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  • 94
    Millipore expand long range dntpack pcr system
    Expand Long Range Dntpack Pcr System, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche long expand range pcr
    Tumor-specific L1 insertions in HCC patients with a history of alcoholism. For each insertion, the chromosomal location and orientation relative to the interrupted gene, if applicable, are shown. L1 insertions are depicted as white rectangles; the truncation point of each insertion is indicated. Target-site duplications (TSDs) are shown as black arrows; deletions of genomic DNA are shown as white triangles. Poly(A) tail length is indicated. Where applicable, untemplated nucleotides at the 5′ L1-genome junction are indicated as a blue rectangle. The location and count of junction-spanning RC-seq reads supporting each insertion are depicted as red and white rectangles. The positions of <t>PCR</t> validation primers are shown as small arrows. Where empty-filled validation was successful, primers are indicated in red. Where junction <t>PCRs</t> were employed, primers are depicted in pink (5′ junction) and blue (3′ junction). Where nested or hemi-nested PCR was necessary, the outer primer(s) are depicted in gray and the inner primers are depicted in color. Agarose gels containing the empty/filled or 5′ and 3′ junction validation products are shown. Templates are indicated above; for nested and hemi-nested PCR reactions, NTC 1st and NTC 2nd depict the no-template control reactions for the first and second rounds of PCR. Red arrows indicate the validating band. ( A ) A 5′ truncated L1 insertion in antisense orientation within the seventh intron of the gene KHDRBS2 , detected in patient HCC.58 tumor. ( B ) KHDRBS2 mRNA abundance relative to TBP measured by qRT-PCR using RNA extracted from 10 HCC patient tumor (red) and nontumor (light blue) sample pairs, including patient HCC.58. Data represent the mean of three technical replicates ± SD. For each patient, values were normalized to the nontumor mean. Significance values were obtained via a two-way ANOVA followed by Tukey's post hoc analysis. ( C , D ) Two additional tumor-specific 5′ truncated L1 insertions, their genomic locations and structural hallmarks, and validating PCR products are depicted as described above.
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    Boehringer Mannheim expand long range pcr kit
    <t>nurf301</t> is required for homeotic gene expression. ( A,B ) UBX protein in imaginal discs of the third thoracic segment (brown staining) is undetectable in nurf301 1 homozygotes. ( C,D ) Antibody staining shows that EN protein (revealed in green), which normally can be detected in the posterior compartment of all imaginal discs, is lost in nurf301 2 mutant larvae. ( E ) Semiquantitative <t>RT–PCR</t> analysis confirms that Ubx and en transcript abundance is reduced between 5- and 25-fold in total RNA isolated from nurf301 mutant animals. Lanes represent fivefold serial dilutions of mutant and wild-type total RNA (lane 1 , 200 ng; lane 2 , 40 ng; lane 3 , 8 ng; and lane 4 , 1.6 ng).
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    ψND5 element positions in C. briggsae mitochondrial genomes . Dashed boxes indicate the two ψND5 elements and open boxes indicate mtDNA genes. Protein-coding genes are indicated by their common abbreviations and tRNA genes are indicated by their respective associated single-letter amino acid codes. ψND5-1 is 214–223 bp, depending on isolate, and ψND5-2 is 325–344 bp. The dashed horizontal line on top indicates <t>DNA</t> sequences that are lost in heteroplasmic ND5 deletion variants and the arrows indicate the primer positions that are employed for conventional <t>PCR</t> assays.
    Expand Long Range Pcr Kit, supplied by Roche, used in various techniques. Bioz Stars score: 90/100, based on 186 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche expand long range dntpack pcr kit
    ψND5 element positions in C. briggsae mitochondrial genomes . Dashed boxes indicate the two ψND5 elements and open boxes indicate mtDNA genes. Protein-coding genes are indicated by their common abbreviations and tRNA genes are indicated by their respective associated single-letter amino acid codes. ψND5-1 is 214–223 bp, depending on isolate, and ψND5-2 is 325–344 bp. The dashed horizontal line on top indicates <t>DNA</t> sequences that are lost in heteroplasmic ND5 deletion variants and the arrows indicate the primer positions that are employed for conventional <t>PCR</t> assays.
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    Roche long range expand high fidelityplus pcr system
    ψND5 element positions in C. briggsae mitochondrial genomes . Dashed boxes indicate the two ψND5 elements and open boxes indicate mtDNA genes. Protein-coding genes are indicated by their common abbreviations and tRNA genes are indicated by their respective associated single-letter amino acid codes. ψND5-1 is 214–223 bp, depending on isolate, and ψND5-2 is 325–344 bp. The dashed horizontal line on top indicates <t>DNA</t> sequences that are lost in heteroplasmic ND5 deletion variants and the arrows indicate the primer positions that are employed for conventional <t>PCR</t> assays.
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    Roche expand long range dntpack
    ψND5 element positions in C. briggsae mitochondrial genomes . Dashed boxes indicate the two ψND5 elements and open boxes indicate mtDNA genes. Protein-coding genes are indicated by their common abbreviations and tRNA genes are indicated by their respective associated single-letter amino acid codes. ψND5-1 is 214–223 bp, depending on isolate, and ψND5-2 is 325–344 bp. The dashed horizontal line on top indicates <t>DNA</t> sequences that are lost in heteroplasmic ND5 deletion variants and the arrows indicate the primer positions that are employed for conventional <t>PCR</t> assays.
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    ψND5 element positions in C. briggsae mitochondrial genomes . Dashed boxes indicate the two ψND5 elements and open boxes indicate mtDNA genes. Protein-coding genes are indicated by their common abbreviations and tRNA genes are indicated by their respective associated single-letter amino acid codes. ψND5-1 is 214–223 bp, depending on isolate, and ψND5-2 is 325–344 bp. The dashed horizontal line on top indicates <t>DNA</t> sequences that are lost in heteroplasmic ND5 deletion variants and the arrows indicate the primer positions that are employed for conventional <t>PCR</t> assays.
    Expand Long Range System, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore expand long range pcr system
    ψND5 element positions in C. briggsae mitochondrial genomes . Dashed boxes indicate the two ψND5 elements and open boxes indicate mtDNA genes. Protein-coding genes are indicated by their common abbreviations and tRNA genes are indicated by their respective associated single-letter amino acid codes. ψND5-1 is 214–223 bp, depending on isolate, and ψND5-2 is 325–344 bp. The dashed horizontal line on top indicates <t>DNA</t> sequences that are lost in heteroplasmic ND5 deletion variants and the arrows indicate the primer positions that are employed for conventional <t>PCR</t> assays.
    Expand Long Range Pcr System, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Boehringer Mannheim expand long range pcr system
    ψND5 element positions in C. briggsae mitochondrial genomes . Dashed boxes indicate the two ψND5 elements and open boxes indicate mtDNA genes. Protein-coding genes are indicated by their common abbreviations and tRNA genes are indicated by their respective associated single-letter amino acid codes. ψND5-1 is 214–223 bp, depending on isolate, and ψND5-2 is 325–344 bp. The dashed horizontal line on top indicates <t>DNA</t> sequences that are lost in heteroplasmic ND5 deletion variants and the arrows indicate the primer positions that are employed for conventional <t>PCR</t> assays.
    Expand Long Range Pcr System, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 88/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche expand long range pcr dntpack
    ψND5 element positions in C. briggsae mitochondrial genomes . Dashed boxes indicate the two ψND5 elements and open boxes indicate mtDNA genes. Protein-coding genes are indicated by their common abbreviations and tRNA genes are indicated by their respective associated single-letter amino acid codes. ψND5-1 is 214–223 bp, depending on isolate, and ψND5-2 is 325–344 bp. The dashed horizontal line on top indicates <t>DNA</t> sequences that are lost in heteroplasmic ND5 deletion variants and the arrows indicate the primer positions that are employed for conventional <t>PCR</t> assays.
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    Deletion of SAMHD1 does not affect <t>mtDNA</t> stability. ( A ) MtDNA copy number in the TA muscle of 5 or 6 wt (filled dots) and SAMHD1 −/− (open dots) 13-wk-old (adult), 1-y-old (old adult), and 2-y-old (aged) animals was determined by qPCR and normalized to the value for adult wt mice. The mean for each group is indicated by a horizontal line. The P values were calculated using Welch’s t test; ns, nonsignificant. ( B ) DNA isolated from embryos and from the TA muscle of pups, adults, 1-y-old (old) adults, or aged animals was linearized with SacI endonuclease and separated on a neutral gel. MtDNA was visualized as above. One undigested sample is shown for comparison (Uncut). ( C ) Long-range <t>PCR</t> to detect deletions in mtDNA from the TA muscle of wt mice of various ages. Full-length product is indicated (FL). Only minor species containing deletions are observed in the mtDNA from old adults and aged animals, as indicated by the vertical line on the right-hand side of the gel. ( D ) Untreated or alkali-treated DNA from skeletal muscle of aged wt and SAMHD1 −/− (ko) mice was analyzed on a denaturing gel, and mtDNA was visualized using a COX1 probe. Each sample lane corresponds to an individual mouse, and dotted lines represent the median. ( E ) The median length of the untreated mtDNA in samples from D is indicated by a horizontal line. The two groups were compared using Welch’s t test (ns, nonsignificant; n = 4). ( F ) The length difference between untreated and alkali-treated mtDNAs shown in D was used to compute the number of rNMPs per single strand of mtDNA. The horizontal lines indicate the median. The P value of the statistically significant difference between the two groups was calculated by Welch’s t test; n = 4. ( G ) Long-range PCR was performed on mtDNA isolated from the TA muscle of adult and aged wt or SAMHD1 −/− (ko) mice. FL, full-length product; the vertical line indicates the size range of mtDNA molecules with deletions. ( H ) Kaplan–Meier survival curve for wt and SAMHD1 −/− (ko) mice. Comparison of the curves by the log-rank (Mantel–Cox) test confirmed no statistically significant difference between the genotypes. The sizes of the bands in the DNA ladder are indicated in kb. See also SI Appendix , Fig. S5 .
    Long Range Pcr The Expand Long Template Pcr System, supplied by Roche, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche expand long range dna polymerase
    Deletion of SAMHD1 does not affect <t>mtDNA</t> stability. ( A ) MtDNA copy number in the TA muscle of 5 or 6 wt (filled dots) and SAMHD1 −/− (open dots) 13-wk-old (adult), 1-y-old (old adult), and 2-y-old (aged) animals was determined by qPCR and normalized to the value for adult wt mice. The mean for each group is indicated by a horizontal line. The P values were calculated using Welch’s t test; ns, nonsignificant. ( B ) DNA isolated from embryos and from the TA muscle of pups, adults, 1-y-old (old) adults, or aged animals was linearized with SacI endonuclease and separated on a neutral gel. MtDNA was visualized as above. One undigested sample is shown for comparison (Uncut). ( C ) Long-range <t>PCR</t> to detect deletions in mtDNA from the TA muscle of wt mice of various ages. Full-length product is indicated (FL). Only minor species containing deletions are observed in the mtDNA from old adults and aged animals, as indicated by the vertical line on the right-hand side of the gel. ( D ) Untreated or alkali-treated DNA from skeletal muscle of aged wt and SAMHD1 −/− (ko) mice was analyzed on a denaturing gel, and mtDNA was visualized using a COX1 probe. Each sample lane corresponds to an individual mouse, and dotted lines represent the median. ( E ) The median length of the untreated mtDNA in samples from D is indicated by a horizontal line. The two groups were compared using Welch’s t test (ns, nonsignificant; n = 4). ( F ) The length difference between untreated and alkali-treated mtDNAs shown in D was used to compute the number of rNMPs per single strand of mtDNA. The horizontal lines indicate the median. The P value of the statistically significant difference between the two groups was calculated by Welch’s t test; n = 4. ( G ) Long-range PCR was performed on mtDNA isolated from the TA muscle of adult and aged wt or SAMHD1 −/− (ko) mice. FL, full-length product; the vertical line indicates the size range of mtDNA molecules with deletions. ( H ) Kaplan–Meier survival curve for wt and SAMHD1 −/− (ko) mice. Comparison of the curves by the log-rank (Mantel–Cox) test confirmed no statistically significant difference between the genotypes. The sizes of the bands in the DNA ladder are indicated in kb. See also SI Appendix , Fig. S5 .
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    Deletion of SAMHD1 does not affect <t>mtDNA</t> stability. ( A ) MtDNA copy number in the TA muscle of 5 or 6 wt (filled dots) and SAMHD1 −/− (open dots) 13-wk-old (adult), 1-y-old (old adult), and 2-y-old (aged) animals was determined by qPCR and normalized to the value for adult wt mice. The mean for each group is indicated by a horizontal line. The P values were calculated using Welch’s t test; ns, nonsignificant. ( B ) DNA isolated from embryos and from the TA muscle of pups, adults, 1-y-old (old) adults, or aged animals was linearized with SacI endonuclease and separated on a neutral gel. MtDNA was visualized as above. One undigested sample is shown for comparison (Uncut). ( C ) Long-range <t>PCR</t> to detect deletions in mtDNA from the TA muscle of wt mice of various ages. Full-length product is indicated (FL). Only minor species containing deletions are observed in the mtDNA from old adults and aged animals, as indicated by the vertical line on the right-hand side of the gel. ( D ) Untreated or alkali-treated DNA from skeletal muscle of aged wt and SAMHD1 −/− (ko) mice was analyzed on a denaturing gel, and mtDNA was visualized using a COX1 probe. Each sample lane corresponds to an individual mouse, and dotted lines represent the median. ( E ) The median length of the untreated mtDNA in samples from D is indicated by a horizontal line. The two groups were compared using Welch’s t test (ns, nonsignificant; n = 4). ( F ) The length difference between untreated and alkali-treated mtDNAs shown in D was used to compute the number of rNMPs per single strand of mtDNA. The horizontal lines indicate the median. The P value of the statistically significant difference between the two groups was calculated by Welch’s t test; n = 4. ( G ) Long-range PCR was performed on mtDNA isolated from the TA muscle of adult and aged wt or SAMHD1 −/− (ko) mice. FL, full-length product; the vertical line indicates the size range of mtDNA molecules with deletions. ( H ) Kaplan–Meier survival curve for wt and SAMHD1 −/− (ko) mice. Comparison of the curves by the log-rank (Mantel–Cox) test confirmed no statistically significant difference between the genotypes. The sizes of the bands in the DNA ladder are indicated in kb. See also SI Appendix , Fig. S5 .
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    Deletion of SAMHD1 does not affect <t>mtDNA</t> stability. ( A ) MtDNA copy number in the TA muscle of 5 or 6 wt (filled dots) and SAMHD1 −/− (open dots) 13-wk-old (adult), 1-y-old (old adult), and 2-y-old (aged) animals was determined by qPCR and normalized to the value for adult wt mice. The mean for each group is indicated by a horizontal line. The P values were calculated using Welch’s t test; ns, nonsignificant. ( B ) DNA isolated from embryos and from the TA muscle of pups, adults, 1-y-old (old) adults, or aged animals was linearized with SacI endonuclease and separated on a neutral gel. MtDNA was visualized as above. One undigested sample is shown for comparison (Uncut). ( C ) Long-range <t>PCR</t> to detect deletions in mtDNA from the TA muscle of wt mice of various ages. Full-length product is indicated (FL). Only minor species containing deletions are observed in the mtDNA from old adults and aged animals, as indicated by the vertical line on the right-hand side of the gel. ( D ) Untreated or alkali-treated DNA from skeletal muscle of aged wt and SAMHD1 −/− (ko) mice was analyzed on a denaturing gel, and mtDNA was visualized using a COX1 probe. Each sample lane corresponds to an individual mouse, and dotted lines represent the median. ( E ) The median length of the untreated mtDNA in samples from D is indicated by a horizontal line. The two groups were compared using Welch’s t test (ns, nonsignificant; n = 4). ( F ) The length difference between untreated and alkali-treated mtDNAs shown in D was used to compute the number of rNMPs per single strand of mtDNA. The horizontal lines indicate the median. The P value of the statistically significant difference between the two groups was calculated by Welch’s t test; n = 4. ( G ) Long-range PCR was performed on mtDNA isolated from the TA muscle of adult and aged wt or SAMHD1 −/− (ko) mice. FL, full-length product; the vertical line indicates the size range of mtDNA molecules with deletions. ( H ) Kaplan–Meier survival curve for wt and SAMHD1 −/− (ko) mice. Comparison of the curves by the log-rank (Mantel–Cox) test confirmed no statistically significant difference between the genotypes. The sizes of the bands in the DNA ladder are indicated in kb. See also SI Appendix , Fig. S5 .
    High Fidelity Expand Long Range Dntpack, supplied by Roche, used in various techniques. Bioz Stars score: 90/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Tumor-specific L1 insertions in HCC patients with a history of alcoholism. For each insertion, the chromosomal location and orientation relative to the interrupted gene, if applicable, are shown. L1 insertions are depicted as white rectangles; the truncation point of each insertion is indicated. Target-site duplications (TSDs) are shown as black arrows; deletions of genomic DNA are shown as white triangles. Poly(A) tail length is indicated. Where applicable, untemplated nucleotides at the 5′ L1-genome junction are indicated as a blue rectangle. The location and count of junction-spanning RC-seq reads supporting each insertion are depicted as red and white rectangles. The positions of PCR validation primers are shown as small arrows. Where empty-filled validation was successful, primers are indicated in red. Where junction PCRs were employed, primers are depicted in pink (5′ junction) and blue (3′ junction). Where nested or hemi-nested PCR was necessary, the outer primer(s) are depicted in gray and the inner primers are depicted in color. Agarose gels containing the empty/filled or 5′ and 3′ junction validation products are shown. Templates are indicated above; for nested and hemi-nested PCR reactions, NTC 1st and NTC 2nd depict the no-template control reactions for the first and second rounds of PCR. Red arrows indicate the validating band. ( A ) A 5′ truncated L1 insertion in antisense orientation within the seventh intron of the gene KHDRBS2 , detected in patient HCC.58 tumor. ( B ) KHDRBS2 mRNA abundance relative to TBP measured by qRT-PCR using RNA extracted from 10 HCC patient tumor (red) and nontumor (light blue) sample pairs, including patient HCC.58. Data represent the mean of three technical replicates ± SD. For each patient, values were normalized to the nontumor mean. Significance values were obtained via a two-way ANOVA followed by Tukey's post hoc analysis. ( C , D ) Two additional tumor-specific 5′ truncated L1 insertions, their genomic locations and structural hallmarks, and validating PCR products are depicted as described above.

    Journal: Genome Research

    Article Title: L1 retrotransposition is a common feature of mammalian hepatocarcinogenesis

    doi: 10.1101/gr.226993.117

    Figure Lengend Snippet: Tumor-specific L1 insertions in HCC patients with a history of alcoholism. For each insertion, the chromosomal location and orientation relative to the interrupted gene, if applicable, are shown. L1 insertions are depicted as white rectangles; the truncation point of each insertion is indicated. Target-site duplications (TSDs) are shown as black arrows; deletions of genomic DNA are shown as white triangles. Poly(A) tail length is indicated. Where applicable, untemplated nucleotides at the 5′ L1-genome junction are indicated as a blue rectangle. The location and count of junction-spanning RC-seq reads supporting each insertion are depicted as red and white rectangles. The positions of PCR validation primers are shown as small arrows. Where empty-filled validation was successful, primers are indicated in red. Where junction PCRs were employed, primers are depicted in pink (5′ junction) and blue (3′ junction). Where nested or hemi-nested PCR was necessary, the outer primer(s) are depicted in gray and the inner primers are depicted in color. Agarose gels containing the empty/filled or 5′ and 3′ junction validation products are shown. Templates are indicated above; for nested and hemi-nested PCR reactions, NTC 1st and NTC 2nd depict the no-template control reactions for the first and second rounds of PCR. Red arrows indicate the validating band. ( A ) A 5′ truncated L1 insertion in antisense orientation within the seventh intron of the gene KHDRBS2 , detected in patient HCC.58 tumor. ( B ) KHDRBS2 mRNA abundance relative to TBP measured by qRT-PCR using RNA extracted from 10 HCC patient tumor (red) and nontumor (light blue) sample pairs, including patient HCC.58. Data represent the mean of three technical replicates ± SD. For each patient, values were normalized to the nontumor mean. Significance values were obtained via a two-way ANOVA followed by Tukey's post hoc analysis. ( C , D ) Two additional tumor-specific 5′ truncated L1 insertions, their genomic locations and structural hallmarks, and validating PCR products are depicted as described above.

    Article Snippet: Empty-filled validation PCRs were carried out using the Roche Expand Long-Range PCR system (Roche) with primers specific to the 5′ and 3′ genomic sequence flanking putative insertions.

    Techniques: Polymerase Chain Reaction, Nested PCR, Quantitative RT-PCR

    ICC patient tumor mutational landscape, including tumor-specific L1 insertions. ( A ) summarizing ICC somatic mutations including copy number gains (red) and losses (blue), intra-chromosomal (colored by chromosome pair) and inter-chromosomal (red = duplication, blue = deletion, green = inversion) rearrangements. Rearrangements shown are those that intersect genes identified in prior ICC studies or present in the COSMIC Cancer Gene Census. L1 insertions are highlighted in red text labels on the outside of the circle, and selected genes with somatic mutations are highlighted with blue labels. Selected somatic mutations (SNVs and INDELs) are shown as yellow dots with highlights superimposed on the copy number variation rings. ( B , C ) Detailed characterization of two intergenic and 5′ truncated tumor-specific L1 insertions. For each insertion, the chromosomal location is shown. L1 insertions are depicted as white rectangles; the truncation point of each insertion is indicated. The location and count of junction-spanning RC-seq and WGS reads supporting each insertion are depicted as red and white rectangles. Target-site duplications are shown as black arrows; deletions of genomic DNA are shown as white triangles. Poly(A) tail length is indicated. The positions of PCR validation primers are shown as small arrows. Where empty-filled validation was successful, primers are indicated in red. Where junction PCRs were employed, primers are depicted in pink (5′ junction) and blue (3′ junction). Where hemi-nested PCR was necessary, the outer primer(s) are depicted in gray and the inner primers are depicted in color. Agarose gels containing the empty/filled or 5′ and 3′ junction validation products are shown. Templates are indicated above ; for hemi-nested PCR reactions, NTC 1st and NTC 2nd depict the no-template control reactions for the first and second rounds of PCR. Red arrows indicate the validating band. The L1 insertion shown in C carried an 18-bp 3′ transduced sequence implicating a donor L1 (shown in blue) located in the TTC28 gene on Chromosome 22. The methylation state of this specific donor L1 ( below ) was determined via targeted bisulfite sequencing of its promoter CpG island (primers are depicted in C as black arrows). Each cartoon represents 50 random, nonidentical sequences matching the donor L1 (black circle, methylated CpG; white circle, unmethylated CpG; “x,” absent) obtained from patient ICC.75 nontumor liver and matched tumor.

    Journal: Genome Research

    Article Title: L1 retrotransposition is a common feature of mammalian hepatocarcinogenesis

    doi: 10.1101/gr.226993.117

    Figure Lengend Snippet: ICC patient tumor mutational landscape, including tumor-specific L1 insertions. ( A ) summarizing ICC somatic mutations including copy number gains (red) and losses (blue), intra-chromosomal (colored by chromosome pair) and inter-chromosomal (red = duplication, blue = deletion, green = inversion) rearrangements. Rearrangements shown are those that intersect genes identified in prior ICC studies or present in the COSMIC Cancer Gene Census. L1 insertions are highlighted in red text labels on the outside of the circle, and selected genes with somatic mutations are highlighted with blue labels. Selected somatic mutations (SNVs and INDELs) are shown as yellow dots with highlights superimposed on the copy number variation rings. ( B , C ) Detailed characterization of two intergenic and 5′ truncated tumor-specific L1 insertions. For each insertion, the chromosomal location is shown. L1 insertions are depicted as white rectangles; the truncation point of each insertion is indicated. The location and count of junction-spanning RC-seq and WGS reads supporting each insertion are depicted as red and white rectangles. Target-site duplications are shown as black arrows; deletions of genomic DNA are shown as white triangles. Poly(A) tail length is indicated. The positions of PCR validation primers are shown as small arrows. Where empty-filled validation was successful, primers are indicated in red. Where junction PCRs were employed, primers are depicted in pink (5′ junction) and blue (3′ junction). Where hemi-nested PCR was necessary, the outer primer(s) are depicted in gray and the inner primers are depicted in color. Agarose gels containing the empty/filled or 5′ and 3′ junction validation products are shown. Templates are indicated above ; for hemi-nested PCR reactions, NTC 1st and NTC 2nd depict the no-template control reactions for the first and second rounds of PCR. Red arrows indicate the validating band. The L1 insertion shown in C carried an 18-bp 3′ transduced sequence implicating a donor L1 (shown in blue) located in the TTC28 gene on Chromosome 22. The methylation state of this specific donor L1 ( below ) was determined via targeted bisulfite sequencing of its promoter CpG island (primers are depicted in C as black arrows). Each cartoon represents 50 random, nonidentical sequences matching the donor L1 (black circle, methylated CpG; white circle, unmethylated CpG; “x,” absent) obtained from patient ICC.75 nontumor liver and matched tumor.

    Article Snippet: Empty-filled validation PCRs were carried out using the Roche Expand Long-Range PCR system (Roche) with primers specific to the 5′ and 3′ genomic sequence flanking putative insertions.

    Techniques: Immunocytochemistry, Polymerase Chain Reaction, Nested PCR, Sequencing, Methylation, Methylation Sequencing

    LCT13 and TFPI-2as expression is linked. ( A ) Schematic diagram of the genomic region in Figure 1 A indicating regions (1–7) analysed by strand-specific RT–PCR (middle). Shown above and below the schematic are the ethidium bromide–stained gels used to visualize the strand-specific RT–PCR. Regions 2–7 are specifically expressed in cancer cell lines (H, HCC-1954 and M, MCF-7), but not normal breast (N), showing that cancer-specific antisense transcription is detectable up to 300 kb away from the TFPI-2 gene and up to the LINE-1 retrotransposon associated with LCT13. ( B ) siRNA knockdown of the LCT13 transcript. 2D densitometry of semiquantitative strand-specific RT–PCR analysis normalized to APRT control reveals an approximate 50% knockdown in LCT13 levels in cells transfected with a pool of three siRNA duplexes directed against LCT13 compared to those transfected with scrambled control siRNAs (left panel). This is paralleled by a 40–50% decrease in the TFPI-2as transcript (right panel).

    Journal: Nucleic Acids Research

    Article Title: Expression of a large LINE-1-driven antisense RNA is linked to epigenetic silencing of the metastasis suppressor gene TFPI-2 in cancer

    doi: 10.1093/nar/gkt438

    Figure Lengend Snippet: LCT13 and TFPI-2as expression is linked. ( A ) Schematic diagram of the genomic region in Figure 1 A indicating regions (1–7) analysed by strand-specific RT–PCR (middle). Shown above and below the schematic are the ethidium bromide–stained gels used to visualize the strand-specific RT–PCR. Regions 2–7 are specifically expressed in cancer cell lines (H, HCC-1954 and M, MCF-7), but not normal breast (N), showing that cancer-specific antisense transcription is detectable up to 300 kb away from the TFPI-2 gene and up to the LINE-1 retrotransposon associated with LCT13. ( B ) siRNA knockdown of the LCT13 transcript. 2D densitometry of semiquantitative strand-specific RT–PCR analysis normalized to APRT control reveals an approximate 50% knockdown in LCT13 levels in cells transfected with a pool of three siRNA duplexes directed against LCT13 compared to those transfected with scrambled control siRNAs (left panel). This is paralleled by a 40–50% decrease in the TFPI-2as transcript (right panel).

    Article Snippet: Generation of constructs and ES cell clones For pTFPI-2as and pTFPI-2pa constructs, a 4.93-kb human genomic DNA fragment including the full-length TFPI-2 gene obtained by long-range PCR (Expand Long PCR kit, Roche) on human genomic DNA with primers HC63f and HC63g and was cloned into the BamHI and KpnI sites of pcDNA3 (Invitrogen) and pcDNA3p(A)for, respectively. pcDNA3p(A)for was derived from pcDNA3 by cloning a 262 bp BGHp(A) fragment, obtained by PCR on pcDNA3 with primers Hind-p(A)-for and Hind-p(A)-rev, into the HindIII site of pcDNA3.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Staining, Transfection

    A human TFPI-2 transgene is sensitive to antisense RNA repression in mouse ES cells. (A) Schematic diagram of constructs introduced into mouse ES cells: pTFPI-2as is designed to transcribe antisense to TFPI-2 from a CMV promoter, while pTFPI-2pa has a poly-A signal insertion downstream of the CMV promoter to block antisense transcription. Arrows indicate direction of transcription. Regions analysed by ChIP are annotated as ‘prom’ and ‘ex-in2’. ( B ) Strand-specific RT–PCR analysis of TFPI-2 antisenese (TFPI-2as) expression in transgenic mouse ES cell lines demonstrates increased levels in pTFPI-2as lines (L2 and L12) relative to pTFPI-2pa cells (L7 and L9), mouse Aprt acts as a positive control for RNA quality and quantity. This correlates with a reduction in TFPI-2 expression as shown by real-time PCR normalized to mouse Gapdh . ( C ) ChIP analysis followed by real-time PCR. Left panel: Antibodies to H3K9me3 reveal localized enrichment of H3K9me3 in the promoter region in the antisense expressing cell line, pTFPI-2as (L2), compared to cells transfected with pTFPI-2pa (L9), which express low levels of TFPI-2as. Right panel: Antibodies to H4K20me3 also show enrichment at the TFPI-2 promoter in pTFPI-2as compared to pTFPI-2pa.

    Journal: Nucleic Acids Research

    Article Title: Expression of a large LINE-1-driven antisense RNA is linked to epigenetic silencing of the metastasis suppressor gene TFPI-2 in cancer

    doi: 10.1093/nar/gkt438

    Figure Lengend Snippet: A human TFPI-2 transgene is sensitive to antisense RNA repression in mouse ES cells. (A) Schematic diagram of constructs introduced into mouse ES cells: pTFPI-2as is designed to transcribe antisense to TFPI-2 from a CMV promoter, while pTFPI-2pa has a poly-A signal insertion downstream of the CMV promoter to block antisense transcription. Arrows indicate direction of transcription. Regions analysed by ChIP are annotated as ‘prom’ and ‘ex-in2’. ( B ) Strand-specific RT–PCR analysis of TFPI-2 antisenese (TFPI-2as) expression in transgenic mouse ES cell lines demonstrates increased levels in pTFPI-2as lines (L2 and L12) relative to pTFPI-2pa cells (L7 and L9), mouse Aprt acts as a positive control for RNA quality and quantity. This correlates with a reduction in TFPI-2 expression as shown by real-time PCR normalized to mouse Gapdh . ( C ) ChIP analysis followed by real-time PCR. Left panel: Antibodies to H3K9me3 reveal localized enrichment of H3K9me3 in the promoter region in the antisense expressing cell line, pTFPI-2as (L2), compared to cells transfected with pTFPI-2pa (L9), which express low levels of TFPI-2as. Right panel: Antibodies to H4K20me3 also show enrichment at the TFPI-2 promoter in pTFPI-2as compared to pTFPI-2pa.

    Article Snippet: Generation of constructs and ES cell clones For pTFPI-2as and pTFPI-2pa constructs, a 4.93-kb human genomic DNA fragment including the full-length TFPI-2 gene obtained by long-range PCR (Expand Long PCR kit, Roche) on human genomic DNA with primers HC63f and HC63g and was cloned into the BamHI and KpnI sites of pcDNA3 (Invitrogen) and pcDNA3p(A)for, respectively. pcDNA3p(A)for was derived from pcDNA3 by cloning a 262 bp BGHp(A) fragment, obtained by PCR on pcDNA3 with primers Hind-p(A)-for and Hind-p(A)-rev, into the HindIII site of pcDNA3.

    Techniques: Construct, Blocking Assay, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Expressing, Transgenic Assay, Positive Control, Real-time Polymerase Chain Reaction, Transfection

    Correlated expression of LCT13 and TFPI-2as transcripts in breast cancer cells. ( A ) Schematic diagram of a 300-kb region of chromosome 7q21.3 including LCT13 and the TFPI-2 gene. Scale is kilobase and indicates the position from the centromere with the value of 0 arbitrarily assigned to the TSS of CALCR . Genes (5′ segment of CALCR , TFPI-2 and GNGT1 ) are indicated as gray arrows. Two LINE-1 elements are present in the region (L1PA2 and L1PA6). Transcriptional orientations are indicated by arrows. LCT13 is a previously identified transcript shown to initiate at an L1ASP by 5′ RACE ( 22 ). TFPI-2as is the fragment analysed by strand-specific RT–PCR to test for the presence of TFPI-2 antisense RNAs. Displayed are the three spliced ESTs isolated from kidney (BG432114) and liver (DW466562 and DW435092) libraries that initiate at the LINE1 antisense promoter like LCT13 and extend past the TFPI-2 gene with a putative alternative transcript GNGT1-005 also annotated. ( B ) Expression of TFPI-2as (upper) and TFPI-2 (lower) in normal breast (N) and in breast cancer cell lines (H, HCC-1954; M, MCF7) analysed by strand specific and real-time RT–PCR, respectively. TFPI-2 expression is reduced in both breast cancer cell lines compared to normal controls (n = 3). TFPI-2 expression levels were normalized to HPRT . ( C ) Expression of TFPI-2as (upper) and TFPI-2 (lower) in a panel of five matched normal and tumour breast tissue analysed as described in B.

    Journal: Nucleic Acids Research

    Article Title: Expression of a large LINE-1-driven antisense RNA is linked to epigenetic silencing of the metastasis suppressor gene TFPI-2 in cancer

    doi: 10.1093/nar/gkt438

    Figure Lengend Snippet: Correlated expression of LCT13 and TFPI-2as transcripts in breast cancer cells. ( A ) Schematic diagram of a 300-kb region of chromosome 7q21.3 including LCT13 and the TFPI-2 gene. Scale is kilobase and indicates the position from the centromere with the value of 0 arbitrarily assigned to the TSS of CALCR . Genes (5′ segment of CALCR , TFPI-2 and GNGT1 ) are indicated as gray arrows. Two LINE-1 elements are present in the region (L1PA2 and L1PA6). Transcriptional orientations are indicated by arrows. LCT13 is a previously identified transcript shown to initiate at an L1ASP by 5′ RACE ( 22 ). TFPI-2as is the fragment analysed by strand-specific RT–PCR to test for the presence of TFPI-2 antisense RNAs. Displayed are the three spliced ESTs isolated from kidney (BG432114) and liver (DW466562 and DW435092) libraries that initiate at the LINE1 antisense promoter like LCT13 and extend past the TFPI-2 gene with a putative alternative transcript GNGT1-005 also annotated. ( B ) Expression of TFPI-2as (upper) and TFPI-2 (lower) in normal breast (N) and in breast cancer cell lines (H, HCC-1954; M, MCF7) analysed by strand specific and real-time RT–PCR, respectively. TFPI-2 expression is reduced in both breast cancer cell lines compared to normal controls (n = 3). TFPI-2 expression levels were normalized to HPRT . ( C ) Expression of TFPI-2as (upper) and TFPI-2 (lower) in a panel of five matched normal and tumour breast tissue analysed as described in B.

    Article Snippet: Generation of constructs and ES cell clones For pTFPI-2as and pTFPI-2pa constructs, a 4.93-kb human genomic DNA fragment including the full-length TFPI-2 gene obtained by long-range PCR (Expand Long PCR kit, Roche) on human genomic DNA with primers HC63f and HC63g and was cloned into the BamHI and KpnI sites of pcDNA3 (Invitrogen) and pcDNA3p(A)for, respectively. pcDNA3p(A)for was derived from pcDNA3 by cloning a 262 bp BGHp(A) fragment, obtained by PCR on pcDNA3 with primers Hind-p(A)-for and Hind-p(A)-rev, into the HindIII site of pcDNA3.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Quantitative RT-PCR

    nurf301 is required for homeotic gene expression. ( A,B ) UBX protein in imaginal discs of the third thoracic segment (brown staining) is undetectable in nurf301 1 homozygotes. ( C,D ) Antibody staining shows that EN protein (revealed in green), which normally can be detected in the posterior compartment of all imaginal discs, is lost in nurf301 2 mutant larvae. ( E ) Semiquantitative RT–PCR analysis confirms that Ubx and en transcript abundance is reduced between 5- and 25-fold in total RNA isolated from nurf301 mutant animals. Lanes represent fivefold serial dilutions of mutant and wild-type total RNA (lane 1 , 200 ng; lane 2 , 40 ng; lane 3 , 8 ng; and lane 4 , 1.6 ng).

    Journal: Genes & Development

    Article Title: Biological functions of the ISWI chromatin remodeling complex NURF

    doi: 10.1101/gad.1032202

    Figure Lengend Snippet: nurf301 is required for homeotic gene expression. ( A,B ) UBX protein in imaginal discs of the third thoracic segment (brown staining) is undetectable in nurf301 1 homozygotes. ( C,D ) Antibody staining shows that EN protein (revealed in green), which normally can be detected in the posterior compartment of all imaginal discs, is lost in nurf301 2 mutant larvae. ( E ) Semiquantitative RT–PCR analysis confirms that Ubx and en transcript abundance is reduced between 5- and 25-fold in total RNA isolated from nurf301 mutant animals. Lanes represent fivefold serial dilutions of mutant and wild-type total RNA (lane 1 , 200 ng; lane 2 , 40 ng; lane 3 , 8 ng; and lane 4 , 1.6 ng).

    Article Snippet: EMS-induced nucleotide changes were determined by amplifying overlapping DNA fragments covering nurf301 using nurf301 -specific primers and an Expand Long Range PCR kit (Boehringer, Mannheim) and DNA isolated from homozygous mutant animals as template.

    Techniques: Expressing, Staining, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Isolation

    ψND5 element positions in C. briggsae mitochondrial genomes . Dashed boxes indicate the two ψND5 elements and open boxes indicate mtDNA genes. Protein-coding genes are indicated by their common abbreviations and tRNA genes are indicated by their respective associated single-letter amino acid codes. ψND5-1 is 214–223 bp, depending on isolate, and ψND5-2 is 325–344 bp. The dashed horizontal line on top indicates DNA sequences that are lost in heteroplasmic ND5 deletion variants and the arrows indicate the primer positions that are employed for conventional PCR assays.

    Journal: BMC Evolutionary Biology

    Article Title: Muller's Ratchet and compensatory mutation in Caenorhabditis briggsae mitochondrial genome evolution

    doi: 10.1186/1471-2148-8-62

    Figure Lengend Snippet: ψND5 element positions in C. briggsae mitochondrial genomes . Dashed boxes indicate the two ψND5 elements and open boxes indicate mtDNA genes. Protein-coding genes are indicated by their common abbreviations and tRNA genes are indicated by their respective associated single-letter amino acid codes. ψND5-1 is 214–223 bp, depending on isolate, and ψND5-2 is 325–344 bp. The dashed horizontal line on top indicates DNA sequences that are lost in heteroplasmic ND5 deletion variants and the arrows indicate the primer positions that are employed for conventional PCR assays.

    Article Snippet: PCR, DNA sequencing and phylogenetics mtDNA sequencing and PCR were performed as previously described [ , ], with the exception that mitochondrial genome sequences were initially amplified as four overlapping PCR products 3–5 kb in size each using the Expand Long Range PCR kit (Roche). e2TAK (Takara) proofreading DNA polymerase was used for all conventional PCRs.

    Techniques: Polymerase Chain Reaction

    Deletion of SAMHD1 does not affect mtDNA stability. ( A ) MtDNA copy number in the TA muscle of 5 or 6 wt (filled dots) and SAMHD1 −/− (open dots) 13-wk-old (adult), 1-y-old (old adult), and 2-y-old (aged) animals was determined by qPCR and normalized to the value for adult wt mice. The mean for each group is indicated by a horizontal line. The P values were calculated using Welch’s t test; ns, nonsignificant. ( B ) DNA isolated from embryos and from the TA muscle of pups, adults, 1-y-old (old) adults, or aged animals was linearized with SacI endonuclease and separated on a neutral gel. MtDNA was visualized as above. One undigested sample is shown for comparison (Uncut). ( C ) Long-range PCR to detect deletions in mtDNA from the TA muscle of wt mice of various ages. Full-length product is indicated (FL). Only minor species containing deletions are observed in the mtDNA from old adults and aged animals, as indicated by the vertical line on the right-hand side of the gel. ( D ) Untreated or alkali-treated DNA from skeletal muscle of aged wt and SAMHD1 −/− (ko) mice was analyzed on a denaturing gel, and mtDNA was visualized using a COX1 probe. Each sample lane corresponds to an individual mouse, and dotted lines represent the median. ( E ) The median length of the untreated mtDNA in samples from D is indicated by a horizontal line. The two groups were compared using Welch’s t test (ns, nonsignificant; n = 4). ( F ) The length difference between untreated and alkali-treated mtDNAs shown in D was used to compute the number of rNMPs per single strand of mtDNA. The horizontal lines indicate the median. The P value of the statistically significant difference between the two groups was calculated by Welch’s t test; n = 4. ( G ) Long-range PCR was performed on mtDNA isolated from the TA muscle of adult and aged wt or SAMHD1 −/− (ko) mice. FL, full-length product; the vertical line indicates the size range of mtDNA molecules with deletions. ( H ) Kaplan–Meier survival curve for wt and SAMHD1 −/− (ko) mice. Comparison of the curves by the log-rank (Mantel–Cox) test confirmed no statistically significant difference between the genotypes. The sizes of the bands in the DNA ladder are indicated in kb. See also SI Appendix , Fig. S5 .

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Elimination of rNMPs from mitochondrial DNA has no effect on its stability

    doi: 10.1073/pnas.1916851117

    Figure Lengend Snippet: Deletion of SAMHD1 does not affect mtDNA stability. ( A ) MtDNA copy number in the TA muscle of 5 or 6 wt (filled dots) and SAMHD1 −/− (open dots) 13-wk-old (adult), 1-y-old (old adult), and 2-y-old (aged) animals was determined by qPCR and normalized to the value for adult wt mice. The mean for each group is indicated by a horizontal line. The P values were calculated using Welch’s t test; ns, nonsignificant. ( B ) DNA isolated from embryos and from the TA muscle of pups, adults, 1-y-old (old) adults, or aged animals was linearized with SacI endonuclease and separated on a neutral gel. MtDNA was visualized as above. One undigested sample is shown for comparison (Uncut). ( C ) Long-range PCR to detect deletions in mtDNA from the TA muscle of wt mice of various ages. Full-length product is indicated (FL). Only minor species containing deletions are observed in the mtDNA from old adults and aged animals, as indicated by the vertical line on the right-hand side of the gel. ( D ) Untreated or alkali-treated DNA from skeletal muscle of aged wt and SAMHD1 −/− (ko) mice was analyzed on a denaturing gel, and mtDNA was visualized using a COX1 probe. Each sample lane corresponds to an individual mouse, and dotted lines represent the median. ( E ) The median length of the untreated mtDNA in samples from D is indicated by a horizontal line. The two groups were compared using Welch’s t test (ns, nonsignificant; n = 4). ( F ) The length difference between untreated and alkali-treated mtDNAs shown in D was used to compute the number of rNMPs per single strand of mtDNA. The horizontal lines indicate the median. The P value of the statistically significant difference between the two groups was calculated by Welch’s t test; n = 4. ( G ) Long-range PCR was performed on mtDNA isolated from the TA muscle of adult and aged wt or SAMHD1 −/− (ko) mice. FL, full-length product; the vertical line indicates the size range of mtDNA molecules with deletions. ( H ) Kaplan–Meier survival curve for wt and SAMHD1 −/− (ko) mice. Comparison of the curves by the log-rank (Mantel–Cox) test confirmed no statistically significant difference between the genotypes. The sizes of the bands in the DNA ladder are indicated in kb. See also SI Appendix , Fig. S5 .

    Article Snippet: MtDNA Deletion Analysis by Long-Range PCR.The Expand Long Template PCR system (Roche) with forward and reverse primers at nt 2,478 to 2,512 and nt 1,933 to 1,906, respectively, was used to amplify an ∼15,800-base pair fragment of mouse mtDNA from 25 ng of total DNA.

    Techniques: Real-time Polymerase Chain Reaction, Mouse Assay, Isolation, Polymerase Chain Reaction