of rv0519c and ml0314c were analysed and the modified primers were reconstructed for the amplification of gene . we followed the above given pcr procedure to amplify the gene with annealing temperatures of 64.5c and 62c . to confirm the sequence of amplified product , the purified pcr products for all three genes were sent for sequencing with specific primers . 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 ( http://eu.idtdna.com/analyzer/applications/oligoanalyzer/ ) . high gc content in the genes of mycobacterium species generate stable secondary structures which often form in the oligos dna that halt the progression of dna polymerase during amplification ( figures 1(a ) and 1(b ) ) . in the present study , the primer properties were evaluated through different most widely used oligo designing tools such as idt , sigma , oc , and manual . current study illustrated the need to balance length and melting temperature with respect to gc content of gene at terminal sites , while designing primers for the pcr . it also emphasised the importance of careful investigation of sequences for gc - rich repeats , giving rise to complicated secondary structures , which could reduce the efficiency of amplification . we successfully amplified the rv0774c gene with normal pcr primers and procedure ( figure 2(a ) , lane 3 ) . on the other hand , no amplification was observed in case of rv0519c gene ( gc content 69% ) under same reaction conditions ( figure 2(a ) , lane 4 ) . attempt had been made to amplify the gene with a long range of annealing temperatures but without success . application of dmso and glycerol was also checked with different concentrations ( 310% ) with no visible effect on the amplification of the rv0519c gene ( data not shown ) . the16s rdna was amplified with dna template ( figure 2(a ) , lane 1 ) . on analysis of terminal regions of the rv0774c and rv0519c genes the presence of stable hairpin structures was not observed in case of rv0774c , despite the high gc content while the high gc content at terminal region of rv0519c gene led to formation of strong hairpin loop structure formation during normal primer designing procedure ( table 1 ) . in normal forward primer the value of g is more towards negative side ( g = 3.67  kcal / mol ) , and a complicated hairpin structure formation was observed in case of standard reverse primer used for amplification of rv0519c gene ( table 1 ) . thus , it was essential to decrease the gc content and change of corresponding nucleotides of oligos according to the wobble hypothesis approach . therefore , we designed the primers by using the degeneracy of codon and checked the hairpin loop formation by integrative dna - oligoanalyser . all the introduced modifications at wobble position were analysed on idt tools for their effectiveness to distort hairpin . out of five changes , one by one and together , we observed that single / double point changes were able to distort the whole secondary structure ( data not shown ) . we introduced small change at nucleotide level , starting with codon cgg to cga and cgt to cga during designing of forward primer sequence while single nucleotide change cgt to cga in case of reverse primer distorted the possible hairpin structure . introduction of small changes at nucleotide level during primer designing reduced the chances of hairpin loop formation ( g = 0.31  kcal / mol ) , resulting in sharp amplification of rv0519c gene through normal pcr procedures ( figure 2(c ) , lane 2 ) . similar problem was observed in amplification of ml0314c gene ( gc content , 60% ) from m. leprae while 16s rdna was successfully amplified from m. leprae genome ( figure 2(b ) , lane 3 ) . primers designed for ml0314c gene from normal procedures demonstrated mismatch annealing temperature as well as strong hairpin loop structure ( table 1 ) . this problem was also tackled successfully by changing nucleotide as well as addition of nucleotide at terminal position , upstream to the restriction sites to sort out the annealing mismatch between the primers . g for reverse primer was changed from 1.45 to 1.19  kcal / mol . the single nucleotide modification at wobble position tcg to tcc distorted the whole hairpin structure . the strategy was successfully confirmed by amplification of ml0314c gene ( figure 2(d ) , lane 1 ) from m. leprae genome . 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 3 ) . this strategy might work successfully for amplification of ppe , pe , and pgrs protein of m. tuberculosis containing large number of proline and aspartate residues that are mostly coded by repeats or stretch of g or c , which increased the chance of hairpin loop formation and annealing mismatch during standard pcr primer designing . by using the wobble hypothesis approach in primer designing , we were able to tackle the ongoing amplification problem associated with complicated gene of mycobacterium with high gc content . in this approach a single modification at base level the resulting gene sequence is different slightly from the canonical sequence of the reference genome without modification in the amino acid sequence of the protein .