A method to amplify and express malaria parasite genes containing an intron without cDNA synthesis.

A method to amplify and express malaria parasite genes containing an intron without cDNA synthesis. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A method to amplify and express malaria parasite genes containing an intron without cDNA synthesis. Ankit Gupta, Ameer Abbas, Manoj Yadav, Vikas Kamalvanshi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7145169/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background The amplification of eukaryotic genes, which contain introns for expression purposes, is very tedious and expensive. The only available lab method is through cDNA synthesis, which has a very low success rate in generating a pure gene fragment lacking any introns. Methods The current paucity of any alternative lab method directed us to devise a strategy to amplify a P. falciparum RPL12mito gene ( Pf RPL12mito) containing an intron through three sequential PCR steps using gDNA and primers with a minimum 15 bp 5′-overhang for the full-length protein expression. Results Sanger sequencing confirmed the absence of an intronic segment and a continuous exonic region in the amplified gene fragment. The gene was successfully expressed as recombinant protein with a 6xHis tag. Conclusions This method provides a novel and cost-effective approach to amplify intron-containing eukaryotic genes, including those from the malaria parasite, directly from genomic DNA for expression purposes. Intron Exon cDNA gene expression PCR amplification P. falciparum Figures Figure 1 Figure 2 Figure 3 Background Splicing is a key step during the expression of eukaryotic genes containing introns which encompasses the removal of introns and joining of adjacent exons (Eickbush 2000 ). In vitro expression of gene containing introns involves the synthesis of cDNA from RNA through reverse transcription using oligo (dT) primers (OKAYAMA 2012 ). The complete removal of genomic DNA (gDNA) contamination from cDNA before pcr amplification is prerequisite for the purpose of either recombinant protein expression or protein overexpression (Sun et al. 2012 ). Mild DNase treatment of cDNA often leaves trace amounts of genomic DNA, leading to the unintended amplification of gene fragments containing intronic regions. In contrast, harsh DNase treatment compromises cDNA quality, resulting in little to no amplification of intron-free gene fragments. Therefore, the amplification of gene fragments without introns from cDNA is mostly expensive, tedious, exhaustive, and unfeasible. The alternative ways are either outsourced gene or dsDNA (G-blocks) synthesis, but both are quite expensive and time-consuming approaches. Therefore, the paucity of alternative methods directed us to develop a plausible method to amplify a gene fragment lacking an intron without cDNA synthesis. Here, we developed a novel method to successfully amplify and express the P. falciparum gene ( rpl12 mito ) without intronic segment through three pcr steps using gDNA and primers with 5′-overhang. Methods Parasite culture P. falciparum (3D7) parasites were cultivated at 37 o C under 5% O 2 , 5% CO 2 , 90% N 2 in O + human erythrocytes, RPMI 1640 (Invitrogen) supplemented with 25 mM HEPES, 50 µg/ml hypoxanthine, 0.5% albumax (Gibco), and 0.23% NaHCO 3 . phenol-chloroform method. Primer design, gDNA isolation and PCR amplification The reference sequence of Plasmodium falciparum RPL12 mito gene (PF3D7_0212200; Pf RPL12 mito ) was retrieved from PlasmoDB site ( http://PlasmoDB.org ) which contained a single intron (147bp) towards 3′-end followed by exon2 (71 bp). The primers were designed based on vector map (SnapGene) of pET28a vector with Pf RPL12 mito sequence containing intron. The strategy was devised to amplify full-length Pf RPL12 mito gene fragment (765 bp) without intron through three sequential pcr steps without synthesizing cDNA (Fig. 1 ). Four sequence-specific primers were developed and designated as p1, p2, p3, p4 which contained 5′-overhang complementary to vector or Pf RPL12 mito gene sequence as given in Table 1 . The length of primers p1, p2, p3 was 45 bp; whereas, p4 primer was 50 bp long. Genomic DNA (gDNA) was isolated from in vitro blood-stage P. falciparum (3D7) parasites (predominantly in trophozoite stage) using Quick-DNA MiniPrep kit (ZYMO Research) and used as template for PCR1-3 as reference (Fig. 2 a). Table 1 Details of primers used for the amplification of Pf RPL12 mito gene lacking intron. The sequence highlighted in red denotes restriction sites, and the underlined refers to 5 ¢ overhangs homologous to vector or gene sequences. Primer Sequence (5 ¢ to 3 ¢ ) GC% Length (base) T m (°C) p1 CAGCAAATGGGTCGC GGATCC ATGAAAAGGAATAAAATACTACTA 40 45 64.4 p2 CGGCTTTTTCAGAAGGTAC ACTTTTTTGTATGTAGAAGGGAGCAC 42.2 45 64.8 p3 CACCTAGTTGTTCAAAACTTTTTTTCATTTCTTCGGCTTTTTCAG 33.3 45 62.2 p4 GTGGTGGTGGTGGTG CTCGAG TTCCAAAATTATTGTTGCACCTAGTTGTT 46 50 68.5 PCR1 amplification (736 bp) was done with primers (p1 and p2) using parasite gDNA (10ng/reaction) and SpeedSTAR HS DNA polymerase (Takara) at an annealing temperature of 62.4°C (Fig. 2 b, lane 1 ). The pcr product was purified by NucleoSpin pcr clean-up kit (Macherey-Nagel) and used as template for PCR2 (769 bp) with primers (p1 and p3) at an annealing temperature of 60.2°C (Fig. 2 b, lane 2 ). At last, PCR3 (807 bp) was done with purified pcr2 as a template using primers (p1 and p4) at an annealing temperature of 62.4°C (Fig. 2 b, lane 3 ). Molecular cloning and recombinant protein expression PCR3 was cloned in pET28a expression vector at BamHI and XhoI sites using HD Infusion cloning kit (Takara) and clones were confirmed through restriction digestion with same restriction enzymes yielding bands at the size of ~ 5.3kb and 771bp (Raman and Martin 2014 ) (Fig. 2 c ) . The sequence of Pf RPL12 mito encompassing exon1 and 2, and lacking intronic segment in plasmid clones was further confirmed by Sanger sequencing (Fig. 2 d ) . The plasmid clone was co-transformed with RIG plasmid into BL21 (DE3) cells to express recombinant Pf RPL12 mito encompassing both n- and c-terminal 6xHis tag respectively. The expression of recombinant Pf RPL12 mito protein (~ 35 kDa) was confirmed by western blotting using anti-His tag antibody (Sigma; 1:1000 dilution) and 3,3-Diaminobenzidine (Sigma) (Fig. 3 ). Results and Discussion Our method strategy bypasses tedious and expensive steps of synthesizing RNA followed by cDNA synthesis. Notably, RNA is quite unstable and a good quality RNA is prerequisite for cDNA synthesis. Mostly, either cDNA contains gDNA contamination even after DNase treatment or vigorous DNase treatment results in the loss of quality cDNA which eventually end up in no or very low-quality amplification of desired gene fragment lacking introns. Therefore, cDNA-based gene amplification either fails or it takes several repetitions for desired protein expression. Other alternatives of cDNA synthesis include dsDNA or gene synthesis, which is quite expensive and time-consuming process. Here, we devised a novel strategy to PCR amplify genes from eukaryotic genomes e.g. Plasmodium falciparum containing introns for protein expression. We successfully amplified Pf RPL12 mito gene fragment encompassing only exons and no intron through sequential pcr steps with primers containing 5′ overhang. Primer p2 was designed in such a way that it contained 3′ end (30 bp) homologous to exon 1 and 5′ overhang (15 bp) homologous to exon 2 segment. PCR1 was the key step for this strategy to pcr amplify a gene fragment lacking intronic segment which was further used as a template for PCR2. Sanger sequencing of resulting full-length Pf RPL12 mito gene fragment confirmed the lack of intron and presence of exon1 and 2 both in frame and in continuation. Furthermore, the western blotting confirmed the successful expression of full-length Pf RPL12 mito protein with 6xHis tag. Conclusion The results suggest that this strategy is very useful to clone genes containing introns for expression analysis, particularly in laboratories with limited resources. However, the strategy has certain limitations, such as it can be used for genes containing a single intron at either end of the gene. Abbreviations cDNA Complementary DNA gDNA Genomic DNA RNA Ribonucleic acid PCR Polymerase chain reaction RPL Ribosomal Protein Large Subunit Pf Plasmodium falciparum Declarations Ethics approval The study includes the work which was duly approved by both the Institutional Ethics Committee (2024 -1- EMP- 8) and the Institutional Biosafety Committee (IBSC-2024-1-EMP-03). Conflict of interest The authors declare that they have no competing interests linked with the work reported in this paper. Acknowledgement The work is funded by a project grant (CST/D-896) received from the Council of Science and Technology (CST), Uttar Pradesh. We also acknowledge the Department of Transfusion Medicine & Blood Bank for providing fresh erythrocytes for Plasmodium culture, and MRU lab at AIIMS, Raebareli. References Eickbush TH (2000) Introns gain ground. Nature 404:940–943. https://doi.org/10.1038/35010246 OKAYAMA H (2012) Functional cDNA expression cloning: Pushing it to the limit. Proceedings of the Japan Academy, Series B 88:102–119. https://doi.org/10.2183/pjab.88.102 Raman M, Martin K (2014) One solution for cloning and mutagenesis: In-Fusion® HD Cloning Plus. Nat Methods 11:iii–v. https://doi.org/10.1038/nmeth.f.373 Sun B, Hou Y-L, Hou W-R, et al (2012) cDNA Cloning, Overexpression, Purification and Pharmacologic Evaluation for Anticancer Activity of Ribosomal Protein L23A Gene (RPL23A) from the Giant Panda. Int J Mol Sci 13:2133–2147. https://doi.org/10.3390/ijms13022133 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7145169","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":489091650,"identity":"bf275711-67c5-49c8-a5c8-e7415ca7231e","order_by":0,"name":"Ankit Gupta","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-0503-2322","institution":"AIIMS Raebareli: All India Institute of Medical Sciences - Raebareli","correspondingAuthor":true,"prefix":"","firstName":"Ankit","middleName":"","lastName":"Gupta","suffix":""},{"id":489091651,"identity":"84e06e1f-cc13-46dc-addc-1219a9f0db14","order_by":1,"name":"Ameer Abbas","email":"","orcid":"","institution":"AIIMS Raebareli: All India Institute of Medical Sciences - Raebareli","correspondingAuthor":false,"prefix":"","firstName":"Ameer","middleName":"","lastName":"Abbas","suffix":""},{"id":489091652,"identity":"ce044af9-e10f-437a-8e3b-e45566ea0f97","order_by":2,"name":"Manoj Yadav","email":"","orcid":"","institution":"AIIMS Raebareli: All India Institute of Medical Sciences - Raebareli","correspondingAuthor":false,"prefix":"","firstName":"Manoj","middleName":"","lastName":"Yadav","suffix":""},{"id":489091653,"identity":"8f85d001-2e37-4516-bed9-e9f31b161f82","order_by":3,"name":"Vikas Kamalvanshi","email":"","orcid":"","institution":"AIIMS Raebareli: All India Institute of Medical Sciences - Raebareli","correspondingAuthor":false,"prefix":"","firstName":"Vikas","middleName":"","lastName":"Kamalvanshi","suffix":""}],"badges":[],"createdAt":"2025-07-17 05:39:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7145169/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7145169/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87398954,"identity":"da11cf66-5bd9-4c13-ae4c-d4b247161498","added_by":"auto","created_at":"2025-07-23 11:23:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":492105,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic depicting the strategy to amplify gene fragment without intronic segment through PCR. Exon 1 and 2 are shown in purple and red respectively, connected by an intron (shown as loop). PCR was done in three steps using various primer pairs (p1-p4).\u003c/p\u003e","description":"","filename":"MethodFig1.png","url":"https://assets-eu.researchsquare.com/files/rs-7145169/v1/2d783a21af9c3d77d800b4bb.png"},{"id":87398952,"identity":"dd564546-65b2-434b-9b60-78678a09a31d","added_by":"auto","created_at":"2025-07-23 11:23:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":572264,"visible":true,"origin":"","legend":"\u003cp\u003ePCR amplification and cloning of a gene fragment lacking intronic segment. (a) PCR amplified gene fragments using genomic DNA (lane 1: 736bp, p1+p2; lane 2: 916bp, p1+p3; lane 3: 954bp, p1+p4). (b) PCR amplicons using our strategy (lane 1: 736bp, p1+p2; lane 2: 769bp, p1+p3; lane 3: 807bp, p1+p4). (c) Cloning of PCR 3 product in pET28a vector as verified through restriction digestion using BamHI and XhoI enzymes. (d) Sequence chromatogram of PCR3 product showing gene sequence of both exon 1 and 2 in continuation, and lacking intronic segment.\u003c/p\u003e","description":"","filename":"MethodFig2.png","url":"https://assets-eu.researchsquare.com/files/rs-7145169/v1/f871f4dc81b4fbc6e160fad8.png"},{"id":87398951,"identity":"8c46e714-2f9c-4cf2-b56c-25632b20c030","added_by":"auto","created_at":"2025-07-23 11:23:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":639673,"visible":true,"origin":"","legend":"\u003cp\u003eWestern blotting of recombinantly expressed \u003cem\u003ePf\u003c/em\u003eRPL12\u003csub\u003emito\u003c/sub\u003e protein (~35kDa) using anti-His antibody. Lane 1: uninduced fraction, lane 2: soluble fraction and lane 3: insoluble fraction.\u003c/p\u003e","description":"","filename":"MethodFig3.png","url":"https://assets-eu.researchsquare.com/files/rs-7145169/v1/72059d2485ea7d0158384ef4.png"},{"id":87399201,"identity":"2258faf4-3890-4b49-b855-5395cb5a44a8","added_by":"auto","created_at":"2025-07-23 11:32:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1666632,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7145169/v1/fe2abbae-6edb-4677-977a-a278628d6960.pdf"}],"financialInterests":"","formattedTitle":"A method to amplify and express malaria parasite genes containing an intron without cDNA synthesis.","fulltext":[{"header":"Background","content":"\u003cp\u003eSplicing is a key step during the expression of eukaryotic genes containing introns which encompasses the removal of introns and joining of adjacent exons (Eickbush \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). \u003cem\u003eIn vitro\u003c/em\u003e expression of gene containing introns involves the synthesis of cDNA from RNA through reverse transcription using oligo (dT) primers (OKAYAMA \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The complete removal of genomic DNA (gDNA) contamination from cDNA before pcr amplification is prerequisite for the purpose of either recombinant protein expression or protein overexpression (Sun et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Mild DNase treatment of cDNA often leaves trace amounts of genomic DNA, leading to the unintended amplification of gene fragments containing intronic regions. In contrast, harsh DNase treatment compromises cDNA quality, resulting in little to no amplification of intron-free gene fragments. Therefore, the amplification of gene fragments without introns from cDNA is mostly expensive, tedious, exhaustive, and unfeasible. The alternative ways are either outsourced gene or dsDNA (G-blocks) synthesis, but both are quite expensive and time-consuming approaches. Therefore, the paucity of alternative methods directed us to develop a plausible method to amplify a gene fragment lacking an intron without cDNA synthesis. Here, we developed a novel method to successfully amplify and express the \u003cem\u003eP. falciparum\u003c/em\u003e gene (\u003cem\u003erpl12\u003c/em\u003e\u003csub\u003e\u003cem\u003emito\u003c/em\u003e\u003c/sub\u003e) without intronic segment through three pcr steps using gDNA and primers with 5′-overhang.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cem\u003eParasite culture\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eP. falciparum\u003c/em\u003e (3D7) parasites were cultivated at 37\u003csup\u003eo\u003c/sup\u003eC under 5% O\u003csub\u003e2\u003c/sub\u003e, 5% CO\u003csub\u003e2\u003c/sub\u003e, 90% N\u003csub\u003e2\u003c/sub\u003e in O\u003csup\u003e+\u003c/sup\u003e human erythrocytes, RPMI 1640 (Invitrogen) supplemented with 25 mM HEPES, 50 \u0026micro;g/ml hypoxanthine, 0.5% albumax (Gibco), and 0.23% NaHCO\u003csub\u003e3\u003c/sub\u003e. phenol-chloroform method.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePrimer design, gDNA isolation and PCR amplification\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe reference sequence of \u003cem\u003ePlasmodium falciparum\u003c/em\u003e RPL12\u003csub\u003emito\u003c/sub\u003e gene (PF3D7_0212200; \u003cem\u003ePf\u003c/em\u003eRPL12\u003csub\u003emito\u003c/sub\u003e) was retrieved from PlasmoDB site (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://PlasmoDB.org\u003c/span\u003e\u003c/span\u003e) which contained a single intron (147bp) towards 3\u0026prime;-end followed by exon2 (71 bp). The primers were designed based on vector map (SnapGene) of pET28a vector with \u003cem\u003ePf\u003c/em\u003eRPL12\u003csub\u003emito\u003c/sub\u003e sequence containing intron. The strategy was devised to amplify full-length \u003cem\u003ePf\u003c/em\u003eRPL12\u003csub\u003emito\u003c/sub\u003e gene fragment (765 bp) without intron through three sequential pcr steps without synthesizing cDNA (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Four sequence-specific primers were developed and designated as p1, p2, p3, p4 which contained 5\u0026prime;-overhang complementary to vector or \u003cem\u003ePf\u003c/em\u003eRPL12\u003csub\u003emito\u003c/sub\u003e gene sequence as given in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. The length of primers p1, p2, p3 was 45 bp; whereas, p4 primer was 50 bp long. Genomic DNA (gDNA) was isolated from \u003cem\u003ein vitro\u003c/em\u003e blood-stage \u003cem\u003eP. falciparum\u003c/em\u003e (3D7) parasites (predominantly in trophozoite stage) using Quick-DNA MiniPrep kit (ZYMO Research) and used as template for PCR1-3 as reference (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:8.0pt;margin-left:0cm;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;font-family:\"Arial\",sans-serif;'\u003eTable 1 \u003c/span\u003e\u003c/strong\u003e\u003cspan style='font-size:16px;font-family:\"Arial\",sans-serif;'\u003eDetails of\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eprimers used for the amplification of\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Arial\",sans-serif;color:black;'\u003ePf\u003c/span\u003e\u003c/em\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Arial\",sans-serif;color:black;'\u003eRPL12\u003csub\u003emito\u0026nbsp;\u003c/sub\u003egene lacking intron. The sequence highlighted in red denotes restriction sites, and the underlined refers to\u0026nbsp;\u003c/span\u003e\u003cspan style='font-size:16px;font-family:\"Arial\",sans-serif;'\u003e5\u003c/span\u003e\u003cspan style=\"font-size:16px;font-family:Symbol;\"\u003e\u0026cent;\u003c/span\u003e\u003cspan style='font-size:16px;font-family:\"Arial\",sans-serif;'\u003e\u0026nbsp;overhangs homologous to vector or gene sequences.\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003ctable style=\"border-collapse: collapse;border: none;width: 614px;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 49.4pt;border: 1pt solid rgb(191, 191, 191);padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;font-family:\"Arial\",sans-serif;'\u003ePrimer\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10cm;border-top: 1pt solid rgb(191, 191, 191);border-right: 1pt solid rgb(191, 191, 191);border-bottom: 1pt solid rgb(191, 191, 191);border-image: initial;border-left: none;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;font-family:\"Arial\",sans-serif;'\u003eSequence (5\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style=\"font-size:13px;font-family:Symbol;\"\u003e\u0026cent;\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;font-family:\"Arial\",sans-serif;'\u003e to 3\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style=\"font-size:13px;font-family:Symbol;\"\u003e\u0026cent;\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;font-family:\"Arial\",sans-serif;'\u003e)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.4pt;border-top: 1pt solid rgb(191, 191, 191);border-right: 1pt solid rgb(191, 191, 191);border-bottom: 1pt solid rgb(191, 191, 191);border-image: initial;border-left: none;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;font-family:\"Arial\",sans-serif;'\u003eGC%\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.65pt;border-top: 1pt solid rgb(191, 191, 191);border-right: 1pt solid rgb(191, 191, 191);border-bottom: 1pt solid rgb(191, 191, 191);border-image: initial;border-left: none;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;font-family:\"Arial\",sans-serif;'\u003eLength (base)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.5pt;border-top: 1pt solid rgb(191, 191, 191);border-right: 1pt solid rgb(191, 191, 191);border-bottom: 1pt solid rgb(191, 191, 191);border-image: initial;border-left: none;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cstrong\u003e\u003cspan style='font-size:13px;font-family:\"Arial\",sans-serif;'\u003eT\u003csub\u003em\u0026nbsp;\u003c/sub\u003e(\u0026deg;C)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 49.4pt;border-right: 1pt solid rgb(191, 191, 191);border-bottom: 1pt solid rgb(191, 191, 191);border-left: 1pt solid rgb(191, 191, 191);border-image: initial;border-top: none;background: rgb(242, 242, 242);padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Arial\",sans-serif;color:black;'\u003ep1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10cm;border-top: none;border-left: none;border-bottom: 1pt solid rgb(191, 191, 191);border-right: 1pt solid rgb(191, 191, 191);background: rgb(242, 242, 242);padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cu\u003e\u003cspan style='font-size:13px;font-family:\"Arial\",sans-serif;color:black;'\u003eCAGCAAATGGGTCGC\u003c/span\u003e\u003c/u\u003e\u003cspan style='font-size:13px;font-family:\"Arial\",sans-serif;color:red;'\u003eGGATCC\u003c/span\u003e\u003cspan style='font-size:13px;font-family:\"Arial\",sans-serif;color:black;'\u003eATGAAAAGGAATAAAATACTACTA\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.4pt;border-top: none;border-left: none;border-bottom: 1pt solid rgb(191, 191, 191);border-right: 1pt solid rgb(191, 191, 191);background: rgb(242, 242, 242);padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Arial\",sans-serif;color:black;'\u003e40\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.65pt;border-top: none;border-left: none;border-bottom: 1pt solid rgb(191, 191, 191);border-right: 1pt solid rgb(191, 191, 191);background: rgb(242, 242, 242);padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Arial\",sans-serif;color:black;'\u003e45\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.5pt;border-top: none;border-left: none;border-bottom: 1pt solid rgb(191, 191, 191);border-right: 1pt solid rgb(191, 191, 191);background: rgb(242, 242, 242);padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Arial\",sans-serif;color:black;'\u003e64.4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 49.4pt;border-right: 1pt solid rgb(191, 191, 191);border-bottom: 1pt solid rgb(191, 191, 191);border-left: 1pt solid rgb(191, 191, 191);border-image: initial;border-top: none;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Arial\",sans-serif;'\u003ep2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10cm;border-top: none;border-left: none;border-bottom: 1pt solid rgb(191, 191, 191);border-right: 1pt solid rgb(191, 191, 191);padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cu\u003e\u003cspan style='font-size:13px;font-family:\"Arial\",sans-serif;'\u003eCGGCTTTTTCAGAAGGTAC\u003c/span\u003e\u003c/u\u003e\u003cspan style='font-size:13px;font-family:\"Arial\",sans-serif;'\u003eACTTTTTTGTATGTAGAAGGGAGCAC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.4pt;border-top: none;border-left: none;border-bottom: 1pt solid rgb(191, 191, 191);border-right: 1pt solid rgb(191, 191, 191);padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Arial\",sans-serif;'\u003e42.2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.65pt;border-top: none;border-left: none;border-bottom: 1pt solid rgb(191, 191, 191);border-right: 1pt solid rgb(191, 191, 191);padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Arial\",sans-serif;'\u003e45\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.5pt;border-top: none;border-left: none;border-bottom: 1pt solid rgb(191, 191, 191);border-right: 1pt solid rgb(191, 191, 191);padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Arial\",sans-serif;'\u003e64.8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 49.4pt;border-right: 1pt solid rgb(191, 191, 191);border-bottom: 1pt solid rgb(191, 191, 191);border-left: 1pt solid rgb(191, 191, 191);border-image: initial;border-top: none;background: rgb(242, 242, 242);padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Arial\",sans-serif;color:black;'\u003ep3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10cm;border-top: none;border-left: none;border-bottom: 1pt solid rgb(191, 191, 191);border-right: 1pt solid rgb(191, 191, 191);background: rgb(242, 242, 242);padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Arial\",sans-serif;color:black;'\u003eCACCTAGTTGTTCAAAACTTTTTTTCATTTCTTCGGCTTTTTCAG\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.4pt;border-top: none;border-left: none;border-bottom: 1pt solid rgb(191, 191, 191);border-right: 1pt solid rgb(191, 191, 191);background: rgb(242, 242, 242);padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Arial\",sans-serif;color:black;'\u003e33.3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.65pt;border-top: none;border-left: none;border-bottom: 1pt solid rgb(191, 191, 191);border-right: 1pt solid rgb(191, 191, 191);background: rgb(242, 242, 242);padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Arial\",sans-serif;color:black;'\u003e45\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.5pt;border-top: none;border-left: none;border-bottom: 1pt solid rgb(191, 191, 191);border-right: 1pt solid rgb(191, 191, 191);background: rgb(242, 242, 242);padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Arial\",sans-serif;color:black;'\u003e62.2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 49.4pt;border-right: 1pt solid rgb(191, 191, 191);border-bottom: 1pt solid rgb(191, 191, 191);border-left: 1pt solid rgb(191, 191, 191);border-image: initial;border-top: none;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Arial\",sans-serif;'\u003ep4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10cm;border-top: none;border-left: none;border-bottom: 1pt solid rgb(191, 191, 191);border-right: 1pt solid rgb(191, 191, 191);padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cu\u003e\u003cspan style='font-size:13px;font-family:\"Arial\",sans-serif;'\u003eGTGGTGGTGGTGGTG\u003c/span\u003e\u003c/u\u003e\u003cspan style='font-size:13px;font-family:\"Arial\",sans-serif;color:red;'\u003eCTCGAG\u003c/span\u003e\u003cspan style='font-size:13px;font-family:\"Arial\",sans-serif;'\u003eTTCCAAAATTATTGTTGCACCTAGTTGTT\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.4pt;border-top: none;border-left: none;border-bottom: 1pt solid rgb(191, 191, 191);border-right: 1pt solid rgb(191, 191, 191);padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Arial\",sans-serif;'\u003e46\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.65pt;border-top: none;border-left: none;border-bottom: 1pt solid rgb(191, 191, 191);border-right: 1pt solid rgb(191, 191, 191);padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Arial\",sans-serif;'\u003e50\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.5pt;border-top: none;border-left: none;border-bottom: 1pt solid rgb(191, 191, 191);border-right: 1pt solid rgb(191, 191, 191);padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Arial\",sans-serif;'\u003e68.5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003ePCR1 amplification (736 bp) was done with primers (p1 and p2) using parasite gDNA (10ng/reaction) and SpeedSTAR HS DNA polymerase (Takara) at an annealing temperature of 62.4\u0026deg;C (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb, \u003cstrong\u003elane 1\u003c/strong\u003e). The pcr product was purified by NucleoSpin pcr clean-up kit (Macherey-Nagel) and used as template for PCR2 (769 bp) with primers (p1 and p3) at an annealing temperature of 60.2\u0026deg;C (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb, \u003cstrong\u003elane 2\u003c/strong\u003e). At last, PCR3 (807 bp) was done with purified pcr2 as a template using primers (p1 and p4) at an annealing temperature of 62.4\u0026deg;C (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb, \u003cstrong\u003elane 3\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMolecular cloning and recombinant protein expression\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePCR3 was cloned in pET28a expression vector at BamHI and XhoI sites using HD Infusion cloning kit (Takara) and clones were confirmed through restriction digestion with same restriction enzymes yielding bands at the size of ~\u0026thinsp;5.3kb and 771bp (Raman and Martin \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec\u003cstrong\u003e)\u003c/strong\u003e. The sequence of \u003cem\u003ePf\u003c/em\u003eRPL12\u003csub\u003emito\u003c/sub\u003e encompassing exon1 and 2, and lacking intronic segment in plasmid clones was further confirmed by Sanger sequencing (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed\u003cstrong\u003e)\u003c/strong\u003e. The plasmid clone was co-transformed with RIG plasmid into BL21 (DE3) cells to express recombinant \u003cem\u003ePf\u003c/em\u003eRPL12\u003csub\u003emito\u003c/sub\u003e encompassing both n- and c-terminal 6xHis tag respectively. The expression of recombinant \u003cem\u003ePf\u003c/em\u003eRPL12\u003csub\u003emito\u003c/sub\u003e protein (~\u0026thinsp;35 kDa) was confirmed by western blotting using anti-His tag antibody (Sigma; 1:1000 dilution) and 3,3-Diaminobenzidine (Sigma) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eOur method strategy bypasses tedious and expensive steps of synthesizing RNA followed by cDNA synthesis. Notably, RNA is quite unstable and a good quality RNA is prerequisite for cDNA synthesis. Mostly, either cDNA contains gDNA contamination even after DNase treatment or vigorous DNase treatment results in the loss of quality cDNA which eventually end up in no or very low-quality amplification of desired gene fragment lacking introns. Therefore, cDNA-based gene amplification either fails or it takes several repetitions for desired protein expression. Other alternatives of cDNA synthesis include dsDNA or gene synthesis, which is quite expensive and time-consuming process.\u003c/p\u003e\u003cp\u003eHere, we devised a novel strategy to PCR amplify genes from eukaryotic genomes e.g. \u003cem\u003ePlasmodium falciparum\u003c/em\u003e containing introns for protein expression. We successfully amplified \u003cem\u003ePf\u003c/em\u003eRPL12\u003csub\u003emito\u003c/sub\u003e gene fragment encompassing only exons and no intron through sequential pcr steps with primers containing 5\u0026prime; overhang. Primer p2 was designed in such a way that it contained 3\u0026prime; end (30 bp) homologous to exon 1 and 5\u0026prime; overhang (15 bp) homologous to exon 2 segment. PCR1 was the key step for this strategy to pcr amplify a gene fragment lacking intronic segment which was further used as a template for PCR2. Sanger sequencing of resulting full-length \u003cem\u003ePf\u003c/em\u003eRPL12\u003csub\u003emito\u003c/sub\u003e gene fragment confirmed the lack of intron and presence of exon1 and 2 both in frame and in continuation. Furthermore, the western blotting confirmed the successful expression of full-length \u003cem\u003ePf\u003c/em\u003eRPL12\u003csub\u003emito\u003c/sub\u003e protein with 6xHis tag.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe results suggest that this strategy is very useful to clone genes containing introns for expression analysis, particularly in laboratories with limited resources. However, the strategy has certain limitations, such as it can be used for genes containing a single intron at either end of the gene.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003ecDNA \u0026nbsp;Complementary DNA\u0026nbsp;\u003c/p\u003e\n\u003cp\u003egDNA \u0026nbsp;Genomic DNA\u003c/p\u003e\n\u003cp\u003eRNA \u0026nbsp; \u0026nbsp;Ribonucleic acid\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePCR \u0026nbsp;Polymerase chain reaction\u003c/p\u003e\n\u003cp\u003eRPL \u0026nbsp;Ribosomal Protein Large Subunit\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePf \u0026nbsp; \u0026nbsp; \u003cem\u003ePlasmodium falciparum\u003c/em\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study includes the work which was duly approved by both the Institutional Ethics Committee (2024 -1- EMP- 8) and the Institutional Biosafety Committee (IBSC-2024-1-EMP-03).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests linked with the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe work is funded by a project grant (CST/D-896) received from the Council of Science and Technology (CST), Uttar Pradesh. We also acknowledge the Department of Transfusion Medicine \u0026amp; Blood Bank for providing fresh erythrocytes for \u003cem\u003ePlasmodium\u0026nbsp;\u003c/em\u003eculture, and MRU lab at AIIMS, Raebareli.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eEickbush TH (2000) Introns gain ground. Nature 404:940\u0026ndash;943. https://doi.org/10.1038/35010246\u003c/li\u003e\n \u003cli\u003eOKAYAMA H (2012) Functional cDNA expression cloning: Pushing it to the limit. Proceedings of the Japan Academy, Series B 88:102\u0026ndash;119. https://doi.org/10.2183/pjab.88.102\u003c/li\u003e\n \u003cli\u003eRaman M, Martin K (2014) One solution for cloning and mutagenesis: In-Fusion\u0026reg; HD Cloning Plus. Nat Methods 11:iii\u0026ndash;v. https://doi.org/10.1038/nmeth.f.373\u003c/li\u003e\n \u003cli\u003eSun B, Hou Y-L, Hou W-R, et al (2012) cDNA Cloning, Overexpression, Purification and Pharmacologic Evaluation for Anticancer Activity of Ribosomal Protein L23A Gene (RPL23A) from the Giant Panda. Int J Mol Sci 13:2133\u0026ndash;2147. https://doi.org/10.3390/ijms13022133\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Intron, Exon, cDNA, gene expression, PCR amplification, P. falciparum","lastPublishedDoi":"10.21203/rs.3.rs-7145169/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7145169/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eThe amplification of eukaryotic genes, which contain introns for expression purposes, is very tedious and expensive. The only available lab method is through cDNA synthesis, which has a very low success rate in generating a pure gene fragment lacking any introns.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eThe current paucity of any alternative lab method directed us to devise a strategy to amplify a \u003cem\u003eP. falciparum\u003c/em\u003e RPL12mito gene (\u003cem\u003ePf\u003c/em\u003eRPL12mito) containing an intron through three sequential PCR steps using gDNA and primers with a minimum 15 bp 5\u0026prime;-overhang for the full-length protein expression.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eSanger sequencing confirmed the absence of an intronic segment and a continuous exonic region in the amplified gene fragment. The gene was successfully expressed as recombinant protein with a 6xHis tag.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eThis method provides a novel and cost-effective approach to amplify intron-containing eukaryotic genes, including those from the malaria parasite, directly from genomic DNA for expression purposes.\u003c/p\u003e","manuscriptTitle":"A method to amplify and express malaria parasite genes containing an intron without cDNA synthesis.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-23 11:23:30","doi":"10.21203/rs.3.rs-7145169/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"86b85e3a-2e59-4c1d-a2a9-5e4c7aa9fa2f","owner":[],"postedDate":"July 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-23T11:23:30+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-23 11:23:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7145169","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7145169","identity":"rs-7145169","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}