Journal: BMC Medical Genetics

Article Title: Novel mutations of PKD genes in the Czech population with autosomal dominant polycystic kidney disease

doi: 10.1186/1471-2350-15-41

Figure Lengend Snippet: The example of HRM analysis in patients 385 and 419 (exon 39). 1 Detection of mutations by HRM. The green curve in the diagram corresponds to the PCR fragment carrying the nonsense mutation c.11172G > A (p.Trp3724X) in patient 385. The red curve refers to the PCR fragment carrying the likely pathogenic substitution c.11248C > G (p.Arg3750Gly) in patient 419. The rest of fragments with blue curves are wild type samples (patients without sequence changes). 2, 3 Confirmation of mutations by direct sequencing in patient 385 (number 2) and patient 419 (number 3). Both sequences are in reverse direction. The letter A corresponds to the mutated sequence; B is the wild type.

Article Snippet: PCR fragments were produced and analyzed by HRM using the LightCycler® 480 (Roche Applied Science).

Techniques: Polymerase Chain Reaction, Mutagenesis, Sequencing

Journal: Journal of Zhejiang University. Science. B

Article Title: Identification of a heat shock cognate protein 70 gene in Chinese soft-shell turtle (Pelodiscus sinensis) and its expression profiles under thermal stress *

doi: 10.1631/jzus.B1100309

Figure Lengend Snippet: Real-time RT-PCR analyses of tHsc70 expression in the liver (a), skeletal muscle (b), lung (c), and heart (d) of P . sinensis after heat stress for 1, 2, and 4 h with 1-h adaptive recovery at 25 °C (Groups V, VI, and VII) or without adaptation

Article Snippet: The polymerase chain reaction (PCR) fragment of tHsc70 was amplified using the primer pair Hsc70-dF and Hsc70-dR (Table ) on a thermal cycler S1000™ (Bio-Rad, Hercules, CA, USA).

Techniques: Quantitative RT-PCR, Expressing

Journal: Journal of Zhejiang University. Science. B

Article Title: Identification of a heat shock cognate protein 70 gene in Chinese soft-shell turtle (Pelodiscus sinensis) and its expression profiles under thermal stress *

doi: 10.1631/jzus.B1100309

Figure Lengend Snippet: Semi-quantitative RT-PCR analysis of tHsc70 expression in the liver, skeletal muscle, lung, and heart of P . sinensis

Article Snippet: The polymerase chain reaction (PCR) fragment of tHsc70 was amplified using the primer pair Hsc70-dF and Hsc70-dR (Table ) on a thermal cycler S1000™ (Bio-Rad, Hercules, CA, USA).

Techniques: Quantitative RT-PCR, Expressing

Journal: Nucleic Acids Research

Article Title: Re-splicing of mature mRNA in cancer cells promotes activation of distant weak alternative splice sites

doi: 10.1093/nar/gks520

Figure Lengend Snippet: Ectopically expressed intronless TSG101 gene (cDNA) is spliced and generates equivalent product as the endogenous aberrant mRNA in cancer cells. ( A ) The structure of the TSG101 mRNA. Red line and triangles indicate the postulated mRNA re-splicing via activated alternative 5′ and 3′ splice sites. Blue triangle indicates the PTC (1112–1114) generated by the postulated splicing. Black flags indicate the open reading frame (ORF; 127–1296). The positions of the PCR primers (P1–P4, P7–P10) are shown. ( B ) Using normal cells (HMEC) and cancer cells (MCF-7), endogenous TSG101 mRNAs were analyzed by RT-PCR (RT) and the genomic DNA was analyzed by PCR (Ge) with primer sets (P1–P4, P7–P10) annealed to the indicated exons. The black and red arrowheads denote the full-length and major aberrant mRNAs, respectively ( Figure 1 ). ( C ) The structures of three reporter TSG101-EGFP plasmids (see (A) for the red and blue triangles). The postulated splicing-dependent EGFP expression of these plasmids is indicated with (−) and (+). The positions of the PCR primer sets (P1–P5, P3–P6) are shown. ( D ) Cancer cells (MCF-7) were transfected with the indicated TSG101-EGFP reporter plasmids and the EGFP expression was analyzed by fluorescence microscopy. ( E ) These ectopically expressed TSG101-EGFP transcripts were analyzed by RT-PCR. The black and red arrowheads indicate the unspliced and spliced products via the alternative splice sites, respectively. Two minor spliced products (indicated with * and **) were generated from different alternative 3′ splice sites, i.e. Δ190–776 (587-nt deletion) and Δ190–1236 (1047-nt deletion), respectively.

Article Snippet: Construction of plasmids The expression plasmids for the TSG101-EGFP fusion proteins (TSG101-EGFP, TSG101-EGFP[+] and TSG101-EGFP[−]; C) were constructed by subcloning the corresponding polymerase chain reaction (PCR) fragments from the TSG101 gene together with PCR fragments from the pBI-EGFP plasmid (Clontech) into the pCXN2 mammalian expression vector ( ).

Techniques: Generated, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Fluorescence, Microscopy

Journal: Nucleic Acids Research

Article Title: Re-splicing of mature mRNA in cancer cells promotes activation of distant weak alternative splice sites

doi: 10.1093/nar/gks520

Figure Lengend Snippet: Ectopically expressed intronless FHIT gene (cDNA) is spliced, generating a product equivalent to the endogenous aberrant mRNA in cancer cells. ( A ) Schematic representation of the FHIT pre-mRNA and the observed aberrant splicing in cancer cells (red line). The aligned sequences of the normal (full-length) and aberrant FHIT mRNAs reveal the skipping of exon 3 to exon 6. ( B ) Using normal cells (HMEC) and cancer cells (MCF-7), endogenous FHIT mRNAs (schematically shown) were analyzed by RT-PCR with the indicated primer sets (P25–P26, P25′–P26′). The full-length mRNA (black arrowhead) and aberrantly spliced mRNA (red arrowhead) are indicated. Black flags indicate the ORF (372–815) and the red line indicates the postulated mRNA re-splicing that skips the whole exon 3 to exon 6 region. ( C ) Cancer cells (MCF-7) were transfected with the FHIT-EGFP reporter plasmid (schematically shown) and the spliced products were analyzed by RT-PCR with the indicated primer sets (P25–P5, P25′–P6). The black and red arrowheads indicate the unspliced and spliced products, respectively. Sequence analysis mapped the spliced sites (red triangles) to the 5′ end of exon 3 and the 3′ end of exon 6 (data not shown).

Article Snippet: Construction of plasmids The expression plasmids for the TSG101-EGFP fusion proteins (TSG101-EGFP, TSG101-EGFP[+] and TSG101-EGFP[−]; C) were constructed by subcloning the corresponding polymerase chain reaction (PCR) fragments from the TSG101 gene together with PCR fragments from the pBI-EGFP plasmid (Clontech) into the pCXN2 mammalian expression vector ( ).

Techniques: Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation, Sequencing

Journal: Nucleic Acids Research

Article Title: Re-splicing of mature mRNA in cancer cells promotes activation of distant weak alternative splice sites

doi: 10.1093/nar/gks520

Figure Lengend Snippet: The lariat RNA containing TSG101 exon 2 to exon 9 was identified by sequencing. ( A ) Detection of the lariat exons by lariat-specific RT-PCR amplification across the branch point with the indicated primer sets (P23–P24, P23′–P24′). The RT-PCR signal remained in the RNase R-digested (+) RNA sample, revealing a closed lariat structure. ( B ) Sequencing analysis of the lariat-specific RT-PCR product. Electropherogram of the sequence containing the branch point (arrow), followed by the alternative 5′ splice site (arrow) in the lariat exons. ( C ) A sequence alignment of the PCR fragment (blue) and the TSG101 gene (black) reveals a 2′–5′ branched connection between the end of the alternative 5′ splice site ( GT ) and the branch point ( A ), which is located upstream from the alternative 3′ splice site ( AG ).

Article Snippet: Construction of plasmids The expression plasmids for the TSG101-EGFP fusion proteins (TSG101-EGFP, TSG101-EGFP[+] and TSG101-EGFP[−]; C) were constructed by subcloning the corresponding polymerase chain reaction (PCR) fragments from the TSG101 gene together with PCR fragments from the pBI-EGFP plasmid (Clontech) into the pCXN2 mammalian expression vector ( ).

Techniques: Sequencing, Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction

Journal: Nucleic Acids Research

Article Title: Re-splicing of mature mRNA in cancer cells promotes activation of distant weak alternative splice sites

doi: 10.1093/nar/gks520

Figure Lengend Snippet: Detection of the lariat RNA consisting of only exons demonstrates the re-splicing of the endogenous TSG101 mRNA. ( A ) Schematic representation of the two postulated pathways leading to the generation of the aberrant mRNA, i.e. a conventional one-step direct aberrant splicing (right) and the proposed two-step process, including the re-splicing of the constitutively spliced mRNA (left). The pre-mRNA [1] and specific splicing products [2–5] from these two pathways were analyzed by RT-PCR with the indicated primers (arrows). ( B ) Detection of the specific splicing products [2–5] by RT-PCR using RNase R-digested (+) or RNase R-undigested (−) endogenous total RNA. The detected RT-PCR signals in the RNase R-digested sample (containing no linear RNAs) indicates an RNA species with either a 5′–2′ lariat or a 5′–3′ circular structure. However, the latter case was ruled out by the identification of a 5′–2′ branched structure ( Figure 4 A). Primers P19, P20, P21 and P22 anneal to introns 6, 7, 7 and 8, respectively. Primers P13/14 and P15/16 anneal to introns 7 and 8, respectively. Primers P11, P12, P17 and P18 anneal to exons 8, 10, 5 and 8, respectively. We used exactly the same PCR cycle numbers to amplify all these RNAs ( Supplementary Materials and Methods ).

Article Snippet: Construction of plasmids The expression plasmids for the TSG101-EGFP fusion proteins (TSG101-EGFP, TSG101-EGFP[+] and TSG101-EGFP[−]; C) were constructed by subcloning the corresponding polymerase chain reaction (PCR) fragments from the TSG101 gene together with PCR fragments from the pBI-EGFP plasmid (Clontech) into the pCXN2 mammalian expression vector ( ).

Techniques: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

Journal: Genes

Article Title: The drnf1 Gene from the Drought-Adapted Cyanobacterium Nostoc flagelliforme Improved Salt Tolerance in Transgenic Synechocystis and Arabidopsis Plant

doi: 10.3390/genes9090441

Figure Lengend Snippet: Identification of transgenic Arabidopsis plants and their salt tolerance phenotypes. ( A ) Identification of transgenes by PCR amplification of genomic DNA using drnf1 gene-specific primers; ( B ) Semi-quantitative RT-PCR analysis of drnf1 transcription. M: B031 DNA ladder (Dingguo, Beijing, China). WT: wild-type plant. 35d1-6: independent transgenic lines; ( C ) Germination performance of transgenic seeds on solid media supplemented with various concentrations of NaCl; ( D ) Growth performance of transgenic plants in 150 mM NaCl solution-watered soils. Three-week-old plantlets (0 d) were subjected to salt stress treatment.

Article Snippet: The drnf1 sequence was amplified by PCR with the primers drnf1 -F and drnf1 -R. The PCR fragment was ligated into pMD18-T vector (Takara, Dalian, China) for sequencing.

Techniques: Transgenic Assay, Polymerase Chain Reaction, Amplification, Quantitative RT-PCR

Journal: Genes

Article Title: The drnf1 Gene from the Drought-Adapted Cyanobacterium Nostoc flagelliforme Improved Salt Tolerance in Transgenic Synechocystis and Arabidopsis Plant

doi: 10.3390/genes9090441

Figure Lengend Snippet: Identification of transgenic drnf1 Synechocystis and its resistance to short-term salt stress. ( A ) Identification of the transgenic strain using drnf1 gene-specific primers by PCR. WT: wild-type strain. drnf1 -O: transgenic strain. M: DNA marker; ( B ) Semi-quantitative RT-PCR detection of drnf1 transcription in the transgenic strain; ( C ) Changes of relative Fv/Fm during salt stresses for 3 h. Data are mean ± s.d. ( n = 5).

Article Snippet: The drnf1 sequence was amplified by PCR with the primers drnf1 -F and drnf1 -R. The PCR fragment was ligated into pMD18-T vector (Takara, Dalian, China) for sequencing.

Techniques: Transgenic Assay, Polymerase Chain Reaction, Marker, Quantitative RT-PCR

Journal: Genes

Article Title: The drnf1 Gene from the Drought-Adapted Cyanobacterium Nostoc flagelliforme Improved Salt Tolerance in Transgenic Synechocystis and Arabidopsis Plant

doi: 10.3390/genes9090441

Figure Lengend Snippet: Salt stress response of Nostoc flagelliforme and induction of drnf1 transcription. ( A ) Physiologically recovered samples were treated by NaCl solutions for 27 h and then transferred to normal solutions for physiological recovery for 20 h. Physiological changes were indexed with the Fv/Fm parameter. Data are mean ± s.d. ( n = 5); ( B ) Semi-quantitative RT-PCR analysis of transcription levels of drnf1 gene under 0.4 M NaCl for different times. 16S rRNA gene was used an internal reference.

Article Snippet: The drnf1 sequence was amplified by PCR with the primers drnf1 -F and drnf1 -R. The PCR fragment was ligated into pMD18-T vector (Takara, Dalian, China) for sequencing.

Techniques: Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Hunting the Extinct Steppe Bison (Bison priscus) Mitochondrial Genome in the Trois-Frères Paleolithic Painted Cave

doi: 10.1371/journal.pone.0128267

Figure Lengend Snippet: Characteristics of the sequences used to assemble the Bison priscus mitochondrial genome. (A) Size distribution of the 3,851 unique Illumina reads. (B) Coverage for each position of the SGE2seq sequence obtained by merging the sequences of Illumina reads and PCR fragments.

Article Snippet: The final sequence, termed SGE2seq, was established using 81 consensus sequences of PCR fragments to complement the information derived from the Illumina reads.

Techniques: Sequencing, Polymerase Chain Reaction

Journal: Plant Physiology

Article Title: Expression of an Expansin Is Associated with Endosperm Weakening during Tomato Seed Germination 1

doi:

Figure Lengend Snippet: Genomic DNA gel-blot analysis of tomato expansin genes. Tomato genomic DNA (10 μg) was digested by Eco RI, Eco RV, and Hin dIII, respectively, and subjected to gel-blot hybridization using gene-specific cDNA probes amplified by PCR using primers corresponding to the 3′-untranslated regions of LeEXP8 (left) and LeEXP10 (right).

Article Snippet: The PCR fragments were used to screen a cDNA library prepared from whole gib-1 seeds imbibed in 100 μ m GA4+7 for 24 h. The cDNAs were labeled with enhanced chemiluminescence (ECL) nucleic acid labeling reagents (ECL kit, Amersham Life Science, Arlington Heights, IL) and were added to prehybridization solution at a final concentration of 10 ng/mL.

Techniques: Western Blot, Hybridization, Amplification, Polymerase Chain Reaction

Journal: G3: Genes|Genomes|Genetics

Article Title: A Nonsense Variant in COL6A1 in Landseer Dogs with Muscular Dystrophy

doi: 10.1534/g3.115.021923

Figure Lengend Snippet: Electropherograms of the COL6A1:c.289G > T variant. A fragment harboring exon 3 and flanking sequences of the COL6A1 gene was amplified by polymerase chain reaction and sequenced with the Sanger method. Shown are representative traces from Landseer dogs with the three different genotypes. The position of the variant is indicated by an arrow.

Article Snippet: Whole-genome sequencing of an affected Landseer dog We prepared a polymerase chain reaction (PCR)-free fragment library with 300-bp insert size and collected 330,331,465 Illumina HiSeq2500 paired-end reads (2 × 100 bp) or roughly 14.2× coverage.

Techniques: Variant Assay, Amplification, Polymerase Chain Reaction