Structured Review

Genewiz plasmid dna
Mbo II digest results. Agarose gel showing Mbo II digests of GAA <t>PCR</t> products of FRDA samples. The expected 170bp (5′) and 120bp (3′) undigested GAA-flanking fragments from normal pure GAA repeat expansion FRDA samples are shown in lanes 2, 3, and 4. These band sizes can be seen in between the 200 and 100bp fragments of the 1 Kb+ <t>DNA</t> ladder markers, which are loaded into lanes 1 and 11 of the gel. Lane 5 shows a large Mbo II band of approximately 600bp that was obtained from the positive interrupted GAA repeat sequence from the “NEP” BAC transgenic mouse that contains approximately 500 triplet repeats with the previously determined interrupted sequence of (GAA) 21 (GGAGAA) 5 (GGAGGAGAA) 70 (GAA) n ( Holloway et al., 2011 ). In addition for this positive sample, we also identified the expected 5′ flanking band of 170bp, together with a smaller band of less than 100bp that we sequenced and we showed to contain a 27bp deletion in the 3′ flanking region. Lane 6 shows an abnormal band of 200bp representing the 80bp duplication in the 3′ GAA flanking region. Lane 7 shows an abnormal band of approximately 100bp representing the 19bp deletion in the 3′ GAA flanking region. Lanes 8, 9, and 10 contain abnormal bands of approximately 300, 100, and 180bp, respectively, that are likely to contain a region of interrupted GAA repeat sequence within the body of one or other of the large FRDA GAA repeat expansions.
Plasmid Dna, supplied by Genewiz, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/plasmid dna/product/Genewiz
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
plasmid dna - by Bioz Stars, 2021-03
86/100 stars

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1) Product Images from "Large Interruptions of GAA Repeat Expansion Mutations in Friedreich Ataxia Are Very Rare"

Article Title: Large Interruptions of GAA Repeat Expansion Mutations in Friedreich Ataxia Are Very Rare

Journal: Frontiers in Cellular Neuroscience

doi: 10.3389/fncel.2018.00443

Mbo II digest results. Agarose gel showing Mbo II digests of GAA PCR products of FRDA samples. The expected 170bp (5′) and 120bp (3′) undigested GAA-flanking fragments from normal pure GAA repeat expansion FRDA samples are shown in lanes 2, 3, and 4. These band sizes can be seen in between the 200 and 100bp fragments of the 1 Kb+ DNA ladder markers, which are loaded into lanes 1 and 11 of the gel. Lane 5 shows a large Mbo II band of approximately 600bp that was obtained from the positive interrupted GAA repeat sequence from the “NEP” BAC transgenic mouse that contains approximately 500 triplet repeats with the previously determined interrupted sequence of (GAA) 21 (GGAGAA) 5 (GGAGGAGAA) 70 (GAA) n ( Holloway et al., 2011 ). In addition for this positive sample, we also identified the expected 5′ flanking band of 170bp, together with a smaller band of less than 100bp that we sequenced and we showed to contain a 27bp deletion in the 3′ flanking region. Lane 6 shows an abnormal band of 200bp representing the 80bp duplication in the 3′ GAA flanking region. Lane 7 shows an abnormal band of approximately 100bp representing the 19bp deletion in the 3′ GAA flanking region. Lanes 8, 9, and 10 contain abnormal bands of approximately 300, 100, and 180bp, respectively, that are likely to contain a region of interrupted GAA repeat sequence within the body of one or other of the large FRDA GAA repeat expansions.
Figure Legend Snippet: Mbo II digest results. Agarose gel showing Mbo II digests of GAA PCR products of FRDA samples. The expected 170bp (5′) and 120bp (3′) undigested GAA-flanking fragments from normal pure GAA repeat expansion FRDA samples are shown in lanes 2, 3, and 4. These band sizes can be seen in between the 200 and 100bp fragments of the 1 Kb+ DNA ladder markers, which are loaded into lanes 1 and 11 of the gel. Lane 5 shows a large Mbo II band of approximately 600bp that was obtained from the positive interrupted GAA repeat sequence from the “NEP” BAC transgenic mouse that contains approximately 500 triplet repeats with the previously determined interrupted sequence of (GAA) 21 (GGAGAA) 5 (GGAGGAGAA) 70 (GAA) n ( Holloway et al., 2011 ). In addition for this positive sample, we also identified the expected 5′ flanking band of 170bp, together with a smaller band of less than 100bp that we sequenced and we showed to contain a 27bp deletion in the 3′ flanking region. Lane 6 shows an abnormal band of 200bp representing the 80bp duplication in the 3′ GAA flanking region. Lane 7 shows an abnormal band of approximately 100bp representing the 19bp deletion in the 3′ GAA flanking region. Lanes 8, 9, and 10 contain abnormal bands of approximately 300, 100, and 180bp, respectively, that are likely to contain a region of interrupted GAA repeat sequence within the body of one or other of the large FRDA GAA repeat expansions.

Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Sequencing, BAC Assay, Transgenic Assay

Mbo II digests of GAA repeat expansions from human FRDA somatic tissues and mouse FRDA intergenerational and somatic tissues. Agarose gels showing Mbo II digests of GAA PCR products of (A) FRDA patient cerebellum tissue samples, (B) YG8sR mouse ear biopsy samples and human FRDA blood samples, and (C) four tissues from one YG8sR mouse. In each case, the expected 170 and 120bp undigested GAA-flanking fragments can be identified in between the 200 and 100bp fragments of the 1 Kb+ DNA ladder marker, which is loaded into the first lane of each gel. (A) Lanes 1–3 show the results from cerebellum tissue samples from three FRDA patients. (B) Lanes 1 and 2 are from FRDA patient blood samples; lanes 3–6 are from ear biopsy samples from 4 GAA repeat expansion-based YG8sR mice of four different generations, and lane 7 is from an ear biopsy sample from the Y47R mouse which has nine GAA repeats. (C) Lanes 1–4 are from brain, cerebellum, heart, and liver tissues of the YG8sR mouse, respectively.
Figure Legend Snippet: Mbo II digests of GAA repeat expansions from human FRDA somatic tissues and mouse FRDA intergenerational and somatic tissues. Agarose gels showing Mbo II digests of GAA PCR products of (A) FRDA patient cerebellum tissue samples, (B) YG8sR mouse ear biopsy samples and human FRDA blood samples, and (C) four tissues from one YG8sR mouse. In each case, the expected 170 and 120bp undigested GAA-flanking fragments can be identified in between the 200 and 100bp fragments of the 1 Kb+ DNA ladder marker, which is loaded into the first lane of each gel. (A) Lanes 1–3 show the results from cerebellum tissue samples from three FRDA patients. (B) Lanes 1 and 2 are from FRDA patient blood samples; lanes 3–6 are from ear biopsy samples from 4 GAA repeat expansion-based YG8sR mice of four different generations, and lane 7 is from an ear biopsy sample from the Y47R mouse which has nine GAA repeats. (C) Lanes 1–4 are from brain, cerebellum, heart, and liver tissues of the YG8sR mouse, respectively.

Techniques Used: Polymerase Chain Reaction, Marker, Mouse Assay

Related Articles

Sequencing:

Article Title: Conserved determinants of lentiviral genome dimerization
Article Snippet: The QuikChange Lightning Site-Directed Mutagenesis protocol (Agilent) was used for the mutagenesis process. .. All of the DNA plasmids were sent to Genewiz sequencing to ensure that the desired mutations were obtained. .. RNA synthesis and purification pUC19 plasmids carrying different RNA clones were amplified in E. coli XL10-Gold ultracompetent cells.

Article Title: Applying EEM- PARAFAC Analysis With Quantitative Real-Time PCR to Monitor Methanogenic Activity of High-Solid Anaerobic Digestion of Rice Straw
Article Snippet: .. The PCR procedure went as follows: initial denaturation at 95°C for 3 min, followed by 40 cycles of denaturing at 95°C for 30 s, annealing at 60°C for 30 s, extending at 72°C for 30 s; a final extension was made at 72°C for 10 min. After molecular cloning, the positive plasmid DNA was identified by sequencing of the samples (GENEWIZ, Inc., China). .. Plasmid DNA was serially diluted 10-fold, from 103 to 109 copies/μL; the 10-μL Q-PCR assay was set up with SYBR Premix Ex TaqTM II (Takara Bio Inc., Dalian, China) that consisted of 0.2 μL of primer (10 μM) and 2 μL of plasmid DNA template.

Article Title: Site-Directed Mutagenesis to Improve Sensitivity of a Synthetic Two-Component Signaling System
Article Snippet: Non-plasmid DNA was precipitated in 3 M KAc pH 4.8, and the supernatants were microfuged with 100% ethanol to form pellets of plasmid DNA. .. Plasmid DNA (800 ng) and sequencing primer N0296 (seq = TCG TCA ACC TCA TTT TGC GCC AG , 25 pmol) were combined with sterile water to a final volume of 15 uL, then sequenced by GENEWIZ (South Plainfield, NJ). .. β-galactosidase Assay NB466 was cultured in liquid media in both the light and dark and assessed for β-galactosidase activity.

Plasmid Preparation:

Article Title: Binding of a glaucoma-associated myocilin variant to the αB-crystallin chaperone impedes protein clearance in trabecular meshwork cells
Article Snippet: Human MYOC plasmids were all in the same vector, which had a minimal CMV promoter. .. CMV–MYOC plasmid cDNAs had been created and sequences confirmed by GeneWiz. .. One plasmid contained cDNA for WT untagged MYOC (accession no. ), and the other plasmids had cDNA for mutant MYOC with either the Y437H mutation or Q368X mutation.

Article Title: Applying EEM- PARAFAC Analysis With Quantitative Real-Time PCR to Monitor Methanogenic Activity of High-Solid Anaerobic Digestion of Rice Straw
Article Snippet: .. The PCR procedure went as follows: initial denaturation at 95°C for 3 min, followed by 40 cycles of denaturing at 95°C for 30 s, annealing at 60°C for 30 s, extending at 72°C for 30 s; a final extension was made at 72°C for 10 min. After molecular cloning, the positive plasmid DNA was identified by sequencing of the samples (GENEWIZ, Inc., China). .. Plasmid DNA was serially diluted 10-fold, from 103 to 109 copies/μL; the 10-μL Q-PCR assay was set up with SYBR Premix Ex TaqTM II (Takara Bio Inc., Dalian, China) that consisted of 0.2 μL of primer (10 μM) and 2 μL of plasmid DNA template.

Article Title: UDiTaS™, a genome editing detection method for indels and genome rearrangements
Article Snippet: Round 1 was performed in a 12 μl reaction volume, consisting of 6 μL of NEBNext® Ultra™ II Q5® Master Mix (New England Biolabs), 0.125 μM forward primer (OLI4610), 0.125 μM reverse primer (OLI4611), and 20 ng of gDNA template. .. Plasmid DNA from 96 colonies per sample was sequenced by Genewiz, Inc. using an M13 reverse primer. .. Analysis of indel rates was done with the Geneious Software package (Biomatters, https://www.geneious.com ).

Article Title: Identification of PTC725, an Orally Bioavailable Small Molecule That Selectively Targets the Hepatitis C Virus NS4B Protein
Article Snippet: E. coli DH5 α-T1R or TOP10 (Invitrogen) was transformed with plasmid DNA. .. Plasmid DNAs from 17 to 20 clones were sequenced at GENEWIZ (South Plainfield, NJ). .. Sequencing data were analyzed using the Sequencher software program (Gene Codes Corporation, Ann Arbor, MI).

Article Title: Analytical Characteristics of a Noninvasive Gene Expression Assay for Pigmented Skin Lesions.
Article Snippet: We previously reported clinical performance of a novel noninvasive and quantitative PCR (qPCR)-based molecular diagnostic assay (the pigmented lesion assay; PLA) that differentiates primary cutaneous melanoma from benign pigmented skin lesions through two target gene signatures, LINC00518 (LINC) and preferentially expressed antigen in melanoma (PRAME). .. We previously reported clinical performance of a novel noninvasive and quantitative PCR (qPCR)-based molecular diagnostic assay (the pigmented lesion assay; PLA) that differentiates primary cutaneous melanoma from benign pigmented skin lesions through two target gene signatures, LINC00518 (LINC) and preferentially expressed antigen in melanoma (PRAME). .. We previously reported clinical performance of a novel noninvasive and quantitative PCR (qPCR)-based molecular diagnostic assay (the pigmented lesion assay; PLA) that differentiates primary cutaneous melanoma from benign pigmented skin lesions through two target gene signatures, LINC00518 (LINC) and preferentially expressed antigen in melanoma (PRAME).

Article Title: Mutant myocilin impacts sarcomere ultrastructure in mouse gastrocnemius muscle
Article Snippet: .. The control vector was CMV-Tag1 (Aligent, 211170) and the CMV-MYOC plasmid cDNAs was constructed and sequenced by GeneWiz (Cambridge, MA). .. One plasmid contained cDNA for normal (wild-type) untagged human MYOC (Accession NM_000261) and the other plasmid had cDNA for MYOC with the Y437H mutation.

Article Title: Site-Directed Mutagenesis to Improve Sensitivity of a Synthetic Two-Component Signaling System
Article Snippet: Non-plasmid DNA was precipitated in 3 M KAc pH 4.8, and the supernatants were microfuged with 100% ethanol to form pellets of plasmid DNA. .. Plasmid DNA (800 ng) and sequencing primer N0296 (seq = TCG TCA ACC TCA TTT TGC GCC AG , 25 pmol) were combined with sterile water to a final volume of 15 uL, then sequenced by GENEWIZ (South Plainfield, NJ). .. β-galactosidase Assay NB466 was cultured in liquid media in both the light and dark and assessed for β-galactosidase activity.

Polymerase Chain Reaction:

Article Title: Applying EEM- PARAFAC Analysis With Quantitative Real-Time PCR to Monitor Methanogenic Activity of High-Solid Anaerobic Digestion of Rice Straw
Article Snippet: .. The PCR procedure went as follows: initial denaturation at 95°C for 3 min, followed by 40 cycles of denaturing at 95°C for 30 s, annealing at 60°C for 30 s, extending at 72°C for 30 s; a final extension was made at 72°C for 10 min. After molecular cloning, the positive plasmid DNA was identified by sequencing of the samples (GENEWIZ, Inc., China). .. Plasmid DNA was serially diluted 10-fold, from 103 to 109 copies/μL; the 10-μL Q-PCR assay was set up with SYBR Premix Ex TaqTM II (Takara Bio Inc., Dalian, China) that consisted of 0.2 μL of primer (10 μM) and 2 μL of plasmid DNA template.

Molecular Cloning:

Article Title: Applying EEM- PARAFAC Analysis With Quantitative Real-Time PCR to Monitor Methanogenic Activity of High-Solid Anaerobic Digestion of Rice Straw
Article Snippet: .. The PCR procedure went as follows: initial denaturation at 95°C for 3 min, followed by 40 cycles of denaturing at 95°C for 30 s, annealing at 60°C for 30 s, extending at 72°C for 30 s; a final extension was made at 72°C for 10 min. After molecular cloning, the positive plasmid DNA was identified by sequencing of the samples (GENEWIZ, Inc., China). .. Plasmid DNA was serially diluted 10-fold, from 103 to 109 copies/μL; the 10-μL Q-PCR assay was set up with SYBR Premix Ex TaqTM II (Takara Bio Inc., Dalian, China) that consisted of 0.2 μL of primer (10 μM) and 2 μL of plasmid DNA template.

Clone Assay:

Article Title: Identification of PTC725, an Orally Bioavailable Small Molecule That Selectively Targets the Hepatitis C Virus NS4B Protein
Article Snippet: E. coli DH5 α-T1R or TOP10 (Invitrogen) was transformed with plasmid DNA. .. Plasmid DNAs from 17 to 20 clones were sequenced at GENEWIZ (South Plainfield, NJ). .. Sequencing data were analyzed using the Sequencher software program (Gene Codes Corporation, Ann Arbor, MI).

Isolation:

Article Title: Analytical Characteristics of a Noninvasive Gene Expression Assay for Pigmented Skin Lesions.
Article Snippet: We previously reported clinical performance of a novel noninvasive and quantitative PCR (qPCR)-based molecular diagnostic assay (the pigmented lesion assay; PLA) that differentiates primary cutaneous melanoma from benign pigmented skin lesions through two target gene signatures, LINC00518 (LINC) and preferentially expressed antigen in melanoma (PRAME). .. We previously reported clinical performance of a novel noninvasive and quantitative PCR (qPCR)-based molecular diagnostic assay (the pigmented lesion assay; PLA) that differentiates primary cutaneous melanoma from benign pigmented skin lesions through two target gene signatures, LINC00518 (LINC) and preferentially expressed antigen in melanoma (PRAME). .. We previously reported clinical performance of a novel noninvasive and quantitative PCR (qPCR)-based molecular diagnostic assay (the pigmented lesion assay; PLA) that differentiates primary cutaneous melanoma from benign pigmented skin lesions through two target gene signatures, LINC00518 (LINC) and preferentially expressed antigen in melanoma (PRAME).

Real-time Polymerase Chain Reaction:

Article Title: Analytical Characteristics of a Noninvasive Gene Expression Assay for Pigmented Skin Lesions.
Article Snippet: We previously reported clinical performance of a novel noninvasive and quantitative PCR (qPCR)-based molecular diagnostic assay (the pigmented lesion assay; PLA) that differentiates primary cutaneous melanoma from benign pigmented skin lesions through two target gene signatures, LINC00518 (LINC) and preferentially expressed antigen in melanoma (PRAME). .. We previously reported clinical performance of a novel noninvasive and quantitative PCR (qPCR)-based molecular diagnostic assay (the pigmented lesion assay; PLA) that differentiates primary cutaneous melanoma from benign pigmented skin lesions through two target gene signatures, LINC00518 (LINC) and preferentially expressed antigen in melanoma (PRAME). .. We previously reported clinical performance of a novel noninvasive and quantitative PCR (qPCR)-based molecular diagnostic assay (the pigmented lesion assay; PLA) that differentiates primary cutaneous melanoma from benign pigmented skin lesions through two target gene signatures, LINC00518 (LINC) and preferentially expressed antigen in melanoma (PRAME).

Construct:

Article Title: Mutant myocilin impacts sarcomere ultrastructure in mouse gastrocnemius muscle
Article Snippet: .. The control vector was CMV-Tag1 (Aligent, 211170) and the CMV-MYOC plasmid cDNAs was constructed and sequenced by GeneWiz (Cambridge, MA). .. One plasmid contained cDNA for normal (wild-type) untagged human MYOC (Accession NM_000261) and the other plasmid had cDNA for MYOC with the Y437H mutation.

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    Genewiz plasmid dna
    Mbo II digest results. Agarose gel showing Mbo II digests of GAA <t>PCR</t> products of FRDA samples. The expected 170bp (5′) and 120bp (3′) undigested GAA-flanking fragments from normal pure GAA repeat expansion FRDA samples are shown in lanes 2, 3, and 4. These band sizes can be seen in between the 200 and 100bp fragments of the 1 Kb+ <t>DNA</t> ladder markers, which are loaded into lanes 1 and 11 of the gel. Lane 5 shows a large Mbo II band of approximately 600bp that was obtained from the positive interrupted GAA repeat sequence from the “NEP” BAC transgenic mouse that contains approximately 500 triplet repeats with the previously determined interrupted sequence of (GAA) 21 (GGAGAA) 5 (GGAGGAGAA) 70 (GAA) n ). In addition for this positive sample, we also identified the expected 5′ flanking band of 170bp, together with a smaller band of less than 100bp that we sequenced and we showed to contain a 27bp deletion in the 3′ flanking region. Lane 6 shows an abnormal band of 200bp representing the 80bp duplication in the 3′ GAA flanking region. Lane 7 shows an abnormal band of approximately 100bp representing the 19bp deletion in the 3′ GAA flanking region. Lanes 8, 9, and 10 contain abnormal bands of approximately 300, 100, and 180bp, respectively, that are likely to contain a region of interrupted GAA repeat sequence within the body of one or other of the large FRDA GAA repeat expansions.
    Plasmid Dna, supplied by Genewiz, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/plasmid dna/product/Genewiz
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    plasmid dna - by Bioz Stars, 2021-03
    86/100 stars
      Buy from Supplier

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    Mbo II digest results. Agarose gel showing Mbo II digests of GAA PCR products of FRDA samples. The expected 170bp (5′) and 120bp (3′) undigested GAA-flanking fragments from normal pure GAA repeat expansion FRDA samples are shown in lanes 2, 3, and 4. These band sizes can be seen in between the 200 and 100bp fragments of the 1 Kb+ DNA ladder markers, which are loaded into lanes 1 and 11 of the gel. Lane 5 shows a large Mbo II band of approximately 600bp that was obtained from the positive interrupted GAA repeat sequence from the “NEP” BAC transgenic mouse that contains approximately 500 triplet repeats with the previously determined interrupted sequence of (GAA) 21 (GGAGAA) 5 (GGAGGAGAA) 70 (GAA) n ). In addition for this positive sample, we also identified the expected 5′ flanking band of 170bp, together with a smaller band of less than 100bp that we sequenced and we showed to contain a 27bp deletion in the 3′ flanking region. Lane 6 shows an abnormal band of 200bp representing the 80bp duplication in the 3′ GAA flanking region. Lane 7 shows an abnormal band of approximately 100bp representing the 19bp deletion in the 3′ GAA flanking region. Lanes 8, 9, and 10 contain abnormal bands of approximately 300, 100, and 180bp, respectively, that are likely to contain a region of interrupted GAA repeat sequence within the body of one or other of the large FRDA GAA repeat expansions.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Large Interruptions of GAA Repeat Expansion Mutations in Friedreich Ataxia Are Very Rare

    doi: 10.3389/fncel.2018.00443

    Figure Lengend Snippet: Mbo II digest results. Agarose gel showing Mbo II digests of GAA PCR products of FRDA samples. The expected 170bp (5′) and 120bp (3′) undigested GAA-flanking fragments from normal pure GAA repeat expansion FRDA samples are shown in lanes 2, 3, and 4. These band sizes can be seen in between the 200 and 100bp fragments of the 1 Kb+ DNA ladder markers, which are loaded into lanes 1 and 11 of the gel. Lane 5 shows a large Mbo II band of approximately 600bp that was obtained from the positive interrupted GAA repeat sequence from the “NEP” BAC transgenic mouse that contains approximately 500 triplet repeats with the previously determined interrupted sequence of (GAA) 21 (GGAGAA) 5 (GGAGGAGAA) 70 (GAA) n ). In addition for this positive sample, we also identified the expected 5′ flanking band of 170bp, together with a smaller band of less than 100bp that we sequenced and we showed to contain a 27bp deletion in the 3′ flanking region. Lane 6 shows an abnormal band of 200bp representing the 80bp duplication in the 3′ GAA flanking region. Lane 7 shows an abnormal band of approximately 100bp representing the 19bp deletion in the 3′ GAA flanking region. Lanes 8, 9, and 10 contain abnormal bands of approximately 300, 100, and 180bp, respectively, that are likely to contain a region of interrupted GAA repeat sequence within the body of one or other of the large FRDA GAA repeat expansions.

    Article Snippet: For each PCR product, plasmid DNA from two independent colonies was Sanger sequenced by Genewiz using T3 and T7 sequencing primers.

    Techniques: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Sequencing, BAC Assay, Transgenic Assay

    Mbo II digests of GAA repeat expansions from human FRDA somatic tissues and mouse FRDA intergenerational and somatic tissues. Agarose gels showing Mbo II digests of GAA PCR products of (A) FRDA patient cerebellum tissue samples, (B) YG8sR mouse ear biopsy samples and human FRDA blood samples, and (C) four tissues from one YG8sR mouse. In each case, the expected 170 and 120bp undigested GAA-flanking fragments can be identified in between the 200 and 100bp fragments of the 1 Kb+ DNA ladder marker, which is loaded into the first lane of each gel. (A) Lanes 1–3 show the results from cerebellum tissue samples from three FRDA patients. (B) Lanes 1 and 2 are from FRDA patient blood samples; lanes 3–6 are from ear biopsy samples from 4 GAA repeat expansion-based YG8sR mice of four different generations, and lane 7 is from an ear biopsy sample from the Y47R mouse which has nine GAA repeats. (C) Lanes 1–4 are from brain, cerebellum, heart, and liver tissues of the YG8sR mouse, respectively.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Large Interruptions of GAA Repeat Expansion Mutations in Friedreich Ataxia Are Very Rare

    doi: 10.3389/fncel.2018.00443

    Figure Lengend Snippet: Mbo II digests of GAA repeat expansions from human FRDA somatic tissues and mouse FRDA intergenerational and somatic tissues. Agarose gels showing Mbo II digests of GAA PCR products of (A) FRDA patient cerebellum tissue samples, (B) YG8sR mouse ear biopsy samples and human FRDA blood samples, and (C) four tissues from one YG8sR mouse. In each case, the expected 170 and 120bp undigested GAA-flanking fragments can be identified in between the 200 and 100bp fragments of the 1 Kb+ DNA ladder marker, which is loaded into the first lane of each gel. (A) Lanes 1–3 show the results from cerebellum tissue samples from three FRDA patients. (B) Lanes 1 and 2 are from FRDA patient blood samples; lanes 3–6 are from ear biopsy samples from 4 GAA repeat expansion-based YG8sR mice of four different generations, and lane 7 is from an ear biopsy sample from the Y47R mouse which has nine GAA repeats. (C) Lanes 1–4 are from brain, cerebellum, heart, and liver tissues of the YG8sR mouse, respectively.

    Article Snippet: For each PCR product, plasmid DNA from two independent colonies was Sanger sequenced by Genewiz using T3 and T7 sequencing primers.

    Techniques: Polymerase Chain Reaction, Marker, Mouse Assay

    Mbo II digest results. Agarose gel showing Mbo II digests of GAA PCR products of FRDA samples. The expected 170bp (5′) and 120bp (3′) undigested GAA-flanking fragments from normal pure GAA repeat expansion FRDA samples are shown in lanes 2, 3, and 4. These band sizes can be seen in between the 200 and 100bp fragments of the 1 Kb+ DNA ladder markers, which are loaded into lanes 1 and 11 of the gel. Lane 5 shows a large Mbo II band of approximately 600bp that was obtained from the positive interrupted GAA repeat sequence from the “NEP” BAC transgenic mouse that contains approximately 500 triplet repeats with the previously determined interrupted sequence of (GAA) 21 (GGAGAA) 5 (GGAGGAGAA) 70 (GAA) n ( Holloway et al., 2011 ). In addition for this positive sample, we also identified the expected 5′ flanking band of 170bp, together with a smaller band of less than 100bp that we sequenced and we showed to contain a 27bp deletion in the 3′ flanking region. Lane 6 shows an abnormal band of 200bp representing the 80bp duplication in the 3′ GAA flanking region. Lane 7 shows an abnormal band of approximately 100bp representing the 19bp deletion in the 3′ GAA flanking region. Lanes 8, 9, and 10 contain abnormal bands of approximately 300, 100, and 180bp, respectively, that are likely to contain a region of interrupted GAA repeat sequence within the body of one or other of the large FRDA GAA repeat expansions.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Large Interruptions of GAA Repeat Expansion Mutations in Friedreich Ataxia Are Very Rare

    doi: 10.3389/fncel.2018.00443

    Figure Lengend Snippet: Mbo II digest results. Agarose gel showing Mbo II digests of GAA PCR products of FRDA samples. The expected 170bp (5′) and 120bp (3′) undigested GAA-flanking fragments from normal pure GAA repeat expansion FRDA samples are shown in lanes 2, 3, and 4. These band sizes can be seen in between the 200 and 100bp fragments of the 1 Kb+ DNA ladder markers, which are loaded into lanes 1 and 11 of the gel. Lane 5 shows a large Mbo II band of approximately 600bp that was obtained from the positive interrupted GAA repeat sequence from the “NEP” BAC transgenic mouse that contains approximately 500 triplet repeats with the previously determined interrupted sequence of (GAA) 21 (GGAGAA) 5 (GGAGGAGAA) 70 (GAA) n ( Holloway et al., 2011 ). In addition for this positive sample, we also identified the expected 5′ flanking band of 170bp, together with a smaller band of less than 100bp that we sequenced and we showed to contain a 27bp deletion in the 3′ flanking region. Lane 6 shows an abnormal band of 200bp representing the 80bp duplication in the 3′ GAA flanking region. Lane 7 shows an abnormal band of approximately 100bp representing the 19bp deletion in the 3′ GAA flanking region. Lanes 8, 9, and 10 contain abnormal bands of approximately 300, 100, and 180bp, respectively, that are likely to contain a region of interrupted GAA repeat sequence within the body of one or other of the large FRDA GAA repeat expansions.

    Article Snippet: For each PCR product, plasmid DNA from two independent colonies was Sanger sequenced by Genewiz using T3 and T7 sequencing primers.

    Techniques: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Sequencing, BAC Assay, Transgenic Assay

    Mbo II digests of GAA repeat expansions from human FRDA somatic tissues and mouse FRDA intergenerational and somatic tissues. Agarose gels showing Mbo II digests of GAA PCR products of (A) FRDA patient cerebellum tissue samples, (B) YG8sR mouse ear biopsy samples and human FRDA blood samples, and (C) four tissues from one YG8sR mouse. In each case, the expected 170 and 120bp undigested GAA-flanking fragments can be identified in between the 200 and 100bp fragments of the 1 Kb+ DNA ladder marker, which is loaded into the first lane of each gel. (A) Lanes 1–3 show the results from cerebellum tissue samples from three FRDA patients. (B) Lanes 1 and 2 are from FRDA patient blood samples; lanes 3–6 are from ear biopsy samples from 4 GAA repeat expansion-based YG8sR mice of four different generations, and lane 7 is from an ear biopsy sample from the Y47R mouse which has nine GAA repeats. (C) Lanes 1–4 are from brain, cerebellum, heart, and liver tissues of the YG8sR mouse, respectively.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Large Interruptions of GAA Repeat Expansion Mutations in Friedreich Ataxia Are Very Rare

    doi: 10.3389/fncel.2018.00443

    Figure Lengend Snippet: Mbo II digests of GAA repeat expansions from human FRDA somatic tissues and mouse FRDA intergenerational and somatic tissues. Agarose gels showing Mbo II digests of GAA PCR products of (A) FRDA patient cerebellum tissue samples, (B) YG8sR mouse ear biopsy samples and human FRDA blood samples, and (C) four tissues from one YG8sR mouse. In each case, the expected 170 and 120bp undigested GAA-flanking fragments can be identified in between the 200 and 100bp fragments of the 1 Kb+ DNA ladder marker, which is loaded into the first lane of each gel. (A) Lanes 1–3 show the results from cerebellum tissue samples from three FRDA patients. (B) Lanes 1 and 2 are from FRDA patient blood samples; lanes 3–6 are from ear biopsy samples from 4 GAA repeat expansion-based YG8sR mice of four different generations, and lane 7 is from an ear biopsy sample from the Y47R mouse which has nine GAA repeats. (C) Lanes 1–4 are from brain, cerebellum, heart, and liver tissues of the YG8sR mouse, respectively.

    Article Snippet: For each PCR product, plasmid DNA from two independent colonies was Sanger sequenced by Genewiz using T3 and T7 sequencing primers.

    Techniques: Polymerase Chain Reaction, Marker, Mouse Assay