smarter race 5 3 kit  (TaKaRa)

Journal: PLOS Neglected Tropical Diseases

Article Title: Identification and genetic characterization of a distinct genotype of Puumala orthohantavirus in Hebei Province, China

doi: 10.1371/journal.pntd.0014250

Figure Lengend Snippet: (A) RT-qPCR detection for the PUUV Guyuan strain in different tissues. (Note: the red curve represents the positive sample; the blue curve represents the positive control.) (B) Capillary electrophoresis results of the full-genome amplification products of the PUUV Guyuan strain.

Article Snippet: The 5’ and 3’ terminal sequences of the PUUV cDNA were amplified and sequenced using the SMARTer RACE 5’/3’ Kit (Takara Bio Inc., Japan), as described previously [ ].

Techniques: Quantitative RT-PCR, Positive Control, Electrophoresis, Amplification

Journal: PLOS Neglected Tropical Diseases

Article Title: Identification and genetic characterization of a distinct genotype of Puumala orthohantavirus in Hebei Province, China

doi: 10.1371/journal.pntd.0014250

Figure Lengend Snippet: A–C: Negative control tissues for the PUUV Guyuan strain (A: lung; B: liver; C: kidney). D–F: Positive tissues for the PUUV Guyuan strain (D: lung; E: liver; F: kidney). Green arrows indicate that granular fluorescence is indicative of PUUV protein antigenic presence. Scale bar = 100 μm and is located in the lower left.

Article Snippet: The 5’ and 3’ terminal sequences of the PUUV cDNA were amplified and sequenced using the SMARTer RACE 5’/3’ Kit (Takara Bio Inc., Japan), as described previously [ ].

Techniques: Negative Control, Fluorescence

Journal: bioRxiv

Article Title: The strictly conserved GGG-tracts in the 5’ and 3’ long terminal repeat of HIV-1 are critical to control multiple steps of HIV-1 replication to prevent acquisition of unwanted mutations in the region

doi: 10.64898/2026.04.24.720579

Figure Lengend Snippet: (A) A schematic represents heterogeneous transcriptional initiation site usage by HIV-1. The strictly conserved GGG-tract is located in the U3/R junction of the 5’ and 3’ LTR of HIV-1 clone DNA (pNL4-3EGFP ΔenvΔnef ). The 5’ leader sequences of HIV-1 RNA whose transcription initiates from the 1st, 2nd and 3rd deoxyguanosine in the tract are also shown (the 3G, 2G and 1G form, respectively). RNAs whose transcription initiates from nucleotides downstream of the tract are defined as the shorter form RNAs. The TATA-box and the GGG-tracts are highlighted by black and blue bold characters, respectively. (B) Nucleotide sequences between the TATA-box and the GGG-tract in the 5’ LTR of the HIV-1 references from the Los Alamos HIV databases ( http://www.hiv.lanl.gov , HIV Sequence Compendium 2023) are shown. Subtypes and accession numbers are shown. Dashes indicate nucleotide identity. The TATA-box and the GGG-tracts are highlighted as black and blue bold characters, respectively. (C) Nucleotide sequences of the CCC-CCC, TTT-TTT and AAA-AAA mutants of NL4-3EGFP ΔenvΔnef are shown. The mutated nucleotides are highlighted as red bold characters. The 5’ tract is highlighted with underlines. (D) Numbers of EGFP-positive cells produced with 1 mL of supernatant containing the VSV-G-pseudotyped NL4-3EGFP ΔenvΔnef wild-type (WT), AAA-AAA, CCC-CCC, or TTT-TTT mutant virus were evaluated. The numbers for WT were arbitrarily set as 100. Results from four independent experiments are shown. Asterisks code for statistical significance compared to WT: *, P<0.01.

Article Snippet: Viral RNA in virus particles were purified using Isogen (Fujifilm Wako) and purified RNAs were subjected to SMARTer® RACE 5’/3’ Kit (Takara Bio USA Inc., San Jose, CA).

Techniques: Sequencing, Produced, Mutagenesis, Virus

Journal: bioRxiv

Article Title: The strictly conserved GGG-tracts in the 5’ and 3’ long terminal repeat of HIV-1 are critical to control multiple steps of HIV-1 replication to prevent acquisition of unwanted mutations in the region

doi: 10.64898/2026.04.24.720579

Figure Lengend Snippet: (A) A schematic represents 5’ RACE analyses and provirus analyses. 293T cells were transfected with an HIV-1 clone plasmid (pNL4-3EGFP ΔenvΔnef WT or mutant plasmid) and pMISSION-VSV-G to produce the VSV-G-pseudotyped HIV-1. Genomic RNAs in the particles were purified and subsequently subjected to 5’ RACE. For provirus analyses, MT-4 cells were exposed with supernatant containing the VSV-G-pseudotyped virus. Genomic DNA containing HIV-1 provirus were purified from MT-4 cells. (B) Results of 5’ RACE analyses are shown. 5’RACE of purified RNA from the VSV-G-pseudotyped virus particles were done for the AAA-AAA mutant virus (n=34 clones). Nucleotides different from the input plasmid for producing particles ( and ) are highlighted as bold characters. Hyphens are used for forms not observed in the analyses. (C) HIV-1 reverse-transcriptase has been reported to have the ability to overcome mismatched 3’ termini between a template RNA and minus-strand strong-stop cDNA (-sscDNA). A schematic based on the knowledge represents predicted reverse-transcription processes to generate unexpected proviral sequences (AC A AAA; the region of the tract is highlighted with an underline) with the 4A form template RNA. Genomic RNAs, DNAs, acquired mutations and tRNAs are drawn in black, blue, red and green, respectively. (D-E) Results of 5’ RACE analyses are shown as described in . The analyses of purified RNA from the particles were done for the CCC-CCC (C; n=34 clones) or the TTT-TTT mutant virus (D; n=34 clones).

Article Snippet: Viral RNA in virus particles were purified using Isogen (Fujifilm Wako) and purified RNAs were subjected to SMARTer® RACE 5’/3’ Kit (Takara Bio USA Inc., San Jose, CA).

Techniques: Transfection, Plasmid Preparation, Mutagenesis, Purification, Virus, Clone Assay, Reverse Transcription

Journal: bioRxiv

Article Title: The strictly conserved GGG-tracts in the 5’ and 3’ long terminal repeat of HIV-1 are critical to control multiple steps of HIV-1 replication to prevent acquisition of unwanted mutations in the region

doi: 10.64898/2026.04.24.720579

Figure Lengend Snippet: (A) A schematic represents 5’ RACE analyses and provirus analyses. 293T cells were transfected with an HIV-1 clone plasmid (pNL4-3EGFP ΔenvΔnef WT or mutant plasmid) and pMISSION-VSV-G to produce the VSV-G-pseudotyped HIV-1. Genomic RNAs in the particles were purified and subsequently subjected to 5’ RACE. For provirus analyses, MT-4 cells were exposed with supernatant containing the VSV-G-pseudotyped virus. Genomic DNA containing HIV-1 provirus were purified from MT-4 cells. (B) Results of 5’ RACE analyses are shown. 5’RACE of purified RNA from the VSV-G-pseudotyped virus particles were done for the AAA-AAA mutant virus (n=34 clones). Nucleotides different from the input plasmid for producing particles ( and ) are highlighted as bold characters. Hyphens are used for forms not observed in the analyses. (C) HIV-1 reverse-transcriptase has been reported to have the ability to overcome mismatched 3’ termini between a template RNA and minus-strand strong-stop cDNA (-sscDNA). A schematic based on the knowledge represents predicted reverse-transcription processes to generate unexpected proviral sequences (AC A AAA; the region of the tract is highlighted with an underline) with the 4A form template RNA. Genomic RNAs, DNAs, acquired mutations and tRNAs are drawn in black, blue, red and green, respectively. (D-E) Results of 5’ RACE analyses are shown as described in . The analyses of purified RNA from the particles were done for the CCC-CCC (C; n=34 clones) or the TTT-TTT mutant virus (D; n=34 clones).

Article Snippet: Five-prime rapid amplification of cDNA end (5’ RACE) analyses of RNAs purified from mutant virus particles revealed multiple RNA variants with 5’ terminal sequences differing from the plasmid used for producing the particles.

Techniques: Transfection, Plasmid Preparation, Mutagenesis, Purification, Virus, Clone Assay, Reverse Transcription

Journal: bioRxiv

Article Title: The strictly conserved GGG-tracts in the 5’ and 3’ long terminal repeat of HIV-1 are critical to control multiple steps of HIV-1 replication to prevent acquisition of unwanted mutations in the region

doi: 10.64898/2026.04.24.720579

Figure Lengend Snippet: (A) Nucleotide sequences of the CCC-AAA, CCC-GGG and CCC-TTT mutants of NL4-3EGFP ΔenvΔnef are shown as described in . (B) Results of 5’ RACE analyses are shown as described in . 5’ RACE analyses of purified RNA from the particles were done as described in for the CCC-AAA (left panels; n=38 clones), the CCC-GGG (center panels; n=40 clones) and the CCC-TTT mutant virus (right panels; n=39 clones).

Article Snippet: Five-prime rapid amplification of cDNA end (5’ RACE) analyses of RNAs purified from mutant virus particles revealed multiple RNA variants with 5’ terminal sequences differing from the plasmid used for producing the particles.

Techniques: Purification, Clone Assay, Mutagenesis, Virus

Journal: bioRxiv

Article Title: The strictly conserved GGG-tracts in the 5’ and 3’ long terminal repeat of HIV-1 are critical to control multiple steps of HIV-1 replication to prevent acquisition of unwanted mutations in the region

doi: 10.64898/2026.04.24.720579

Figure Lengend Snippet: (A) Nucleotide sequences of the GGG-AAA, GGG-CCC and GGG-TTT mutants of NL4-3EGFP ΔenvΔnef are shown as described in . (B-D) Results of 5’ RACE analyses are shown as described in . 5’ RACE analyses of purified RNA from the particles were done as described in for the GGG-AAA (B; n=43 clones), the GGG-CCC (C; n=40 clones) and the GGG-TTT mutant virus (D; n=38 clones).

Article Snippet: Five-prime rapid amplification of cDNA end (5’ RACE) analyses of RNAs purified from mutant virus particles revealed multiple RNA variants with 5’ terminal sequences differing from the plasmid used for producing the particles.

Techniques: Purification, Clone Assay, Mutagenesis, Virus