polymerase chain reaction pcr amplified dna fragments  (TaKaRa)

 
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    Structured Review

    TaKaRa polymerase chain reaction pcr amplified dna fragments
    Polymerase Chain Reaction Pcr Amplified Dna Fragments, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Polymerase Chain Reaction:

    Article Title: MCAF1 and synergistic activation of the transcription of Epstein-Barr virus lytic genes by Rta and Zta
    Article Snippet: .. Plasmids pEGFP-MCAF1-DM1, pEGFP-MCAF1-M and pEGFP-MCAF1-DM2 were constructed by inserting polymerase chain reaction (PCR)-amplified DNA fragments that encoded the MCAF1 regions from amino acid 562 to 817, 818 to 1153 and 1154 to 1270, respectively, into the ScaI–XhoI sites in pEGFP-C1 (Clontech). .. Plasmids pEGFP-ZN, pEGFP-ZM and pEGFP-ZC, which expressed truncated Zta, were constructed by inserting PCR-amplified fragments that encoded the amino acid regions from 1 to 86, 87 to 166 and 167 to 245, respectively, into the EcoRI–SalI sites in pEGFP-C1.

    Construct:

    Article Title: MCAF1 and synergistic activation of the transcription of Epstein-Barr virus lytic genes by Rta and Zta
    Article Snippet: .. Plasmids pEGFP-MCAF1-DM1, pEGFP-MCAF1-M and pEGFP-MCAF1-DM2 were constructed by inserting polymerase chain reaction (PCR)-amplified DNA fragments that encoded the MCAF1 regions from amino acid 562 to 817, 818 to 1153 and 1154 to 1270, respectively, into the ScaI–XhoI sites in pEGFP-C1 (Clontech). .. Plasmids pEGFP-ZN, pEGFP-ZM and pEGFP-ZC, which expressed truncated Zta, were constructed by inserting PCR-amplified fragments that encoded the amino acid regions from 1 to 86, 87 to 166 and 167 to 245, respectively, into the EcoRI–SalI sites in pEGFP-C1.

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    TaKaRa eco ri pcr amplified fragment
    Interaction between calreticulin and <t>TMV</t> MP and effect of calreticulin overexpression on TMV MP subcellular localization. A, Interaction in yeast two-hybrid system. Lane 1, AtCRT1 + TMV MP; lane 2, AtCRT1 + lamin C; lane 3, AtCRT1 lacking signal peptide + TMV MP. B, <t>RT-PCR</t> analysis of ZmCRT1 overexpression in transgenic N. benthamiana . Lane 1, Plants transgenic for ZmCRT1; lane 2, wild-type plants. Top and bottom bands correspond to the ZmCRT1- and actin-specific products. Fragment size in base pairs is indicated on the right. C, Western-blot analysis of ZmCRT1 overexpression in transgenic plants. Lane 1, Plants transgenic for ZmCRT1; lane 2, wild-type plants. Molecular mass expressed in thousands of Daltons is indicated on the right. D, Interaction in renatured-blot overlay assay. Lane 1, Plants transgenic for ZmCRT1 + TMV MP; lane 2, wild-type plants + TMV MP; lane 3, plants transgenic for ZmCRT1 + VirD2. Arrowhead indicates the position of calreticulin. Molecular mass expressed in thousands of Daltons is indicated on the right. E, GFP-TMV MP transiently expressed in wild-type N. benthamiana . F, GFP-TMV MP transiently expressed in transgenic N. benthamiana plants overexpressing ZmCRT1. GFP signal is in green; plastid autofluorescence is in red. Images in E and F are projections of several confocal sections; plastid autofluorescence in E was filtered out. Bars = 10 μ m.
    Eco Ri Pcr Amplified Fragment, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    TaKaRa dna fragments
    Knock-out of <t>LIG1</t> or LIG3 reduced HBV cccDNA production in HBV stable cell line. (A) Confirmation of LIG1 and LIG3 knock-out in HepDES19-based LIG1 K.O. cells and LIG3 K.O. cells by Western blot, respectively. (B) HepDES19-based control K.O. cells, LIG1 K.O. cells, and LIG3 K.O. cells were cultured in tet-free medium for 12 days, then the intracellular HBV core <t>DNA</t> was analyzed by Southern blot. (C) HBV Hirt DNA samples extracted from the above cell cultures were heat denatured and digested by PSAD to remove rcDNA, then cccDNA was quantified by qPCR, normalized by mitochondrial DNA and plotted as relative levels (% of control) (mean±SD, n = 3).
    Dna Fragments, supplied by TaKaRa, used in various techniques. Bioz Stars score: 94/100, based on 525 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    TaKaRa pcr amplified cdna fragments
    <t>RT-PCR</t> amplification of five cassava eIF4E ORFs with gene-specific primers. Total RNA was extracted from TMS60444 cassava line. The first-strand <t>cDNA</t> was synthesized using SuperScript ™ III Reverse Transcriptase (Invitrogen) and PCR was performed using Phusion High-Fidelity DNA polymerase (New England Biolabs) and primers indicated in Table 1 . Lane 1: DNA marker; Lane 2: 016601 CDS amplified with primers 016601F and 016601R; Lane 3: 016620 CDS amplified with primers 016620F and 016620R; Lane 4: 015501 CDS amplified with 015501F and 015501R, Lane 5: 013223 CDS amplified with primers 013223lF and 013223R, Lane 6, 013223 CDS amplified with primers 013223sF and 013223R; Lane 7: 013732 CDS amplified with 013732lF and 013732R; Lane 8: 013732 CDS amplified with 013732sF and 013732R; and Lane 9: negative water control.
    Pcr Amplified Cdna Fragments, supplied by TaKaRa, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    TaKaRa internal pgabca2 cdna fragments
    Mutations affecting <t>PgABCA2</t> protein in Cry2Ab-resistant pink bollworm from Arizona and India. ( a ) The predicted PgABCA2 protein includes amino (N) and carboxyl (C) termini (pink), two transmembrane domains (TMD1 and TMD2), each consisting of 6 transmembrane regions (TM; orange), three extracellular loops (ECL; green), two intracellular loops (ICL; blue), and two nucleotide-binding domains (NBD; purple). Mutations affecting transcripts of resistant pink bollworm: Circles show premature stop codons from India (red), Arizona (yellow), or both (red and yellow). Triangles show in-frame indels from India (red) or Arizona (yellow). Numbers indicate the affected amino acids. ( b ) Full-length and partial PgABCA2 cDNAs were obtained by direct PCR sequencing, DNA sequencing of <t>cDNA</t> clones, and/or PacBio ® DNA sequencing from susceptible (APHIS-S) and resistant, laboratory-selected (Bt4-R2) pink bollworm from Arizona, USA and India field-selected resistant populations (AM, CK, GAP, KT, and RK). The linear schematic (top) shows the predicted translated domain structure of the 5,187-bp full-length PgABCA2 coding sequence. The predicted protein includes amino- and carboxyl-termini (pink), transmembrane regions TM1-TM12 (orange), intracellular loops ICL1-ICL5 (blue), and extracellular loops ECL1-ECL6 (green). The domain structure connected to the exons that encode the respective domains is shown by dotted gray lines. Each predicted domain is numbered, with ECLs on top and ICLs numbered on bottom of protein schematic. Putative exons 1–31 are numbered, with grey exons indicating regions determined by direct PCR sequencing. Exons colored in light blue were further verified by sequencing cDNA clones (either by Sanger or PacBio ® sequencing). Red bars indicate disruption sites within the full-length coding sequence and the red triangles indicate the location of premature stop codons shown to scale based on the linear schematic of the translated domain structure. Unique cDNA variants are indicated as a, b, c, etc.
    Internal Pgabca2 Cdna Fragments, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Interaction between calreticulin and TMV MP and effect of calreticulin overexpression on TMV MP subcellular localization. A, Interaction in yeast two-hybrid system. Lane 1, AtCRT1 + TMV MP; lane 2, AtCRT1 + lamin C; lane 3, AtCRT1 lacking signal peptide + TMV MP. B, RT-PCR analysis of ZmCRT1 overexpression in transgenic N. benthamiana . Lane 1, Plants transgenic for ZmCRT1; lane 2, wild-type plants. Top and bottom bands correspond to the ZmCRT1- and actin-specific products. Fragment size in base pairs is indicated on the right. C, Western-blot analysis of ZmCRT1 overexpression in transgenic plants. Lane 1, Plants transgenic for ZmCRT1; lane 2, wild-type plants. Molecular mass expressed in thousands of Daltons is indicated on the right. D, Interaction in renatured-blot overlay assay. Lane 1, Plants transgenic for ZmCRT1 + TMV MP; lane 2, wild-type plants + TMV MP; lane 3, plants transgenic for ZmCRT1 + VirD2. Arrowhead indicates the position of calreticulin. Molecular mass expressed in thousands of Daltons is indicated on the right. E, GFP-TMV MP transiently expressed in wild-type N. benthamiana . F, GFP-TMV MP transiently expressed in transgenic N. benthamiana plants overexpressing ZmCRT1. GFP signal is in green; plastid autofluorescence is in red. Images in E and F are projections of several confocal sections; plastid autofluorescence in E was filtered out. Bars = 10 μ m.

    Journal: Plant Physiology

    Article Title: Effects of Calreticulin on Viral Cell-to-Cell Movement 1

    doi: 10.1104/pp.105.064386

    Figure Lengend Snippet: Interaction between calreticulin and TMV MP and effect of calreticulin overexpression on TMV MP subcellular localization. A, Interaction in yeast two-hybrid system. Lane 1, AtCRT1 + TMV MP; lane 2, AtCRT1 + lamin C; lane 3, AtCRT1 lacking signal peptide + TMV MP. B, RT-PCR analysis of ZmCRT1 overexpression in transgenic N. benthamiana . Lane 1, Plants transgenic for ZmCRT1; lane 2, wild-type plants. Top and bottom bands correspond to the ZmCRT1- and actin-specific products. Fragment size in base pairs is indicated on the right. C, Western-blot analysis of ZmCRT1 overexpression in transgenic plants. Lane 1, Plants transgenic for ZmCRT1; lane 2, wild-type plants. Molecular mass expressed in thousands of Daltons is indicated on the right. D, Interaction in renatured-blot overlay assay. Lane 1, Plants transgenic for ZmCRT1 + TMV MP; lane 2, wild-type plants + TMV MP; lane 3, plants transgenic for ZmCRT1 + VirD2. Arrowhead indicates the position of calreticulin. Molecular mass expressed in thousands of Daltons is indicated on the right. E, GFP-TMV MP transiently expressed in wild-type N. benthamiana . F, GFP-TMV MP transiently expressed in transgenic N. benthamiana plants overexpressing ZmCRT1. GFP signal is in green; plastid autofluorescence is in red. Images in E and F are projections of several confocal sections; plastid autofluorescence in E was filtered out. Bars = 10 μ m.

    Article Snippet: For a translational fusion of TMV MP to the N terminus of CFP (TMV MP-CFP), the coding sequence of TMV MP was first cloned as a Sal I- Eco RI PCR-amplified fragment into the Xho I- Eco RI sites of pSAT1-MCS , and, into the Sal I- Bam HI sites of the resulting construct, the coding sequence of CFP (derived from pECFP-N1; CLONTECH Laboratories, Mountain View, CA) was then cloned as a Sal I-BglII PCR-amplified fragment.

    Techniques: Over Expression, Reverse Transcription Polymerase Chain Reaction, Transgenic Assay, Western Blot, Overlay Assay

    Knock-out of LIG1 or LIG3 reduced HBV cccDNA production in HBV stable cell line. (A) Confirmation of LIG1 and LIG3 knock-out in HepDES19-based LIG1 K.O. cells and LIG3 K.O. cells by Western blot, respectively. (B) HepDES19-based control K.O. cells, LIG1 K.O. cells, and LIG3 K.O. cells were cultured in tet-free medium for 12 days, then the intracellular HBV core DNA was analyzed by Southern blot. (C) HBV Hirt DNA samples extracted from the above cell cultures were heat denatured and digested by PSAD to remove rcDNA, then cccDNA was quantified by qPCR, normalized by mitochondrial DNA and plotted as relative levels (% of control) (mean±SD, n = 3).

    Journal: PLoS Pathogens

    Article Title: The role of host DNA ligases in hepadnavirus covalently closed circular DNA formation

    doi: 10.1371/journal.ppat.1006784

    Figure Lengend Snippet: Knock-out of LIG1 or LIG3 reduced HBV cccDNA production in HBV stable cell line. (A) Confirmation of LIG1 and LIG3 knock-out in HepDES19-based LIG1 K.O. cells and LIG3 K.O. cells by Western blot, respectively. (B) HepDES19-based control K.O. cells, LIG1 K.O. cells, and LIG3 K.O. cells were cultured in tet-free medium for 12 days, then the intracellular HBV core DNA was analyzed by Southern blot. (C) HBV Hirt DNA samples extracted from the above cell cultures were heat denatured and digested by PSAD to remove rcDNA, then cccDNA was quantified by qPCR, normalized by mitochondrial DNA and plotted as relative levels (% of control) (mean±SD, n = 3).

    Article Snippet: For T7E1-based indel assay, primers used to amplify DNA fragments containing LIG1-sgRNA1 and LIG3-sgRNA1 targeting region were listed in , indel mutations were detected by Guide-it Mutation Detection Kit (Clontech) according to manufacturer’s manual.

    Techniques: Knock-Out, Stable Transfection, Western Blot, Cell Culture, Southern Blot, Real-time Polymerase Chain Reaction

    Knock-out of LIG1 and LIG3 reduced DHBV cccDNA in HepDG10 cells. HepDG10 control and ligase single knock-out (K.O.) cells (A) and double knock-down cells (B) were cultured in tet-free medium for 6 days. The levels of LIG1 and LIG3 in each cell lines were determined by Western blot with β-actin serving as loading control. DHBV core DNA and cccDNA were analyzed by Southern blot. The loading amount of Hirt DNA was normalized based on the relative levels of RC DNA (% of control) on core DNA Southern blot. The data presented here are representative of two independent experiments.

    Journal: PLoS Pathogens

    Article Title: The role of host DNA ligases in hepadnavirus covalently closed circular DNA formation

    doi: 10.1371/journal.ppat.1006784

    Figure Lengend Snippet: Knock-out of LIG1 and LIG3 reduced DHBV cccDNA in HepDG10 cells. HepDG10 control and ligase single knock-out (K.O.) cells (A) and double knock-down cells (B) were cultured in tet-free medium for 6 days. The levels of LIG1 and LIG3 in each cell lines were determined by Western blot with β-actin serving as loading control. DHBV core DNA and cccDNA were analyzed by Southern blot. The loading amount of Hirt DNA was normalized based on the relative levels of RC DNA (% of control) on core DNA Southern blot. The data presented here are representative of two independent experiments.

    Article Snippet: For T7E1-based indel assay, primers used to amplify DNA fragments containing LIG1-sgRNA1 and LIG3-sgRNA1 targeting region were listed in , indel mutations were detected by Guide-it Mutation Detection Kit (Clontech) according to manufacturer’s manual.

    Techniques: Knock-Out, Cell Culture, Western Blot, Southern Blot

    Ectopic expression of LIG1 and LIG3 restored DHBV cccDNA formation in ligase knock-out cells. HepDG10-based control knock-out cells and LIG1 knock-out cells (A) or LIG3 knock-out cells (B) were transfected with control vector or plasmid expressing sgRNA-resistant LIG1 (A) or LIG3 (B), and cultured in tet-free medium to induce DHBV replication. Cells were harvested at day 6 post induction, LIG1 and LIG3 expression levels were detected by Western blot, viral core DNA and cccDNA were analyzed by Southern blot. The loading amount of Hirt DNA was normalized by the relative levels of RC DNA (% of control) on core DNA Southern blot. The presented results are representative of two experimental trials.

    Journal: PLoS Pathogens

    Article Title: The role of host DNA ligases in hepadnavirus covalently closed circular DNA formation

    doi: 10.1371/journal.ppat.1006784

    Figure Lengend Snippet: Ectopic expression of LIG1 and LIG3 restored DHBV cccDNA formation in ligase knock-out cells. HepDG10-based control knock-out cells and LIG1 knock-out cells (A) or LIG3 knock-out cells (B) were transfected with control vector or plasmid expressing sgRNA-resistant LIG1 (A) or LIG3 (B), and cultured in tet-free medium to induce DHBV replication. Cells were harvested at day 6 post induction, LIG1 and LIG3 expression levels were detected by Western blot, viral core DNA and cccDNA were analyzed by Southern blot. The loading amount of Hirt DNA was normalized by the relative levels of RC DNA (% of control) on core DNA Southern blot. The presented results are representative of two experimental trials.

    Article Snippet: For T7E1-based indel assay, primers used to amplify DNA fragments containing LIG1-sgRNA1 and LIG3-sgRNA1 targeting region were listed in , indel mutations were detected by Guide-it Mutation Detection Kit (Clontech) according to manufacturer’s manual.

    Techniques: Expressing, Knock-Out, Transfection, Plasmid Preparation, Cell Culture, Western Blot, Southern Blot

    Knock-down of LIG1 or LIG3 reduced HBV cccDNA production in HBV in vitro infection system. (A) The knock-down levels of LIG1 and LIG3 in lentiviral shRNA transduced HepG2-NTCP12 cells were assessed by Western blot. (B) The indicated control and LIG1/3 knock-down HepG2-NTCP12 cells were infected by HBV in the presence of 10 μM of 3TC for 8 days. The extracted intracellular HBV cccDNA were directly analyzed by Southern blot (top panel), or heat denatured at 85°C for 5 min, followed by EcoRI linearization and Southern blot analysis (bottom panel). (C) In addition, cccDNA was quantitated by qPCR, normalized by mitochondrial DNA and plotted as relative levels (% of control) (mean±SD, n = 3). (D) The intracellular core protein (HBc) expression was detected by immunofluorescence microscopy. Cell nuclei were counterstained with DAPI. Each image is a representative of five different microscopic fields, the percentage of HBc-positive cells was indicated (Mean ± SD).

    Journal: PLoS Pathogens

    Article Title: The role of host DNA ligases in hepadnavirus covalently closed circular DNA formation

    doi: 10.1371/journal.ppat.1006784

    Figure Lengend Snippet: Knock-down of LIG1 or LIG3 reduced HBV cccDNA production in HBV in vitro infection system. (A) The knock-down levels of LIG1 and LIG3 in lentiviral shRNA transduced HepG2-NTCP12 cells were assessed by Western blot. (B) The indicated control and LIG1/3 knock-down HepG2-NTCP12 cells were infected by HBV in the presence of 10 μM of 3TC for 8 days. The extracted intracellular HBV cccDNA were directly analyzed by Southern blot (top panel), or heat denatured at 85°C for 5 min, followed by EcoRI linearization and Southern blot analysis (bottom panel). (C) In addition, cccDNA was quantitated by qPCR, normalized by mitochondrial DNA and plotted as relative levels (% of control) (mean±SD, n = 3). (D) The intracellular core protein (HBc) expression was detected by immunofluorescence microscopy. Cell nuclei were counterstained with DAPI. Each image is a representative of five different microscopic fields, the percentage of HBc-positive cells was indicated (Mean ± SD).

    Article Snippet: For T7E1-based indel assay, primers used to amplify DNA fragments containing LIG1-sgRNA1 and LIG3-sgRNA1 targeting region were listed in , indel mutations were detected by Guide-it Mutation Detection Kit (Clontech) according to manufacturer’s manual.

    Techniques: In Vitro, Infection, shRNA, Western Blot, Southern Blot, Real-time Polymerase Chain Reaction, Expressing, Immunofluorescence, Microscopy

    RT-PCR amplification of five cassava eIF4E ORFs with gene-specific primers. Total RNA was extracted from TMS60444 cassava line. The first-strand cDNA was synthesized using SuperScript ™ III Reverse Transcriptase (Invitrogen) and PCR was performed using Phusion High-Fidelity DNA polymerase (New England Biolabs) and primers indicated in Table 1 . Lane 1: DNA marker; Lane 2: 016601 CDS amplified with primers 016601F and 016601R; Lane 3: 016620 CDS amplified with primers 016620F and 016620R; Lane 4: 015501 CDS amplified with 015501F and 015501R, Lane 5: 013223 CDS amplified with primers 013223lF and 013223R, Lane 6, 013223 CDS amplified with primers 013223sF and 013223R; Lane 7: 013732 CDS amplified with 013732lF and 013732R; Lane 8: 013732 CDS amplified with 013732sF and 013732R; and Lane 9: negative water control.

    Journal: PLoS ONE

    Article Title: Variations of five eIF4E genes across cassava accessions exhibiting tolerant and susceptible responses to cassava brown streak disease

    doi: 10.1371/journal.pone.0181998

    Figure Lengend Snippet: RT-PCR amplification of five cassava eIF4E ORFs with gene-specific primers. Total RNA was extracted from TMS60444 cassava line. The first-strand cDNA was synthesized using SuperScript ™ III Reverse Transcriptase (Invitrogen) and PCR was performed using Phusion High-Fidelity DNA polymerase (New England Biolabs) and primers indicated in Table 1 . Lane 1: DNA marker; Lane 2: 016601 CDS amplified with primers 016601F and 016601R; Lane 3: 016620 CDS amplified with primers 016620F and 016620R; Lane 4: 015501 CDS amplified with 015501F and 015501R, Lane 5: 013223 CDS amplified with primers 013223lF and 013223R, Lane 6, 013223 CDS amplified with primers 013223sF and 013223R; Lane 7: 013732 CDS amplified with 013732lF and 013732R; Lane 8: 013732 CDS amplified with 013732sF and 013732R; and Lane 9: negative water control.

    Article Snippet: Cloning and sequencing analysis of cloned eIF4E transcripts The PCR-amplified cDNA fragments were first digested with Eco RI and Bam HI and cloned into the plasmid pGADT7 (Clontech, Mountain View, CA) previously digested with the same two enzymes.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Synthesized, Polymerase Chain Reaction, Marker

    Mutations affecting PgABCA2 protein in Cry2Ab-resistant pink bollworm from Arizona and India. ( a ) The predicted PgABCA2 protein includes amino (N) and carboxyl (C) termini (pink), two transmembrane domains (TMD1 and TMD2), each consisting of 6 transmembrane regions (TM; orange), three extracellular loops (ECL; green), two intracellular loops (ICL; blue), and two nucleotide-binding domains (NBD; purple). Mutations affecting transcripts of resistant pink bollworm: Circles show premature stop codons from India (red), Arizona (yellow), or both (red and yellow). Triangles show in-frame indels from India (red) or Arizona (yellow). Numbers indicate the affected amino acids. ( b ) Full-length and partial PgABCA2 cDNAs were obtained by direct PCR sequencing, DNA sequencing of cDNA clones, and/or PacBio ® DNA sequencing from susceptible (APHIS-S) and resistant, laboratory-selected (Bt4-R2) pink bollworm from Arizona, USA and India field-selected resistant populations (AM, CK, GAP, KT, and RK). The linear schematic (top) shows the predicted translated domain structure of the 5,187-bp full-length PgABCA2 coding sequence. The predicted protein includes amino- and carboxyl-termini (pink), transmembrane regions TM1-TM12 (orange), intracellular loops ICL1-ICL5 (blue), and extracellular loops ECL1-ECL6 (green). The domain structure connected to the exons that encode the respective domains is shown by dotted gray lines. Each predicted domain is numbered, with ECLs on top and ICLs numbered on bottom of protein schematic. Putative exons 1–31 are numbered, with grey exons indicating regions determined by direct PCR sequencing. Exons colored in light blue were further verified by sequencing cDNA clones (either by Sanger or PacBio ® sequencing). Red bars indicate disruption sites within the full-length coding sequence and the red triangles indicate the location of premature stop codons shown to scale based on the linear schematic of the translated domain structure. Unique cDNA variants are indicated as a, b, c, etc.

    Journal: Scientific Reports

    Article Title: ABC transporter mis-splicing associated with resistance to Bt toxin Cry2Ab in laboratory- and field-selected pink bollworm

    doi: 10.1038/s41598-018-31840-5

    Figure Lengend Snippet: Mutations affecting PgABCA2 protein in Cry2Ab-resistant pink bollworm from Arizona and India. ( a ) The predicted PgABCA2 protein includes amino (N) and carboxyl (C) termini (pink), two transmembrane domains (TMD1 and TMD2), each consisting of 6 transmembrane regions (TM; orange), three extracellular loops (ECL; green), two intracellular loops (ICL; blue), and two nucleotide-binding domains (NBD; purple). Mutations affecting transcripts of resistant pink bollworm: Circles show premature stop codons from India (red), Arizona (yellow), or both (red and yellow). Triangles show in-frame indels from India (red) or Arizona (yellow). Numbers indicate the affected amino acids. ( b ) Full-length and partial PgABCA2 cDNAs were obtained by direct PCR sequencing, DNA sequencing of cDNA clones, and/or PacBio ® DNA sequencing from susceptible (APHIS-S) and resistant, laboratory-selected (Bt4-R2) pink bollworm from Arizona, USA and India field-selected resistant populations (AM, CK, GAP, KT, and RK). The linear schematic (top) shows the predicted translated domain structure of the 5,187-bp full-length PgABCA2 coding sequence. The predicted protein includes amino- and carboxyl-termini (pink), transmembrane regions TM1-TM12 (orange), intracellular loops ICL1-ICL5 (blue), and extracellular loops ECL1-ECL6 (green). The domain structure connected to the exons that encode the respective domains is shown by dotted gray lines. Each predicted domain is numbered, with ECLs on top and ICLs numbered on bottom of protein schematic. Putative exons 1–31 are numbered, with grey exons indicating regions determined by direct PCR sequencing. Exons colored in light blue were further verified by sequencing cDNA clones (either by Sanger or PacBio ® sequencing). Red bars indicate disruption sites within the full-length coding sequence and the red triangles indicate the location of premature stop codons shown to scale based on the linear schematic of the translated domain structure. Unique cDNA variants are indicated as a, b, c, etc.

    Article Snippet: We used the primer combinations 1pgABCA2-5 + 8pgABCA2-3 and 4pgABCA2-5 + 27pgABCA2-3 (Supplementary Table ) to amplify two internal PgABCA2 cDNA fragments from APHIS-S. PCR was performed with 2.5 U Takara ExTaq Premix (Takara Bio USA Inc., Mountain View, CA), 2 μM of each sense and antisense primer, and 0.3 µg cDNA using a Biometra TProfessional gradient Thermocycler (Biometra, Germany) at: 95 °C for 3 min (1 cycle); 40 cycles of 95 °C for 45 s, 50 °C for 1 min and 72 °C for 2 min; then 72 °C for 5 min. Nested PCR amplification was carried using one microliter of primary PCR product using the primer combinations 2pgABCA2-5 + 6pgABCA2-3 and 15pgABCA2-5 + 23pgABCA2-3, respectively (Supplementary Table ).

    Techniques: Binding Assay, Polymerase Chain Reaction, Sequencing, DNA Sequencing, Clone Assay