Research Article Primer Based Approach for PCR ...downloads.hindawi.com/journals/mbi/2014/937308.pdfResearch Article Primer Based Approach for PCR Amplification of High GC Content

Research ArticlePrimer Based Approach for PCR Amplification of High GCContent Gene Mycobacterium Gene as a Model

Arbind Kumar and Jagdeep Kaur

Department of Biotechnology Panjab University Sector 14 Chandigarh 160014 India

Correspondence should be addressed to Jagdeep Kaur jagsekhonyahoocom

Received 21 August 2013 Revised 27 November 2013 Accepted 28 January 2014 Published 24 March 2014

Academic Editor Alessandro Desideri

Copyright copy 2014 A Kumar and J Kaur This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The genome ofMycobacterium is rich in GC content and poses problem in amplification of some genes especially those rich in theGC content in terminal regions by standardroutine PCR procedures Attempts have been made to amplify three GC rich genes ofMycobacterium sp (Rv0519c andRv0774c fromM tuberculosis andML0314c fromM leprae) Out of these three genesRv0774c genewas amplified with normal primers under standard PCR conditions while no amplification was observed in case of Rv0519c andML0314c genes In the present investigation a modified primer based approach was successfully used for amplification of GC richsequence of Rv0519c through codon optimization without changing the native amino acid sequence The strategy was successfullyconfirmed by redesigning the standard primers with similar modifications followed by amplification ofML0314c gene

1 Introduction

Polymerase chain reaction (PCR) based cloning of gene ofinterest with high GC content is a long recognized problemPCR is a most sensitive tool and various factors have to beoptimized for amplification of gene of interest Primer is oneof the precise control elements in this process Designing ofprimers directly influences the result of standardized cloningprocedures High GC content of the gene generates com-plication during primer designing like mismatch and highannealing temperature self-dimer formation and secondarystructure Sometimes amplification of gene is not routinelyachieved by normal PCR techniques The most prominentproblem associated is hairpin loop which directly interferesduring annealing of primers on difficult DNA template thatleads to no amplification Different strategies have beenproposed to sort out this problem Use of DMSO and glyc-erol was reported to reduce the annealing temperature anddenaturation temperature increase the chances of breakageof secondary structure and increase the efficiency of amplifi-cation [1ndash5] The whole genome sequence of Mycobacteriumtuberculosis was deciphered by Cole et al [6] The genes ofM tuberculosis are being cloned and expressed in E coli cells

in order to identify their possible role in Mycobacteriumlife The Mycobacterium genome has very high GC content(66) which raised the possibility of hairpin structure in thegenomic structure From genome sequence analysis it wasobserved that PPE PE and PGRS multigene family codefor proteins of approximately 110ndash80 amino acids rich inproline and glutamic acid at N-terminal position Prolineand glutamic acid residues are mainly coded by triplet ofGC bases in Mycobacterium genome Most of the genesfor membrane proteins of M tuberculosis were rich in GCcontent at terminal regions Presence of high GC contentincreased the annealing temperature beyond the extensiontemperature (72∘C) and also repeated stretches generate thehairpin structure In such cases effectiveness and repro-ducibility of PCR amplification depend on detailed analysisof the possible secondary structures of the oligonucleotideprimers as well as formation of self-dimers and cross-dimerswith other interrelating oligonucleotides [7] Though theseproblems have been considered by several investigators nosystematic details are available to approach this problem

In an attempt to clone GC rich genes (Rv0519c andRv0774c from M tuberculosis and ML0314c from M leprae)from Mycobacterium sp we designed primers by using

Hindawi Publishing CorporationMolecular Biology InternationalVolume 2014 Article ID 937308 7 pageshttpdxdoiorg1011552014937308

2 Molecular Biology International

GCC

AT

GC

T A C GG

GTG

CG

GG G A T C T G T G

CCATGCTA C G

GGT

GCG

G GAT

C T G T G

G A T C C A T T G T

CT

AC

GA C

GA

GG

C

Base modification at wobble positions

PCR PCRPolymerase

Polymerization Polymerization

5998400

59984003

998400

3998400

(a) (b)

Figure 1 Prediction of secondary structure (a) Presence of secondary structures in the primers designed by standard primer designingSecondary structure halts the progression of polymerase (b)Modification of bases at wobble position distorts secondary structure and allowsmoving of polymerase Base modifications are shown in light red circle and the possible hydrogen bond is shown in square box

standardmethod for gene amplificationRv0774c andRv0519cgenes demonstrated 100 nucleotide identity in M tuber-culosis H37Rv and M tuberculosis H37Ra Therefore M tu-berculosis H37Ra chromosomal DNA was used as templatefor amplification of these two genes We could amplifyRv0774c gene but Rv0519c and ML0314c genes having highGC content at terminal regionwere not amplified by standardPCR procedures Therefore an attempt has been made inthe present investigation to standardize the conditions andingredients that favor the amplification of GC rich sequences

2 Materials and Methods

21 Materials E coliDH5120572 cells and pET-28a were procuredfrom Invitrogen Taq polymerase and dNTPs were purchasedfrom Fermentas USA Restriction enzymes were purchasedfromNewEngland Biolabs KanamycinMiddlebrookmediaOADC and tween 80 were purchased from Hi-Media IndiaThe strain Mycobacterium tuberculosis H37Ra was a kindgift from Director of National Institute of Leprosy and

Other Mycobacterial Diseases Agra India Genomic DNAfor Mycobacterium leprosy was a kind gift from Dr MallikaLavania of Stanley Browne Laboratory the Leprosy MissionNandnagri Shahdara New Delhi

22 Mycobacterium Genomic DNA Isolation M tuberculosisH37Ra strains were routinely cultured for one week onmiddlebrook 005 Tween 80 enriched with OADC Oneweek grown M tuberculosis H37Ra cells (15mL) were har-vested by centrifugation at 5000timesg for 20min Harvestedpellet was resuspended in 400120583L Tris-EDTA buffer (100mMTris10mM EDTA pH 8) The cells were lysed by puttingthe sample in boiling water bath for 5min followed bycooling on ice for 5min Forty microliters of 20mgmLlysozyme and 5 120583L of proteinase K were added After 1 h ofincubation at 37∘C solution A (56120583L of 10 SDS 64 120583Lof CTAB-NaCl) was added to the reaction mixture After2 h of incubation at 65∘C proteins were removed by 2-3times of washing with phenol chloroform isoamyl alcohol(25 24 1) The genomic DNA was precipitated with 06

Molecular Biology International 3

16

S rD

NA

100

bp M

Rv0774c

Rv0519c

(a)

100

bp M

Rv0519c

(b)

16

S rD

NA

100

bp M

ML0

314c

(c)

100

bp M

ML0

314c

(d)

Figure 2 Amplification pattern of 16S rDNA Rv0519 and Rv0774cgenes from M tuberculosis (Figures 2(a) and 2(b)) ML0314c and16S rDNA fromM leprae (Figures 2(c) and 2(d)) (a) Amplificationof 16S rDNA Rv0519 Rv0774c genes with standard primers (b)amplification of Rv0519 with modified primers (c) amplificationof 16S rDNA and ML0314c genes with standard primers and (d)amplification ofML0314c with modified primers

volume of isopropanol at room temperature for 1 h TheprecipitatedDNAwas dried and dissolved in sterile water andstored at minus20∘C

23 Modification of Primers by Codon Optimization Degen-eracy of codon is normally used to overcome the existingproblem including change of base at wobble position specific

for coding sequence of Mycobacterium genome Designedforward primer ofRv0519c contributes about 64GCcontentand stretches of GC led to generation of complicated hairpinstructure with high value of free energy change ΔG Bycarefully examining the hairpin structure introduction ofthe small basepair distorted the whole secondary structureThe incorporation we opted in the primer sequence wasas follows guanine (G) base turned into adenosine (A) atwobble position of third codon CGG and thymine (T) toadenine (A) in codon CGT (Table 1) Similarly in reverseprimer of Rv0519c primer sequence the adenosine (A) basewas turned into thymine (T) at wobble position of last sixthcodon CGA Mycobacterium leprae genome sequence alsohas high guanine and cytosine stretches Reverse primersequence of ML0314c of leprae gene was also modifiedGuanine was turned into cytosine at wobble position ofTCG codon The effect of modification was analysed by IDToligoanalyzer tools

24 Amplification of GC Rich Sequences of Mycobacteriumsp The primers were designed based on the genomic se-quence deposited in GenBank under accession numberNC 0009623 and sequence of ML0314c was retrieved fromLeprosy database (httpmycobrowserepflchleprosyhtml)PCR amplifications were performed with Taq polymerase(Fermentas USA) PCR was carried out in 25120583L reactionvolumes containing 75 ng genomic DNA templates 25mMof dNTP mix 4mM MgSO

4 10 120583M of each primer sets

1 U120583L Taq polymerase and 1X Tris Buffer containing KCl(Fermentas USA) and 5 DMSO (vv) The sequences ofboth forward and reverse primers are listed in Table 1 Thesesets of primers were used to clone Rv0774c and Rv0519cgenes from isolated genomic DNA ofM tuberculosis H37Ra(Table 1) and ML0314c gene from M leprae genomic DNAas follows denaturation for 4min at 94∘C then 30 cyclesconsisting of 50 s at 94∘C 40 s at 633∘C and 2min at72∘C and then 7min at 72∘C for final extension 16S rDNAwas used as positive control PCR product was analyzedon 15 agarose gel and purified with RBC column DNAextraction kit Amplification of Rv0519c and ML0314c wasnot achieved through normal PCR procedures The genesequences of Rv0519c and ML0314c were analysed and themodified primers were reconstructed for the amplification ofgene We followed the above given PCR procedure to amplifythe gene with annealing temperatures of 645∘C and 62∘CPCRproducts were analyzed on 15 agarose gel and purifiedTo confirm the sequence of amplified product the purifiedPCR products for all three genes were sent for sequencingwith specific primers

3 Results and Discussion

31 Problem with Amplification of Mycobacterium GeneStrategies for the cloning of complicated DNA sequences areof the most significance and it has to be optimised throughsimple procedures For PCR based cloning of genes primeris one of the crucial factors for successful amplificationof the genes to be cloned Length of primer and annealed


Table 1 Comparison of standard (normal) andmodified primer sequences and its secondary structure for amplification ofGC rich sequences

Gene Primers (51015840-31015840) GCcontent Secondary structure Δ119866

(Kcalmol)Gene

annealed at

16s DNAF-GAG GAA GGT GGG GAT GAC GT 60 mdash mdash 58∘C

R-AGG CCC GGG AAC GTA TTC AC 60

Rv0774c

NF-GGATCCATGATGCCCGCATGCCAGA 60

GTAC

CT

AG

G

GCA

CCCG

TA

GC

T

AG

A

C

5998400

3998400

10

20

minus092

635∘C

NR-AAG CTT TTA ACC TGT GAG CAG CGG CGC 556

CAAT T T T

CGC A G

CGG

CGC

AA

AG

C T GTG

5998400

3998400

10

20

minus129

Rv0519

NF-GGATCC ATG CTA CGG CGT GGC TGTG 64

AT

C

G

T

A

C

C

T

AG

G

CG

G

C

G

T

G

GC

TG

TG

5998400

3998400

10

20

minus367

mdash

NR-AAGCTTCTAGGACGCCATCCA AGCCAG 556

CG

C

A

G C

C

A

A

G

C

C

A

G

GATC T

T

C

G

A

A

C

A

T

5998400

3998400

10

20

minus187

MF-GGA TCC ATG CTA CGA CGA GGC TGT G 60

GC

A

T

CGT

AC

C

TAG

G

T

GT

G

G

C

G

AC

GA

5998400

3998400

10

20

minus031

645∘C

MR-AAGCTTCTAGGACGCCAT CCA TGC CAG 556

GC

A

G

GA

TC

T

T

CG

AA

CC

A

T

C

C

C

A

G

C

CA

G

5998400 3

998400

1020

minus125


Table 1 Continued


(Kcalmol)Gene

annealed at

ML0314c

NF-GAATTCTTGGCCATCAGCGCCGGAG 60

TA

C

C

G

CA

G

CG

C

C

G

GA

G

GAA

T

T

CT

T G

5998400

3998400

10

20

minus097

mdash

NR-AAGCTT CTA GAG GCT TTG CCT GTC TCT A 464

GCC

TG

TC

TC

T

A

TTT

CG

GAG

ATCTTCG A A

5998400

3998400

10

20

minus145

MF-AGAATTCTTGGCCATCAGCGCCGGAG 577

T CA

G

CG

C

C

G

G

A

GGA

AT

T

CT

T G

G

C

CA

5998400

3998400

10

20

minus097

62∘C

MR-GG AAG CTT CTA GAG CCT TTG CCT GTC TCT A 533

TTT

G

C

C

CG

T

C

TC

TAA

AG

CTTC

TAG

A G

G

C

5998400

3998400

10

20

minus119

NF normal forward NR normal reverse MF modified forward MR modified reverseData evaluation by oligo-analyzer tools on IDT integrative DNA technology

matches increase the specificity of the reaction but itmay not always be an authentic reason to get desiredamplicons With the development of sequencing technologymany tools have been developed to design primersBut the noticeable points with these databases were thevariability of primers properties like annealing temperatureprediction of secondary structure and so forth Successfulamplifications were performed even with primer pairsthat were generated through integrative oligoanlysertools at annealing temperature close to predicted value(httpeuidtdnacomanalyzerapplicationsoligoanalyzer)High GC content in the genes of Mycobacterium speciesgenerate stable secondary structures which often form in theoligos DNA that halt the progression of DNA polymeraseduring amplification (Figures 1(a) and 1(b)) In the presentstudy two genes ofM tuberculosiswere selected and primerswere designedThe primer properties were evaluated throughdifferent most widely used oligo designing tools such as IDTSigma OC and manual Lots of variations were observedin annealing temperature of the same primer sequenceCurrent study illustrated the need to balance length and

melting temperature with respect to GC content of geneat terminal sites while designing primers for the PCR Italso emphasised the importance of careful investigation ofsequences for GC-rich repeats giving rise to complicatedsecondary structures which could reduce the efficiency ofamplification [8] We successfully amplified the Rv0774cgene with normal PCR primers and procedure (Figure 2(a)lane 3) On the other hand no amplification was observed incase of Rv0519c gene (GC content 69) under same reactionconditions (Figure 2(a) lane 4) Attempt had been made toamplify the gene with a long range of annealing temperaturesbut without success Application of DMSO and glycerol wasalso checked with different concentrations (3ndash10) with novisible effect on the amplification of the Rv0519c gene (datanot shown)The16S rDNA was amplified with DNA template(Figure 2(a) lane 1)

On analysis of terminal regions of the Rv0774c andRv0519c genes we observed very high GC content (Table 1)The presence of stable hairpin structures was not observedin case of Rv0774c despite the high GC content while thehigh GC content at terminal region of Rv0519c gene led to


18bp 74∘C

Base change at wobble position

Solved primer annealing mismatch

Low annealing temperaturerandom binding

High annealing temperatureno binding

Annealing temperatureclose to one primerbinding

Nonspecific amplification

No amplification Very less strand synthesis not visible

Gene sequence amplified

18bp 61∘C 18bp 64∘C 18bp 61∘C

5998400

5998400

5998400

5998400

3998400

3998400

3998400

3998400

5998400

5998400

3998400

3998400

5998400

3998400 5

998400

3998400

Figure 3 Schematic presentation of the annealing primer mismatch and use of wobble hypothesis approach to sort out this problem

formation of strong hairpin loop structure formation duringnormal primer designing procedure (Table 1) In normalforward primer the value ofΔG ismore towards negative side(ΔG = minus367 Kcalmol) and a complicated hairpin structureformation was observed in case of standard reverse primerused for amplification of Rv0519c gene (Table 1) Such highminusΔG value and complicated hairpin structure might createproblem during amplification proceduresThus it was essen-tial to decrease the GC content and change of correspondingnucleotides of oligos according to the wobble hypothesisapproach Therefore we designed the primers by using thedegeneracy of codon and checked the hairpin loop forma-tion by integrative DNA-oligoanalyser All the introducedmodifications at wobble position were analysed on IDT toolsfor their effectiveness to distort hairpin Out of five changesone by one and together we observed that singledoublepoint changes were able to distort the whole secondarystructure (data not shown) We introduced small change atnucleotide level starting with codon CGG to CGA and CGTto CGA during designing of forward primer sequence whilesingle nucleotide change CGT to CGA in case of reverseprimer distorted the possible hairpin structure Introductionof small changes at nucleotide level during primer design-ing reduced the chances of hairpin loop formation (ΔG =minus031 Kcalmol) resulting in sharp amplification of Rv0519cgene through normal PCR procedures (Figure 2(c) lane 2)Similar problem was observed in amplification of ML0314cgene (GC content 60) fromM leprae while 16S rDNA wassuccessfully amplified from M leprae genome (Figure 2(b)lane 3) Primers designed for ML0314c gene from normalprocedures demonstrated mismatch annealing temperatureas well as strong hairpin loop structure (Table 1) Similarapproachwas used tomodify the forward and reverse primers

forML0314c geneThis problem was also tackled successfullyby changing nucleotide as well as addition of nucleotide atterminal position upstream to the restriction sites to sortout the annealing mismatch between the primers ΔG forreverse primer was changed from minus145 to minus119 KcalmolThe single nucleotide modification at wobble position TCGto TCC distorted the whole hairpin structure The strategywas successfully confirmed by amplification ofML0314c gene(Figure 2(d) lane 1) from M leprae genome Sequencing ofPCR products confirmed the amplification of specific genes

A major benefit of this protocol was to resolve theproblem of Tm mismatch as well as existence of secondarystructure in the primer pairs of high GC rich sequencesBy using this approach we could adjust the Tm mismatchwithout increasing the length of primers which may beresponsible for dimerization The high Tm of primers whichbecome a setback in carrying out PCR was easily solved bythis method (Figure 3)This strategy might work successfullyfor amplification of PPE PE and PGRS protein of Mtuberculosis containing large number of proline and aspartateresidues that are mostly coded by repeats or stretch of G orC which increased the chance of hairpin loop formation andannealing mismatch during standard PCR primer designing

4 Conclusion

By using the wobble hypothesis approach in primer design-ing we were able to tackle the ongoing amplification problemassociated with complicated gene of Mycobacterium withhigh GC content In this approach a single modification atbase level distorted the secondary structure and resolvedthe primer Tm mismatch The resulting gene sequence is


different slightly from the canonical sequence of the referencegenome without modification in the amino acid sequence ofthe protein

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

Authors duly acknowledge theDepartment of BiotechnologyCouncil of Scientific and Industrial Research (CSIR) IndianCouncil of Medical Research (ICMR) India for providingfinancial assistance to Jagdeep Kaur and SRF to ArbindKumar

References

[1] P R Winship ldquoAn improved method for directly sequencingPCR amplified material using dimethyl sulphoxiderdquo NucleicAcids Research vol 17 no 3 pp 1266ndash1269 1989

[2] B Bachmann W Luke and G Hunsmann ldquoImprovement ofPCR amplified DNA sequencing with the aid of detergentsrdquoNucleic Acids Research vol 18 no 5 pp 1309ndash1314 1990

[3] D Pomp and J F Medrano ldquoOrganic solvents as facilitators ofpolymerase chain reactionrdquo BioTechniques vol 10 no 1 pp 58ndash59 1991

[4] K Varadaraj and D M Skinner ldquoDenaturants or cosolventsimprove the specificity of PCR amplification of a G + C-richDNA using genetically engineered DNA polymerasesrdquo Genevol 140 no 1 pp 1ndash5 1994

[5] T Weissensteiner and J S Lanchbury ldquoStrategy for controllingpreferential amplification and avoiding false negatives in PCRtypingrdquo BioTechniques vol 21 no 6 pp 1102ndash1108 1996

[6] S T Cole R Brosch J Parkhill et al ldquoDeciphering the biologyof Mycobacterium tuberculosis from the complete genomesequencerdquo Nature vol 393 no 6685 pp 537ndash544 1998

[7] S Chavali A Mahajan R Tabassum S Maiti and D Bharad-waj ldquoOligonucleotide properties determination and primerdesigning a critical examination of predictionsrdquoBioinformaticsvol 21 no 20 pp 3918ndash3925 2005

[8] D Tang H Jiang Y Zhang Y Li X Zhang and T ZhouldquoCloning and sequencing of the complicated rDNA gene familyof Bos taurusrdquo Czech Journal of Animal Science vol 51 no 10pp 425ndash428 2006

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Advances in

Virolog y

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Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


GCC

AT

GC

T A C GG

GTG

CG

GG G A T C T G T G

CCATGCTA C G

GGT

GCG

G GAT

C T G T G

G A T C C A T T G T

CT

AC

GA C

GA

GG

C

Base modification at wobble positions

PCR PCRPolymerase

Polymerization Polymerization

5998400

59984003

998400

3998400

(a) (b)

Figure 1 Prediction of secondary structure (a) Presence of secondary structures in the primers designed by standard primer designingSecondary structure halts the progression of polymerase (b)Modification of bases at wobble position distorts secondary structure and allowsmoving of polymerase Base modifications are shown in light red circle and the possible hydrogen bond is shown in square box

standardmethod for gene amplificationRv0774c andRv0519cgenes demonstrated 100 nucleotide identity in M tuber-culosis H37Rv and M tuberculosis H37Ra Therefore M tu-berculosis H37Ra chromosomal DNA was used as templatefor amplification of these two genes We could amplifyRv0774c gene but Rv0519c and ML0314c genes having highGC content at terminal regionwere not amplified by standardPCR procedures Therefore an attempt has been made inthe present investigation to standardize the conditions andingredients that favor the amplification of GC rich sequences

2 Materials and Methods

21 Materials E coliDH5120572 cells and pET-28a were procuredfrom Invitrogen Taq polymerase and dNTPs were purchasedfrom Fermentas USA Restriction enzymes were purchasedfromNewEngland Biolabs KanamycinMiddlebrookmediaOADC and tween 80 were purchased from Hi-Media IndiaThe strain Mycobacterium tuberculosis H37Ra was a kindgift from Director of National Institute of Leprosy and

Other Mycobacterial Diseases Agra India Genomic DNAfor Mycobacterium leprosy was a kind gift from Dr MallikaLavania of Stanley Browne Laboratory the Leprosy MissionNandnagri Shahdara New Delhi

22 Mycobacterium Genomic DNA Isolation M tuberculosisH37Ra strains were routinely cultured for one week onmiddlebrook 005 Tween 80 enriched with OADC Oneweek grown M tuberculosis H37Ra cells (15mL) were har-vested by centrifugation at 5000timesg for 20min Harvestedpellet was resuspended in 400120583L Tris-EDTA buffer (100mMTris10mM EDTA pH 8) The cells were lysed by puttingthe sample in boiling water bath for 5min followed bycooling on ice for 5min Forty microliters of 20mgmLlysozyme and 5 120583L of proteinase K were added After 1 h ofincubation at 37∘C solution A (56120583L of 10 SDS 64 120583Lof CTAB-NaCl) was added to the reaction mixture After2 h of incubation at 65∘C proteins were removed by 2-3times of washing with phenol chloroform isoamyl alcohol(25 24 1) The genomic DNA was precipitated with 06


16

S rD

NA

100

bp M

Rv0774c

Rv0519c

(a)

100

bp M

Rv0519c

(b)

16

S rD

NA

100

bp M

ML0

314c

(c)

100

bp M

ML0

314c

(d)













(Kcalmol)Gene

annealed at



Rv0774c


GTAC

CT

AG

G

GCA

CCCG

TA

GC

T

AG

A

C

5998400

3998400

10

20

minus092

635∘C


CAAT T T T

CGC A G

CGG

CGC

AA

AG

C T GTG

5998400

3998400

10

20

minus129

Rv0519


AT

C

G

T

A

C

C

T

AG

G

CG

G

C

G

T

G

GC

TG

TG

5998400

3998400

10

20

minus367

mdash


CG

C

A

G C

C

A

A

G

C

C

A

G

GATC T

T

C

G

A

A

C

A

T

5998400

3998400

10

20

minus187


GC

A

T

CGT

AC

C

TAG

G

T

GT

G

G

C

G

AC

GA

5998400

3998400

10

20

minus031

645∘C


GC

A

G

GA

TC

T

T

CG

AA

CC

A

T

C

C

C

A

G

C

CA

G

5998400 3

998400

1020

minus125


Table 1 Continued


(Kcalmol)Gene

annealed at

ML0314c


TA

C

C

G

CA

G

CG

C

C

G

GA

G

GAA

T

T

CT

T G

5998400

3998400

10

20

minus097

mdash


GCC

TG

TC

TC

T

A

TTT

CG

GAG

ATCTTCG A A

5998400

3998400

10

20

minus145


T CA

G

CG

C

C

G

G

A

GGA

AT

T

CT

T G

G

C

CA

5998400

3998400

10

20

minus097

62∘C


TTT

G

C

C

CG

T

C

TC

TAA

AG

CTTC

TAG

A G

G

C

5998400

3998400

10

20

minus119






18bp 74∘C









18bp 61∘C 18bp 64∘C 18bp 61∘C

5998400

5998400

5998400

5998400

3998400

3998400

3998400

3998400

5998400

5998400

3998400

3998400

5998400

3998400 5

998400

3998400





4 Conclusion






Acknowledgment


References
















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


16

S rD

NA

100

bp M

Rv0774c

Rv0519c

(a)

100

bp M

Rv0519c

(b)

16

S rD

NA

100

bp M

ML0

314c

(c)

100

bp M

ML0

314c

(d)













(Kcalmol)Gene

annealed at



Rv0774c


GTAC

CT

AG

G

GCA

CCCG

TA

GC

T

AG

A

C

5998400

3998400

10

20

minus092

635∘C


CAAT T T T

CGC A G

CGG

CGC

AA

AG

C T GTG

5998400

3998400

10

20

minus129

Rv0519


AT

C

G

T

A

C

C

T

AG

G

CG

G

C

G

T

G

GC

TG

TG

5998400

3998400

10

20

minus367

mdash


CG

C

A

G C

C

A

A

G

C

C

A

G

GATC T

T

C

G

A

A

C

A

T

5998400

3998400

10

20

minus187


GC

A

T

CGT

AC

C

TAG

G

T

GT

G

G

C

G

AC

GA

5998400

3998400

10

20

minus031

645∘C


GC

A

G

GA

TC

T

T

CG

AA

CC

A

T

C

C

C

A

G

C

CA

G

5998400 3

998400

1020

minus125


Table 1 Continued


(Kcalmol)Gene

annealed at

ML0314c


TA

C

C

G

CA

G

CG

C

C

G

GA

G

GAA

T

T

CT

T G

5998400

3998400

10

20

minus097

mdash


GCC

TG

TC

TC

T

A

TTT

CG

GAG

ATCTTCG A A

5998400

3998400

10

20

minus145


T CA

G

CG

C

C

G

G

A

GGA

AT

T

CT

T G

G

C

CA

5998400

3998400

10

20

minus097

62∘C


TTT

G

C

C

CG

T

C

TC

TAA

AG

CTTC

TAG

A G

G

C

5998400

3998400

10

20

minus119






18bp 74∘C









18bp 61∘C 18bp 64∘C 18bp 61∘C

5998400

5998400

5998400

5998400

3998400

3998400

3998400

3998400

5998400

5998400

3998400

3998400

5998400

3998400 5

998400

3998400





4 Conclusion






Acknowledgment


References
















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




(Kcalmol)Gene

annealed at



Rv0774c


GTAC

CT

AG

G

GCA

CCCG

TA

GC

T

AG

A

C

5998400

3998400

10

20

minus092

635∘C


CAAT T T T

CGC A G

CGG

CGC

AA

AG

C T GTG

5998400

3998400

10

20

minus129

Rv0519


AT

C

G

T

A

C

C

T

AG

G

CG

G

C

G

T

G

GC

TG

TG

5998400

3998400

10

20

minus367

mdash


CG

C

A

G C

C

A

A

G

C

C

A

G

GATC T

T

C

G

A

A

C

A

T

5998400

3998400

10

20

minus187


GC

A

T

CGT

AC

C

TAG

G

T

GT

G

G

C

G

AC

GA

5998400

3998400

10

20

minus031

645∘C


GC

A

G

GA

TC

T

T

CG

AA

CC

A

T

C

C

C

A

G

C

CA

G

5998400 3

998400

1020

minus125


Table 1 Continued


(Kcalmol)Gene

annealed at

ML0314c


TA

C

C

G

CA

G

CG

C

C

G

GA

G

GAA

T

T

CT

T G

5998400

3998400

10

20

minus097

mdash


GCC

TG

TC

TC

T

A

TTT

CG

GAG

ATCTTCG A A

5998400

3998400

10

20

minus145


T CA

G

CG

C

C

G

G

A

GGA

AT

T

CT

T G

G

C

CA

5998400

3998400

10

20

minus097

62∘C


TTT

G

C

C

CG

T

C

TC

TAA

AG

CTTC

TAG

A G

G

C

5998400

3998400

10

20

minus119






18bp 74∘C









18bp 61∘C 18bp 64∘C 18bp 61∘C

5998400

5998400

5998400

5998400

3998400

3998400

3998400

3998400

5998400

5998400

3998400

3998400

5998400

3998400 5

998400

3998400





4 Conclusion






Acknowledgment


References
















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table 1 Continued


(Kcalmol)Gene

annealed at

ML0314c


TA

C

C

G

CA

G

CG

C

C

G

GA

G

GAA

T

T

CT

T G

5998400

3998400

10

20

minus097

mdash


GCC

TG

TC

TC

T

A

TTT

CG

GAG

ATCTTCG A A

5998400

3998400

10

20

minus145


T CA

G

CG

C

C

G

G

A

GGA

AT

T

CT

T G

G

C

CA

5998400

3998400

10

20

minus097

62∘C


TTT

G

C

C

CG

T

C

TC

TAA

AG

CTTC

TAG

A G

G

C

5998400

3998400

10

20

minus119






18bp 74∘C









18bp 61∘C 18bp 64∘C 18bp 61∘C

5998400

5998400

5998400

5998400

3998400

3998400

3998400

3998400

5998400

5998400

3998400

3998400

5998400

3998400 5

998400

3998400





4 Conclusion






Acknowledgment


References
















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


18bp 74∘C









18bp 61∘C 18bp 64∘C 18bp 61∘C

5998400

5998400

5998400

5998400

3998400

3998400

3998400

3998400

5998400

5998400

3998400

3998400

5998400

3998400 5

998400

3998400





4 Conclusion






Acknowledgment


References
















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





Acknowledgment


References
















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Primer Based Approach for PCR ...downloads.hindawi.com/journals/mbi/2014/937308.pdfResearch Article Primer Based Approach for PCR Amplification of High GC Content