Journal: BMC Genetics

Article Title: DNA sequence polymorphisms within the bovine guanine nucleotide-binding protein Gs subunit alpha (Gsα)-encoding ( GNAS ) genomic imprinting domain are associated with performance traits

doi: 10.1186/1471-2156-12-4

Figure Lengend Snippet: Sequencing traces of NESP55 gene for genomic DNA and cDNA from both maternal and foetal tissues . The genomic DNA from the mother of each foetus allowed genotyping of the maternal genotype, while the genomic DNA from each foetus allowed genotyping of the each foetus. Arrows indicate the exonic SNP, denoted R (A/G), which was used for both DNA genotyping and to analyse allele-specific expression status of NESP55 transcripts (cDNA) across tissues from 8 and 10 weeks old foetuses. Column number 4 presents the genotypes for all samples/animals tested. Column 5 indicates whether monoallelic ( i.e . uniparental) expression was detected.

Article Snippet: All sequencing reactions were performed commercially by GATC Biotech Ltd. (Constance, Germany) using the primer sequences listed above and resulting DNA sequence traces were analysed using the LaserGene Package (DNASTAR, WI, USA).

Techniques: Sequencing, Expressing

Journal: Scientific Reports

Article Title: Exon-skipping and mRNA decay in human liver tissue: molecular consequences of pathogenic bile salt export pump mutations

doi: 10.1038/srep24827

Figure Lengend Snippet: Sequencing of all 27 coding exons with adjacent intron regions of ABCB11 from gDNA revealed two relevant variants for child 1 (upper panel) and child 2 (lower panel) compared to reference sequence NM_003742.2 (Gene ID: 8647, first translated exon denoted as exon 1, adenine from ATG counted as c.1). Both children were compound heterozygous for the donor splice-site mutation c.150 + 3A > C as well as one exonic mutation. Child 1 had a duplication of GAGAT in exon 21 (c.2783_2787dup5) resulting in a frameshift with a premature termination codon (p.K930fs79X). Child 2 had a nucleotide exchange in exon 20 (c.2494C > T) leading to the missense mutation p.R832C. Corresponding sequences on nucleotide (c.) and protein (p.) level are shown in detail below. IVS4: intervening sequence, intron surrounded by coding exons 3 and 4.

Article Snippet: To quantify the ratio of exon 3 inclusion and exclusion, sequencing traces for each base were separated by CodonCode Aligner (V.4.2.5, CodonCode Corporation, Dedham, MA, USA).

Techniques: Sequencing, Mutagenesis

Journal: Scientific Reports

Article Title: Exon-skipping and mRNA decay in human liver tissue: molecular consequences of pathogenic bile salt export pump mutations

doi: 10.1038/srep24827

Figure Lengend Snippet: For child 1, the ABCB11 splice-site mutation c.150 + 3A > C (red) is inherited by the mother whereas c.2783_2787dup5 (yellow) is transmitted by the father. ( a ) These mutations were detectable by sequencing of gDNA isolated from explanted liver of child 1. ( b , c ) For mRNA analysis, PCR forward primers ex2/4_for and ex3_for were used together with ex22/23_rev . Sequencing of BSEP exon 21 of both PCR products revealed the wildtype sequence. The GAGAT duplication (c.2783_2787dup5) was not detectable in mRNA transcripts with or without exon 3. ex: exon, for: forward primer, gDNA: genomic DNA, mRNA: messenger RNA, rev: reverse primer.

Article Snippet: To quantify the ratio of exon 3 inclusion and exclusion, sequencing traces for each base were separated by CodonCode Aligner (V.4.2.5, CodonCode Corporation, Dedham, MA, USA).

Techniques: Mutagenesis, Sequencing, Isolation

Journal: Scientific Reports

Article Title: Exon-skipping and mRNA decay in human liver tissue: molecular consequences of pathogenic bile salt export pump mutations

doi: 10.1038/srep24827

Figure Lengend Snippet: RNA from human liver tissue was used for reverse transcription and subsequent PCR. Reverse sequencing results are depicted in reverse complement. PCR product sequencing of control liver tissue displays clear peaks of BSEP exons 2, 3 and 4 (n = 14). In contrast, PCR product sequencing of child 1 and 2 shows overlapping peaks of BSEP exons 2 and 3, proving co-existence of mRNA transcripts with and without BSEP exon 3. Peak areas of each nucleotide within the overlap were measured and revealed amounts of 36.9 ± 8.2% for transcripts including exon 3 whereas in 63.1 ± 8.2% of transcripts exon 3 is missing in case of child 1. For child 2, higher amounts of transcripts with exon 3 (62.9 ± 7.7%) compared to transcripts without exon 3 (37.1 ± 7.7%) were calculated. Percentages are given as mean values with standard deviations, *significantly different with p < 0.0001 proved by the student’s t-test. ex: exon, excl.: excluding, incl.: including.

Article Snippet: To quantify the ratio of exon 3 inclusion and exclusion, sequencing traces for each base were separated by CodonCode Aligner (V.4.2.5, CodonCode Corporation, Dedham, MA, USA).

Techniques: Reverse Transcription, Sequencing, Control

Journal: Scientific Reports

Article Title: Exon-skipping and mRNA decay in human liver tissue: molecular consequences of pathogenic bile salt export pump mutations

doi: 10.1038/srep24827

Figure Lengend Snippet: The splice-site mutation c.150 + 3A > C on the paternal allele is shown in red. The missense mutation c.2494C > T (p.R832C) inherited by the mother is displayed in purple. RNA from the liver biopsy was used for reverse transcription. ( a , b ) PCR forward primers ex2/4_for and ex3_for were combined with ex20/21_rev . Exon 20 was sequenced from both PCR products. Appearance of cytosine (C) or thymine (T) at position 2494 indicates the extent of exon-skipping caused by c.150 + 3A > C. ( a ) When primer ex2/4_for was used and hence exon 3 was absent, the main sequencing peak at c.2494 results from C. ( b ) In contrast, presence of exon 3 is associated with T to larger and C to lower amounts at that position. Occurrence of both alleles using ex3_for confirms that c.150 + 3A > C causes only partial exon-skipping in the patients’ liver tissues. for: forward primer, gDNA: genomic DNA, mRNA: messenger RNA, rev: reverse primer.

Article Snippet: To quantify the ratio of exon 3 inclusion and exclusion, sequencing traces for each base were separated by CodonCode Aligner (V.4.2.5, CodonCode Corporation, Dedham, MA, USA).

Techniques: Mutagenesis, Reverse Transcription, Sequencing