Structured Review

Roche long expand range pcr
Long Expand Range Pcr, supplied by Roche, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/long expand range pcr/product/Roche
Average 90 stars, based on 1 article reviews
Price from $9.99 to $1999.99
long expand range pcr - by Bioz Stars, 2020-07
90/100 stars

Related Products / Commonly Used Together

cg7691 genomic fragment

Images

Related Articles

Polymerase Chain Reaction:

Article Title: Hemotin, a Regulator of Phagocytosis Encoded by a Small ORF and Conserved across Metazoans
Article Snippet: .. A CG7691 Genomic Fragment spanning 11.8 Kb was constructed by amplifying independently four sequential genomic fragments by Long Expand Range PCR (Roche, Basel, Switzerland) each containing the EcoRI-Kpn1, KpnI-NotI, NotI-XbaI and XbaI-AscI restriction sites respectively and then cloned in TOPO vector (Invitrogen). ..

Clone Assay:

Article Title: Hemotin, a Regulator of Phagocytosis Encoded by a Small ORF and Conserved across Metazoans
Article Snippet: .. A CG7691 Genomic Fragment spanning 11.8 Kb was constructed by amplifying independently four sequential genomic fragments by Long Expand Range PCR (Roche, Basel, Switzerland) each containing the EcoRI-Kpn1, KpnI-NotI, NotI-XbaI and XbaI-AscI restriction sites respectively and then cloned in TOPO vector (Invitrogen). ..

Construct:

Article Title: Hemotin, a Regulator of Phagocytosis Encoded by a Small ORF and Conserved across Metazoans
Article Snippet: .. A CG7691 Genomic Fragment spanning 11.8 Kb was constructed by amplifying independently four sequential genomic fragments by Long Expand Range PCR (Roche, Basel, Switzerland) each containing the EcoRI-Kpn1, KpnI-NotI, NotI-XbaI and XbaI-AscI restriction sites respectively and then cloned in TOPO vector (Invitrogen). ..

Plasmid Preparation:

Article Title: Hemotin, a Regulator of Phagocytosis Encoded by a Small ORF and Conserved across Metazoans
Article Snippet: .. A CG7691 Genomic Fragment spanning 11.8 Kb was constructed by amplifying independently four sequential genomic fragments by Long Expand Range PCR (Roche, Basel, Switzerland) each containing the EcoRI-Kpn1, KpnI-NotI, NotI-XbaI and XbaI-AscI restriction sites respectively and then cloned in TOPO vector (Invitrogen). ..

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 92
    Roche long range pcr
    Confirmation and characterization of the rearrangements . (A) Confirmation of the deletion of exons 1A/1B-2 by long-range <t>PCR</t> and sequencing of the breakpoints. (B) Confirmation of the deletion of exons 5–14 by long-range PCR and sequencing of the breakpoints. (C) Confirmation of the deletion of exons 11–12 by long-range PCR and sequencing of the breakpoints. Lanes 1+, 2+, carriers of the deletion; lane C-, negative control (wt); lane B, blank; lane M, marker (Ready-Load™ 1 Kb <t>DNA</t> Ladder, Invitrogen).
    Long Range Pcr, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 170 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/long range pcr/product/Roche
    Average 92 stars, based on 170 article reviews
    Price from $9.99 to $1999.99
    long range pcr - by Bioz Stars, 2020-07
    92/100 stars
      Buy from Supplier

    94
    Roche expand long template pcr system
    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-week-old (adult), 1-year-old (old adult), and 2-year-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, non-significant. b) DNA isolated from embryos and from the TA muscle of pups, adults, 1-year-old (old) adults, or aged animals was linearized with SacI endonuclease and separated on a neutral gel. MtDNA was visualized as above. Full-length mtDNA is indicated (FL); the asterisk denotes a higher-migrating species resistant to cleavage. 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 Fig. 5d is indicated by a horizontal line. The two groups were compared using Welch’s t-test (ns, non-significant; n = 4). f) The length difference between untreated and alkali-treated mtDNAs shown in Fig. 5d 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 Fig. S5.
    Expand Long Template Pcr System, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 1004 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/expand long template pcr system/product/Roche
    Average 94 stars, based on 1004 article reviews
    Price from $9.99 to $1999.99
    expand long template pcr system - by Bioz Stars, 2020-07
    94/100 stars
      Buy from Supplier

    Image Search Results


    Confirmation and characterization of the rearrangements . (A) Confirmation of the deletion of exons 1A/1B-2 by long-range PCR and sequencing of the breakpoints. (B) Confirmation of the deletion of exons 5–14 by long-range PCR and sequencing of the breakpoints. (C) Confirmation of the deletion of exons 11–12 by long-range PCR and sequencing of the breakpoints. Lanes 1+, 2+, carriers of the deletion; lane C-, negative control (wt); lane B, blank; lane M, marker (Ready-Load™ 1 Kb DNA Ladder, Invitrogen).

    Journal: BMC Medical Genetics

    Article Title: High occurrence of BRCA1 intragenic rearrangements in hereditary breast and ovarian cancer syndrome in the Czech Republic

    doi: 10.1186/1471-2350-8-32

    Figure Lengend Snippet: Confirmation and characterization of the rearrangements . (A) Confirmation of the deletion of exons 1A/1B-2 by long-range PCR and sequencing of the breakpoints. (B) Confirmation of the deletion of exons 5–14 by long-range PCR and sequencing of the breakpoints. (C) Confirmation of the deletion of exons 11–12 by long-range PCR and sequencing of the breakpoints. Lanes 1+, 2+, carriers of the deletion; lane C-, negative control (wt); lane B, blank; lane M, marker (Ready-Load™ 1 Kb DNA Ladder, Invitrogen).

    Article Snippet: Confirmation and characterization of the rearrangements Positive results detected by MLPA of two independently drawn samples of genomic DNA were confirmed by long-range PCR (Expand Long Template PCR System, Roche Applied Science), conducted in accordance with the manufacturer's instructions.

    Techniques: Polymerase Chain Reaction, Sequencing, Negative Control, Marker

    Confirmation and characterization of the rearrangements . (A) Confirmation of the deletion of exons 18–19 by long-range PCR and sequencing of the breakpoints. (B) Confirmation of the deletion of exon 20 and sequencing of the breakpoints.Lanes 1+, 2+, carriers of the deletion; lane C-, negative control (wt); lane B, blank; lane M, marker (Ready-Load™ 1 Kb DNA Ladder, Invitrogen).

    Journal: BMC Medical Genetics

    Article Title: High occurrence of BRCA1 intragenic rearrangements in hereditary breast and ovarian cancer syndrome in the Czech Republic

    doi: 10.1186/1471-2350-8-32

    Figure Lengend Snippet: Confirmation and characterization of the rearrangements . (A) Confirmation of the deletion of exons 18–19 by long-range PCR and sequencing of the breakpoints. (B) Confirmation of the deletion of exon 20 and sequencing of the breakpoints.Lanes 1+, 2+, carriers of the deletion; lane C-, negative control (wt); lane B, blank; lane M, marker (Ready-Load™ 1 Kb DNA Ladder, Invitrogen).

    Article Snippet: Confirmation and characterization of the rearrangements Positive results detected by MLPA of two independently drawn samples of genomic DNA were confirmed by long-range PCR (Expand Long Template PCR System, Roche Applied Science), conducted in accordance with the manufacturer's instructions.

    Techniques: Polymerase Chain Reaction, Sequencing, Negative Control, Marker

    Confirmation and characterization of the rearrangements . Confirmation of the deletion of the exons 21–22 by long-range PCR and sequencing of the breakpoints. The deletion/insertion event was characterized as g.77128_80906del3779ins236. Lanes 1+, 2+, carriers of the deletion; lane C-, negative control (wt); lane B, blank; lane M, marker (Ready-Load™ 1 Kb DNA Ladder, Invitrogen).

    Journal: BMC Medical Genetics

    Article Title: High occurrence of BRCA1 intragenic rearrangements in hereditary breast and ovarian cancer syndrome in the Czech Republic

    doi: 10.1186/1471-2350-8-32

    Figure Lengend Snippet: Confirmation and characterization of the rearrangements . Confirmation of the deletion of the exons 21–22 by long-range PCR and sequencing of the breakpoints. The deletion/insertion event was characterized as g.77128_80906del3779ins236. Lanes 1+, 2+, carriers of the deletion; lane C-, negative control (wt); lane B, blank; lane M, marker (Ready-Load™ 1 Kb DNA Ladder, Invitrogen).

    Article Snippet: Confirmation and characterization of the rearrangements Positive results detected by MLPA of two independently drawn samples of genomic DNA were confirmed by long-range PCR (Expand Long Template PCR System, Roche Applied Science), conducted in accordance with the manufacturer's instructions.

    Techniques: Polymerase Chain Reaction, Sequencing, Negative Control, Marker

    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

    PCR amplification of fnr region from different MG1655 isolates. The fnr region was amplified from the CGSC isolate of MG1655 (CGSC 6300; lane 1) and the isolate obtained from M. Singer and C. Gross (NCM3430; lane 2) (see Materials and Methods). The sizes of the molecular standards in lane 3 are noted to the right. The genes deleted in the CGSC isolate (b1332 to b1344) are, respectively, ynaJ (open reading frame conserved in E. coli and Salmonella enterica ), uspE ( ydaA ), fnr (Crp family activator of anaerobic respiratory gene transcription), ogt ( O -6-alkylguanine-DNA/cysteine-protein methyltransferase), abgT ( ydaH ; p ), abgB ( ydaI ; p ), abgA ( ydaJ ; p ), abgR ( ydaK ; p ), ydaL (open reading frame conserved in enterobacteria), ydaM (open reading frame conserved in E. coli ), ydaN (open reading frame conserved in enterobacteria), dbpA (ATP-dependent RNA helicase), and ydaO (open reading frame conserved in enterobacteria). The deletion is flanked by tns5_4 (b1331), which codes for IS 5 transposase, and ydaP (b1345), a rac prophage which codes for a putative prophage integrase.

    Journal: Journal of Bacteriology

    Article Title: Physiological Studies of Escherichia coli Strain MG1655: Growth Defects and Apparent Cross-Regulation of Gene Expression

    doi: 10.1128/JB.185.18.5611-5626.2003

    Figure Lengend Snippet: PCR amplification of fnr region from different MG1655 isolates. The fnr region was amplified from the CGSC isolate of MG1655 (CGSC 6300; lane 1) and the isolate obtained from M. Singer and C. Gross (NCM3430; lane 2) (see Materials and Methods). The sizes of the molecular standards in lane 3 are noted to the right. The genes deleted in the CGSC isolate (b1332 to b1344) are, respectively, ynaJ (open reading frame conserved in E. coli and Salmonella enterica ), uspE ( ydaA ), fnr (Crp family activator of anaerobic respiratory gene transcription), ogt ( O -6-alkylguanine-DNA/cysteine-protein methyltransferase), abgT ( ydaH ; p ), abgB ( ydaI ; p ), abgA ( ydaJ ; p ), abgR ( ydaK ; p ), ydaL (open reading frame conserved in enterobacteria), ydaM (open reading frame conserved in E. coli ), ydaN (open reading frame conserved in enterobacteria), dbpA (ATP-dependent RNA helicase), and ydaO (open reading frame conserved in enterobacteria). The deletion is flanked by tns5_4 (b1331), which codes for IS 5 transposase, and ydaP (b1345), a rac prophage which codes for a putative prophage integrase.

    Article Snippet: The entire fnr region was amplified by long-range PCR according to the manufacturer's instructions (Expand PCR Long Range; Boehringer-Roche) with 40 ng of genomic DNA, 3.75 U of Expand polymerase, 1× reaction buffer 2, 500 μM deoxynucleoside triphosphate mix, and 300 nM each primer in a 50-μl reaction) with forward primer b1331 and reverse primer b1345 at 94°C for 120 s and then for 10 cycles at 94°C for 10 s, 60°C for 30 s, and 68°C for 15 min, and 15 additional cycles with an increase of 20 s per cycle at 68°C (see Fig. ).

    Techniques: Polymerase Chain Reaction, Amplification

    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-week-old (adult), 1-year-old (old adult), and 2-year-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, non-significant. b) DNA isolated from embryos and from the TA muscle of pups, adults, 1-year-old (old) adults, or aged animals was linearized with SacI endonuclease and separated on a neutral gel. MtDNA was visualized as above. Full-length mtDNA is indicated (FL); the asterisk denotes a higher-migrating species resistant to cleavage. 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 Fig. 5d is indicated by a horizontal line. The two groups were compared using Welch’s t-test (ns, non-significant; n = 4). f) The length difference between untreated and alkali-treated mtDNAs shown in Fig. 5d 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 Fig. S5.

    Journal: bioRxiv

    Article Title: The physiological level of rNMPs present in mtDNA does not compromise its stability

    doi: 10.1101/746719

    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-week-old (adult), 1-year-old (old adult), and 2-year-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, non-significant. b) DNA isolated from embryos and from the TA muscle of pups, adults, 1-year-old (old) adults, or aged animals was linearized with SacI endonuclease and separated on a neutral gel. MtDNA was visualized as above. Full-length mtDNA is indicated (FL); the asterisk denotes a higher-migrating species resistant to cleavage. 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 Fig. 5d is indicated by a horizontal line. The two groups were compared using Welch’s t-test (ns, non-significant; n = 4). f) The length difference between untreated and alkali-treated mtDNAs shown in Fig. 5d 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 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 2,478–2,512 and nt 1,933–1,906, respectively, was used to amplify a ~15,800 bp fragment of mouse mtDNA from 25 ng of total DNA.

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