long range pcr Search Results


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  • 99
    Thermo Fisher long range pcr
    Bacterial taxonomic composition of nasopharyngeal samples in cases and controls. (A) Graphs show the mean proportion of the most frequent bacterial genera as inferred by <t>PCR</t> amplification and pyrosequencing of the <t>16S</t> rRNA gene. The mean proportions were calculated based on 1,000 sequences per sample. Those bacteria at a proportion lower than 1% are indicated as “Others” and were particularly abundant in cases. (B) Shows a description of those low-frequency bacteria in patients with IPD, which include many oral organisms (bacteria at a proportion
    Long Range Pcr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 723 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Stratagene long range pcr
    Long-range <t>PCR.</t> (A) Primers KG09 and KG10R (arrows above the hemolysin operon) were used to test for the presence of an hlyA 1 -containing PCR fragment amplified from hlyA 1 to the <t>cnf</t> 1 gene on the chromosome of the following strains: CP9 (lane 1), CP9 cnf 1 (lane 2), CP9Δ hlyA 1 :: cat (lane 3), and CP9 cnf 1 Δ hlyA 1 :: cat (lane 4). PCR products (∼6.7 kb) were present for CP9 and CP9 cnf 1 but not for CP9Δ hlyA 1 :: cat and CP9 cnf 1 Δ hlyA 1 :: cat , as shown in the top panel of the ethidium bromide-stained agarose gel on the left. Primers KG03F and KG10R were used to verify the replacement of hlyA 1 with cat upstream of the cnf 1 gene in mutant strains CP9Δ hlyA 1 :: cat and CP9 cnf 1 Δ hlyA 1 :: cat (∼5.0-kb amplified PCR product from cat to cnf 1 in the bottom panel of the ethidium bromide-stained agarose gel). (B) Western blot analysis with polyclonal anti-CNF1 serum as a probe was used to verify that wild-type levels of CNF1 (lane 1) were produced by CP9Δ hlyA 1 :: cat (lane 3) and that, as expected, strains CP9 cnf 1 and CP9 cnf 1 Δ hlyA 1 :: cat did not produce CNF1 (lanes 2 and 4, respectively).
    Long Range Pcr, supplied by Stratagene, used in various techniques. Bioz Stars score: 92/100, based on 189 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Boehringer Mannheim long range pcr
    ( A ) Results of <t>RT-PCR</t> analyses. Lane M contains a size marker VI (Boehringer Mannheim). Lanes 1, 4, 7, and 12 are negative controls in which the cDNA was replaced by sterile water. Normal fragments are obtained from the patient's unaffected GRAF allele (lane 2) and from the cell line Mono-Mac6 (lane 3). A <t>MLL</t> / GRAF fusion mRNA is detected in the sample with a t(5;11)(q31;q23) (lane 5) but not in the cell line lacking this translocation (lane 6). Normal fragments are obtained from the patient's unaffected MLL allele (lane 8) and the cell line Mono-Mac6 (lane 9). The additional fragments in lanes 5, 8, and 9 are generated by MLL splice variants. Further analysis reveals that the reciprocal GRAF / MLL fragment is neither present in the patient's sample (lane 10) nor in the cell line (lane 11). Control amplifications with primers specific for the ABL gene are shown in lanes 15 (patient sample) and 16 (cell line). ( B ) Long-range PCR results of genomic DNA. Lanes M contain the size markers III and VI (Boehringer Mannheim). Lane 1 is a negative control. A MLL / GRAF fusion product is detected in the patient with the t(5;11)(q31;23) (lane 2) but not in the control cell line Mono-Mac6 (lane 3). Lanes 4, 5, 9, and 17 are negative controls. A normal 8-kb fragment that covers the breakpoint cluster region of the unaffected MLL alleles in the patient with the t(5;11)(q31;q23) (lane 6), in a healthy individual (lane 7), and in the Mono-Mac cell line (lane 8) is seen. No reciprocal GRAF / MLL gene fragment is detected in any of these samples (lanes 10–13), whereas in all of them an approximately 13-kb long intron of GRAF becomes evident (lanes 14–16). ( C ) Sequence and schematic representation of the inverted duplication of MLL within the genomic MLL / GRAF fusion. Numbering of nucleotides within the breakpoint region of MLL . The horizontal arrows indicate the positions of the primers used for amplification of the genomic MLL / GRAF fusion seen in lane 2 of B .
    Long Range Pcr, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 88 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen long range pcr kit
    ( A ) Results of <t>RT-PCR</t> analyses. Lane M contains a size marker VI (Boehringer Mannheim). Lanes 1, 4, 7, and 12 are negative controls in which the cDNA was replaced by sterile water. Normal fragments are obtained from the patient's unaffected GRAF allele (lane 2) and from the cell line Mono-Mac6 (lane 3). A <t>MLL</t> / GRAF fusion mRNA is detected in the sample with a t(5;11)(q31;q23) (lane 5) but not in the cell line lacking this translocation (lane 6). Normal fragments are obtained from the patient's unaffected MLL allele (lane 8) and the cell line Mono-Mac6 (lane 9). The additional fragments in lanes 5, 8, and 9 are generated by MLL splice variants. Further analysis reveals that the reciprocal GRAF / MLL fragment is neither present in the patient's sample (lane 10) nor in the cell line (lane 11). Control amplifications with primers specific for the ABL gene are shown in lanes 15 (patient sample) and 16 (cell line). ( B ) Long-range PCR results of genomic DNA. Lanes M contain the size markers III and VI (Boehringer Mannheim). Lane 1 is a negative control. A MLL / GRAF fusion product is detected in the patient with the t(5;11)(q31;23) (lane 2) but not in the control cell line Mono-Mac6 (lane 3). Lanes 4, 5, 9, and 17 are negative controls. A normal 8-kb fragment that covers the breakpoint cluster region of the unaffected MLL alleles in the patient with the t(5;11)(q31;q23) (lane 6), in a healthy individual (lane 7), and in the Mono-Mac cell line (lane 8) is seen. No reciprocal GRAF / MLL gene fragment is detected in any of these samples (lanes 10–13), whereas in all of them an approximately 13-kb long intron of GRAF becomes evident (lanes 14–16). ( C ) Sequence and schematic representation of the inverted duplication of MLL within the genomic MLL / GRAF fusion. Numbering of nucleotides within the breakpoint region of MLL . The horizontal arrows indicate the positions of the primers used for amplification of the genomic MLL / GRAF fusion seen in lane 2 of B .
    Long Range Pcr Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 301 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Pacific Biosciences long range pcr
    Barcoding schemes. Direct versus two‐step sample barcoding. A : In the direct barcoding scheme the sample specific barcodes, indicated by the blue and red for patients 1 and 2, respectively, are attached to the gene‐specific sequences (black arrows) and are introduced in a single <t>PCR</t> reaction. B : For the two‐step procedure for each individual, the region of interest is first amplified with a pair of gene‐specific primers with M13 forward (green) and reverse (purple) sequence tails. A symmetrical sample barcode, indicated by blue and red for patients 1 and 2, respectively, is introduced in a second PCR using a set of M13 barcode primers. A 5' padding sequence (black) is present on the index primers for both the direct and two‐step barcoding schemes to give all fragment identical end sequences to avoid ligation biases during the <t>SMRT</t> bell library preparation.
    Long Range Pcr, supplied by Pacific Biosciences, used in various techniques. Bioz Stars score: 92/100, based on 79 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Macrogen long range pcr
    Barcoding schemes. Direct versus two‐step sample barcoding. A : In the direct barcoding scheme the sample specific barcodes, indicated by the blue and red for patients 1 and 2, respectively, are attached to the gene‐specific sequences (black arrows) and are introduced in a single <t>PCR</t> reaction. B : For the two‐step procedure for each individual, the region of interest is first amplified with a pair of gene‐specific primers with M13 forward (green) and reverse (purple) sequence tails. A symmetrical sample barcode, indicated by blue and red for patients 1 and 2, respectively, is introduced in a second PCR using a set of M13 barcode primers. A 5' padding sequence (black) is present on the index primers for both the direct and two‐step barcoding schemes to give all fragment identical end sequences to avoid ligation biases during the <t>SMRT</t> bell library preparation.
    Long Range Pcr, supplied by Macrogen, used in various techniques. Bioz Stars score: 92/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa long range polymerase chain reaction
    Barcoding schemes. Direct versus two‐step sample barcoding. A : In the direct barcoding scheme the sample specific barcodes, indicated by the blue and red for patients 1 and 2, respectively, are attached to the gene‐specific sequences (black arrows) and are introduced in a single <t>PCR</t> reaction. B : For the two‐step procedure for each individual, the region of interest is first amplified with a pair of gene‐specific primers with M13 forward (green) and reverse (purple) sequence tails. A symmetrical sample barcode, indicated by blue and red for patients 1 and 2, respectively, is introduced in a second PCR using a set of M13 barcode primers. A 5' padding sequence (black) is present on the index primers for both the direct and two‐step barcoding schemes to give all fragment identical end sequences to avoid ligation biases during the <t>SMRT</t> bell library preparation.
    Long Range Polymerase Chain Reaction, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    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).
    Expand Long Range Pcr Kit, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 88/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega gotaq long range pcr master mix
    <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).
    Gotaq Long Range Pcr Master Mix, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Pacific Biosciences long range pcr amplification
    Deletion breakpoint identification. The chromosome 7p14.3 genomic region is illustrated with tracks for the proband chromosomal microarray (CMA) results, genomic <t>PCR</t> mapping amplicon locations (1–9; green: amplified; red: did not amplify), and copy number variants detected among healthy individuals in the Database of Genomic Variants (DGV; blue: duplication; red: deletion) ( a ). Unambiguous breakpoint mapping was performed by long-read single molecule real-time <t>(SMRT)</t> sequencing (PacBio) of long-range PCR products that amplified across the deleted interval in the proband ( b ). These SMRT sequencing data were also aligned to a modified human genome reference that excluded the identified 72.8 kb deletion (chr7:33130616–33203409) ( c ), confirming that there were no other sequence alterations at the breakpoint locations. The precise deletion breakpoints were subsequently confirmed in the proband and both carrier parents by Sanger sequencing of the long-range PCR amplicons ( d )
    Long Range Pcr Amplification, supplied by Pacific Biosciences, used in various techniques. Bioz Stars score: 89/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher long range phusion pcr
    Deletion breakpoint identification. The chromosome 7p14.3 genomic region is illustrated with tracks for the proband chromosomal microarray (CMA) results, genomic <t>PCR</t> mapping amplicon locations (1–9; green: amplified; red: did not amplify), and copy number variants detected among healthy individuals in the Database of Genomic Variants (DGV; blue: duplication; red: deletion) ( a ). Unambiguous breakpoint mapping was performed by long-read single molecule real-time <t>(SMRT)</t> sequencing (PacBio) of long-range PCR products that amplified across the deleted interval in the proband ( b ). These SMRT sequencing data were also aligned to a modified human genome reference that excluded the identified 72.8 kb deletion (chr7:33130616–33203409) ( c ), confirming that there were no other sequence alterations at the breakpoint locations. The precise deletion breakpoints were subsequently confirmed in the proband and both carrier parents by Sanger sequencing of the long-range PCR amplicons ( d )
    Long Range Phusion Pcr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher sequalprep long range pcr kit
    Deletion breakpoint identification. The chromosome 7p14.3 genomic region is illustrated with tracks for the proband chromosomal microarray (CMA) results, genomic <t>PCR</t> mapping amplicon locations (1–9; green: amplified; red: did not amplify), and copy number variants detected among healthy individuals in the Database of Genomic Variants (DGV; blue: duplication; red: deletion) ( a ). Unambiguous breakpoint mapping was performed by long-read single molecule real-time <t>(SMRT)</t> sequencing (PacBio) of long-range PCR products that amplified across the deleted interval in the proband ( b ). These SMRT sequencing data were also aligned to a modified human genome reference that excluded the identified 72.8 kb deletion (chr7:33130616–33203409) ( c ), confirming that there were no other sequence alterations at the breakpoint locations. The precise deletion breakpoints were subsequently confirmed in the proband and both carrier parents by Sanger sequencing of the long-range PCR amplicons ( d )
    Sequalprep Long Range Pcr Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher elongase long range pcr enzyme kit
    Deletion breakpoint identification. The chromosome 7p14.3 genomic region is illustrated with tracks for the proband chromosomal microarray (CMA) results, genomic <t>PCR</t> mapping amplicon locations (1–9; green: amplified; red: did not amplify), and copy number variants detected among healthy individuals in the Database of Genomic Variants (DGV; blue: duplication; red: deletion) ( a ). Unambiguous breakpoint mapping was performed by long-read single molecule real-time <t>(SMRT)</t> sequencing (PacBio) of long-range PCR products that amplified across the deleted interval in the proband ( b ). These SMRT sequencing data were also aligned to a modified human genome reference that excluded the identified 72.8 kb deletion (chr7:33130616–33203409) ( c ), confirming that there were no other sequence alterations at the breakpoint locations. The precise deletion breakpoints were subsequently confirmed in the proband and both carrier parents by Sanger sequencing of the long-range PCR amplicons ( d )
    Elongase Long Range Pcr Enzyme Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher extensor long range pcr enzyme master mix
    Deletion breakpoint identification. The chromosome 7p14.3 genomic region is illustrated with tracks for the proband chromosomal microarray (CMA) results, genomic <t>PCR</t> mapping amplicon locations (1–9; green: amplified; red: did not amplify), and copy number variants detected among healthy individuals in the Database of Genomic Variants (DGV; blue: duplication; red: deletion) ( a ). Unambiguous breakpoint mapping was performed by long-read single molecule real-time <t>(SMRT)</t> sequencing (PacBio) of long-range PCR products that amplified across the deleted interval in the proband ( b ). These SMRT sequencing data were also aligned to a modified human genome reference that excluded the identified 72.8 kb deletion (chr7:33130616–33203409) ( c ), confirming that there were no other sequence alterations at the breakpoint locations. The precise deletion breakpoints were subsequently confirmed in the proband and both carrier parents by Sanger sequencing of the long-range PCR amplicons ( d )
    Extensor Long Range Pcr Enzyme Master Mix, supplied by Thermo Fisher, 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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    95
    TaKaRa la pcr genome dna set
    Identification of SMN1 variants. (a) qPCR analysis of four retrotransposon‐free SMN genomic regions in the intron 1 (I1), in the exon 3–intron 3 junction (E3I3), in the intron 5–exon 6 junction (I5E6), and ~1 kb downstream from exon 8 (+1 kb). Compared with four copies of SMN genes that are present in controls, we found that the patient has three copies at the I1, I5E6, and +1 kb loci and two copies at the E3I3 loci. The mother has three copies through the I1 to I5E6 loci and two copies at the +1 kb loci. The father has three copies only at the I5E6 loci. (b) Schematic representation of SMN1/2 exon (E)/intron (I) structure. Positions of sequence differences between SMN1 and SMN2 are represented by black vertical bars. The black triangles denote sequence‐specific variants in exons 7, 8 targeted by MLPA probes in routine testing. Locations of Alus in the breakpoint candidate regions in the intron 1 and 5, including the causal AluSp in the intron 1 and AluSq in the intron 5 indicated by vertical text, and primers binding sites for Alu <t>PCR</t> indicated by black arrowheads are shown below the scheme of the SMN structure. Position of the PCR4 spanning exons 5–8 that showed absence of SMN1 sequence‐specific variants indicating disruption of both SMN1 alleles in the patient is represented by yellow box. Range of the paternal deletion of exons 2a‐5 is represented by red box. (c) <t>DNA</t> sequence trace of the Alu PCR, Alu_259_4A, showing a double sequence caused by presence of AluSq wt in intron 5 together with a sequence originating from the intron 1 AluSp . Red arrows indicate the addition of AluSp ‐specific sequence in an Alu PCR product. (d) PCR genotyping of the SMN1Δ(2a‐5) variant showed presence of the deletion‐spanning amplification product in the patient (P) and father (F), but not in mother (M) and control (C). (e) DNA sequence trace of the breakpoint junction‐specific PCR and detail of the Δ2a‐5 breakpoint junction show the new Alu ‐ Alu chimeric element originating from the recombination between the AluSp in the intron 1 and AluSq in the intron 5. A breakpoint microhomology of the AluSp and AluSq is marked with a black box. (f) Schematic representation of SMN1 and SMN2 in the family members. Pink‐marked boxes represent maternal alleles (M) and blue boxes paternal alleles (F). The red crosses denote identified deletions and the dashed vertical lines denote loci of the qPCR (I1, E3I3, I5E6, and +1 kb) and MLPA (exon 7‐E7, exon 8‐E8) probes used for deletion mapping. The black junctions on the box terminals indicate a cis configuration of SMN1 and SMN2 alleles. The model shows (a) a whole deletion of one SMN1 allele in the patient (P) inherited from her mother and detected by the combination of the qPCR and MLPA; (b) a deletion of the second SMN1 allele in the patient inherited from her father and detected by the E3I3 qPCR and transcript analysis (Figure 1a ); and (c) deletion of one copy of one SMN2 allele in the mother detected by the MLPA and the + 1kb qPCR
    La Pcr Genome Dna Set, supplied by TaKaRa, used in various techniques. Bioz Stars score: 95/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Merck & Co long range pcr kit
    Identification of SMN1 variants. (a) qPCR analysis of four retrotransposon‐free SMN genomic regions in the intron 1 (I1), in the exon 3–intron 3 junction (E3I3), in the intron 5–exon 6 junction (I5E6), and ~1 kb downstream from exon 8 (+1 kb). Compared with four copies of SMN genes that are present in controls, we found that the patient has three copies at the I1, I5E6, and +1 kb loci and two copies at the E3I3 loci. The mother has three copies through the I1 to I5E6 loci and two copies at the +1 kb loci. The father has three copies only at the I5E6 loci. (b) Schematic representation of SMN1/2 exon (E)/intron (I) structure. Positions of sequence differences between SMN1 and SMN2 are represented by black vertical bars. The black triangles denote sequence‐specific variants in exons 7, 8 targeted by MLPA probes in routine testing. Locations of Alus in the breakpoint candidate regions in the intron 1 and 5, including the causal AluSp in the intron 1 and AluSq in the intron 5 indicated by vertical text, and primers binding sites for Alu <t>PCR</t> indicated by black arrowheads are shown below the scheme of the SMN structure. Position of the PCR4 spanning exons 5–8 that showed absence of SMN1 sequence‐specific variants indicating disruption of both SMN1 alleles in the patient is represented by yellow box. Range of the paternal deletion of exons 2a‐5 is represented by red box. (c) <t>DNA</t> sequence trace of the Alu PCR, Alu_259_4A, showing a double sequence caused by presence of AluSq wt in intron 5 together with a sequence originating from the intron 1 AluSp . Red arrows indicate the addition of AluSp ‐specific sequence in an Alu PCR product. (d) PCR genotyping of the SMN1Δ(2a‐5) variant showed presence of the deletion‐spanning amplification product in the patient (P) and father (F), but not in mother (M) and control (C). (e) DNA sequence trace of the breakpoint junction‐specific PCR and detail of the Δ2a‐5 breakpoint junction show the new Alu ‐ Alu chimeric element originating from the recombination between the AluSp in the intron 1 and AluSq in the intron 5. A breakpoint microhomology of the AluSp and AluSq is marked with a black box. (f) Schematic representation of SMN1 and SMN2 in the family members. Pink‐marked boxes represent maternal alleles (M) and blue boxes paternal alleles (F). The red crosses denote identified deletions and the dashed vertical lines denote loci of the qPCR (I1, E3I3, I5E6, and +1 kb) and MLPA (exon 7‐E7, exon 8‐E8) probes used for deletion mapping. The black junctions on the box terminals indicate a cis configuration of SMN1 and SMN2 alleles. The model shows (a) a whole deletion of one SMN1 allele in the patient (P) inherited from her mother and detected by the combination of the qPCR and MLPA; (b) a deletion of the second SMN1 allele in the patient inherited from her father and detected by the E3I3 qPCR and transcript analysis (Figure 1a ); and (c) deletion of one copy of one SMN2 allele in the mother detected by the MLPA and the + 1kb qPCR
    Long Range Pcr Kit, supplied by Merck & Co, used in various techniques. Bioz Stars score: 90/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Bacterial taxonomic composition of nasopharyngeal samples in cases and controls. (A) Graphs show the mean proportion of the most frequent bacterial genera as inferred by PCR amplification and pyrosequencing of the 16S rRNA gene. The mean proportions were calculated based on 1,000 sequences per sample. Those bacteria at a proportion lower than 1% are indicated as “Others” and were particularly abundant in cases. (B) Shows a description of those low-frequency bacteria in patients with IPD, which include many oral organisms (bacteria at a proportion

    Journal: Frontiers in Microbiology

    Article Title: Nasopharyngeal Microbiota in Children With Invasive Pneumococcal Disease: Identification of Bacteria With Potential Disease-Promoting and Protective Effects

    doi: 10.3389/fmicb.2019.00011

    Figure Lengend Snippet: Bacterial taxonomic composition of nasopharyngeal samples in cases and controls. (A) Graphs show the mean proportion of the most frequent bacterial genera as inferred by PCR amplification and pyrosequencing of the 16S rRNA gene. The mean proportions were calculated based on 1,000 sequences per sample. Those bacteria at a proportion lower than 1% are indicated as “Others” and were particularly abundant in cases. (B) Shows a description of those low-frequency bacteria in patients with IPD, which include many oral organisms (bacteria at a proportion

    Article Snippet: PCR amplification of the 16S rRNA gene was performed using high fidelity Extensor Long Range PCR Enzyme (Thermo Fisher Scientific, Massachusetts, USA), with the degenerate universal bacterial primers of 16S rRNA gene 8F (5′-CAGAGTTTGATCMTGGCTCAG-3′) and 785R (5′-GGCCVGGGTATCTAATCC-3′) (Simón-Soro et al., ) and the following cycling conditions to minimize amplification biases (Simón-Soro et al., ): 2 min at 94°C, followed by 30 cycles of 10s at 94°C, 30s at 52°C, 90s at 68°C, and a final step of 7 min at 68°C.

    Techniques: Polymerase Chain Reaction, Amplification

    Long-range PCR. (A) Primers KG09 and KG10R (arrows above the hemolysin operon) were used to test for the presence of an hlyA 1 -containing PCR fragment amplified from hlyA 1 to the cnf 1 gene on the chromosome of the following strains: CP9 (lane 1), CP9 cnf 1 (lane 2), CP9Δ hlyA 1 :: cat (lane 3), and CP9 cnf 1 Δ hlyA 1 :: cat (lane 4). PCR products (∼6.7 kb) were present for CP9 and CP9 cnf 1 but not for CP9Δ hlyA 1 :: cat and CP9 cnf 1 Δ hlyA 1 :: cat , as shown in the top panel of the ethidium bromide-stained agarose gel on the left. Primers KG03F and KG10R were used to verify the replacement of hlyA 1 with cat upstream of the cnf 1 gene in mutant strains CP9Δ hlyA 1 :: cat and CP9 cnf 1 Δ hlyA 1 :: cat (∼5.0-kb amplified PCR product from cat to cnf 1 in the bottom panel of the ethidium bromide-stained agarose gel). (B) Western blot analysis with polyclonal anti-CNF1 serum as a probe was used to verify that wild-type levels of CNF1 (lane 1) were produced by CP9Δ hlyA 1 :: cat (lane 3) and that, as expected, strains CP9 cnf 1 and CP9 cnf 1 Δ hlyA 1 :: cat did not produce CNF1 (lanes 2 and 4, respectively).

    Journal: Infection and Immunity

    Article Title: Hemolysin of Uropathogenic Escherichia coli Evokes Extensive Shedding of the Uroepithelium and Hemorrhage in Bladder Tissue within the First 24 Hours after Intraurethral Inoculation of Mice

    doi: 10.1128/IAI.00075-08

    Figure Lengend Snippet: Long-range PCR. (A) Primers KG09 and KG10R (arrows above the hemolysin operon) were used to test for the presence of an hlyA 1 -containing PCR fragment amplified from hlyA 1 to the cnf 1 gene on the chromosome of the following strains: CP9 (lane 1), CP9 cnf 1 (lane 2), CP9Δ hlyA 1 :: cat (lane 3), and CP9 cnf 1 Δ hlyA 1 :: cat (lane 4). PCR products (∼6.7 kb) were present for CP9 and CP9 cnf 1 but not for CP9Δ hlyA 1 :: cat and CP9 cnf 1 Δ hlyA 1 :: cat , as shown in the top panel of the ethidium bromide-stained agarose gel on the left. Primers KG03F and KG10R were used to verify the replacement of hlyA 1 with cat upstream of the cnf 1 gene in mutant strains CP9Δ hlyA 1 :: cat and CP9 cnf 1 Δ hlyA 1 :: cat (∼5.0-kb amplified PCR product from cat to cnf 1 in the bottom panel of the ethidium bromide-stained agarose gel). (B) Western blot analysis with polyclonal anti-CNF1 serum as a probe was used to verify that wild-type levels of CNF1 (lane 1) were produced by CP9Δ hlyA 1 :: cat (lane 3) and that, as expected, strains CP9 cnf 1 and CP9 cnf 1 Δ hlyA 1 :: cat did not produce CNF1 (lanes 2 and 4, respectively).

    Article Snippet: The location of the hlyA 1 mutation in the hly operon adjacent to the cnf 1 locus was confirmed by performing long-range PCR with Pfu TURBO (Stratagene, La Jolla, CA) and the following primer sets: KG03F-KG10R and KG09F-KG10R (Fig. ).

    Techniques: Polymerase Chain Reaction, Amplification, Staining, Agarose Gel Electrophoresis, Mutagenesis, Western Blot, Produced

    ( A ) Results of RT-PCR analyses. Lane M contains a size marker VI (Boehringer Mannheim). Lanes 1, 4, 7, and 12 are negative controls in which the cDNA was replaced by sterile water. Normal fragments are obtained from the patient's unaffected GRAF allele (lane 2) and from the cell line Mono-Mac6 (lane 3). A MLL / GRAF fusion mRNA is detected in the sample with a t(5;11)(q31;q23) (lane 5) but not in the cell line lacking this translocation (lane 6). Normal fragments are obtained from the patient's unaffected MLL allele (lane 8) and the cell line Mono-Mac6 (lane 9). The additional fragments in lanes 5, 8, and 9 are generated by MLL splice variants. Further analysis reveals that the reciprocal GRAF / MLL fragment is neither present in the patient's sample (lane 10) nor in the cell line (lane 11). Control amplifications with primers specific for the ABL gene are shown in lanes 15 (patient sample) and 16 (cell line). ( B ) Long-range PCR results of genomic DNA. Lanes M contain the size markers III and VI (Boehringer Mannheim). Lane 1 is a negative control. A MLL / GRAF fusion product is detected in the patient with the t(5;11)(q31;23) (lane 2) but not in the control cell line Mono-Mac6 (lane 3). Lanes 4, 5, 9, and 17 are negative controls. A normal 8-kb fragment that covers the breakpoint cluster region of the unaffected MLL alleles in the patient with the t(5;11)(q31;q23) (lane 6), in a healthy individual (lane 7), and in the Mono-Mac cell line (lane 8) is seen. No reciprocal GRAF / MLL gene fragment is detected in any of these samples (lanes 10–13), whereas in all of them an approximately 13-kb long intron of GRAF becomes evident (lanes 14–16). ( C ) Sequence and schematic representation of the inverted duplication of MLL within the genomic MLL / GRAF fusion. Numbering of nucleotides within the breakpoint region of MLL . The horizontal arrows indicate the positions of the primers used for amplification of the genomic MLL / GRAF fusion seen in lane 2 of B .

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

    Article Title: The human GRAF gene is fused to MLL in a unique t(5;11)(q31;q23) and both alleles are disrupted in three cases of myelodysplastic syndrome/acute myeloid leukemia with a deletion 5q

    doi:

    Figure Lengend Snippet: ( A ) Results of RT-PCR analyses. Lane M contains a size marker VI (Boehringer Mannheim). Lanes 1, 4, 7, and 12 are negative controls in which the cDNA was replaced by sterile water. Normal fragments are obtained from the patient's unaffected GRAF allele (lane 2) and from the cell line Mono-Mac6 (lane 3). A MLL / GRAF fusion mRNA is detected in the sample with a t(5;11)(q31;q23) (lane 5) but not in the cell line lacking this translocation (lane 6). Normal fragments are obtained from the patient's unaffected MLL allele (lane 8) and the cell line Mono-Mac6 (lane 9). The additional fragments in lanes 5, 8, and 9 are generated by MLL splice variants. Further analysis reveals that the reciprocal GRAF / MLL fragment is neither present in the patient's sample (lane 10) nor in the cell line (lane 11). Control amplifications with primers specific for the ABL gene are shown in lanes 15 (patient sample) and 16 (cell line). ( B ) Long-range PCR results of genomic DNA. Lanes M contain the size markers III and VI (Boehringer Mannheim). Lane 1 is a negative control. A MLL / GRAF fusion product is detected in the patient with the t(5;11)(q31;23) (lane 2) but not in the control cell line Mono-Mac6 (lane 3). Lanes 4, 5, 9, and 17 are negative controls. A normal 8-kb fragment that covers the breakpoint cluster region of the unaffected MLL alleles in the patient with the t(5;11)(q31;q23) (lane 6), in a healthy individual (lane 7), and in the Mono-Mac cell line (lane 8) is seen. No reciprocal GRAF / MLL gene fragment is detected in any of these samples (lanes 10–13), whereas in all of them an approximately 13-kb long intron of GRAF becomes evident (lanes 14–16). ( C ) Sequence and schematic representation of the inverted duplication of MLL within the genomic MLL / GRAF fusion. Numbering of nucleotides within the breakpoint region of MLL . The horizontal arrows indicate the positions of the primers used for amplification of the genomic MLL / GRAF fusion seen in lane 2 of B .

    Article Snippet: After amplification of both the genomic unrearranged MLL and the MLL / GRAF fusion by long-range PCR we digested the PCR products by Dde I or Tru91 (Boehringer Mannheim).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Marker, Translocation Assay, Generated, Polymerase Chain Reaction, Negative Control, Sequencing, Amplification

    ( A ) Insertion of 52-bp (capital letters) derived from intron 13 into the final cDNA found in patient #7. The surrounding intronic sequences are shown in lowercase letters. This leads to a reading frame shift followed by a premature stop codon. The GAP domain of Graf is substantially shortened. The intronic regions that were sequenced in patient #7 and 12 healthy controls are indicated by arrows. The splice branch site consensus sequence is shown as follows: Y represents T or C, R either A or G. ( B ) Schematic representation of both cDNA fragments that were coamplified by universal primers I and IV for assessment of their relative amount. Primers II and III amplify the aberrantly spliced fragment only. ( C ) Nested PCR analysis using the first-round primers I and IV and the second-round primers II and III. M, molecular weight marker. Lane 1, negative control. Four of 15 healthy blood donors expressed the aberrantly spliced fragment in their mononuclear cells (lanes 2 and 4–6) because a faint PCR product was seen. Lane 12, positive control. ( D ) Single-round PCR analysis using primers I and IV. Lane 6, two differently sized PCR products are seen from the cDNA of patient #7 even after only one round of PCR. Positive plasmid controls containing the 52-bp insertion (lane 12) or not (lane 13). In each RT-PCR, 2 μg of total RNA was subjected to cDNA synthesis and processed in parallel.

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

    Article Title: The human GRAF gene is fused to MLL in a unique t(5;11)(q31;q23) and both alleles are disrupted in three cases of myelodysplastic syndrome/acute myeloid leukemia with a deletion 5q

    doi:

    Figure Lengend Snippet: ( A ) Insertion of 52-bp (capital letters) derived from intron 13 into the final cDNA found in patient #7. The surrounding intronic sequences are shown in lowercase letters. This leads to a reading frame shift followed by a premature stop codon. The GAP domain of Graf is substantially shortened. The intronic regions that were sequenced in patient #7 and 12 healthy controls are indicated by arrows. The splice branch site consensus sequence is shown as follows: Y represents T or C, R either A or G. ( B ) Schematic representation of both cDNA fragments that were coamplified by universal primers I and IV for assessment of their relative amount. Primers II and III amplify the aberrantly spliced fragment only. ( C ) Nested PCR analysis using the first-round primers I and IV and the second-round primers II and III. M, molecular weight marker. Lane 1, negative control. Four of 15 healthy blood donors expressed the aberrantly spliced fragment in their mononuclear cells (lanes 2 and 4–6) because a faint PCR product was seen. Lane 12, positive control. ( D ) Single-round PCR analysis using primers I and IV. Lane 6, two differently sized PCR products are seen from the cDNA of patient #7 even after only one round of PCR. Positive plasmid controls containing the 52-bp insertion (lane 12) or not (lane 13). In each RT-PCR, 2 μg of total RNA was subjected to cDNA synthesis and processed in parallel.

    Article Snippet: After amplification of both the genomic unrearranged MLL and the MLL / GRAF fusion by long-range PCR we digested the PCR products by Dde I or Tru91 (Boehringer Mannheim).

    Techniques: Derivative Assay, Sequencing, Nested PCR, Molecular Weight, Marker, Negative Control, Polymerase Chain Reaction, Positive Control, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction

    Barcoding schemes. Direct versus two‐step sample barcoding. A : In the direct barcoding scheme the sample specific barcodes, indicated by the blue and red for patients 1 and 2, respectively, are attached to the gene‐specific sequences (black arrows) and are introduced in a single PCR reaction. B : For the two‐step procedure for each individual, the region of interest is first amplified with a pair of gene‐specific primers with M13 forward (green) and reverse (purple) sequence tails. A symmetrical sample barcode, indicated by blue and red for patients 1 and 2, respectively, is introduced in a second PCR using a set of M13 barcode primers. A 5' padding sequence (black) is present on the index primers for both the direct and two‐step barcoding schemes to give all fragment identical end sequences to avoid ligation biases during the SMRT bell library preparation.

    Journal: Human Mutation

    Article Title: Flexible and Scalable Full‐Length CYP2D6 Long Amplicon PacBio Sequencing

    doi: 10.1002/humu.23166

    Figure Lengend Snippet: Barcoding schemes. Direct versus two‐step sample barcoding. A : In the direct barcoding scheme the sample specific barcodes, indicated by the blue and red for patients 1 and 2, respectively, are attached to the gene‐specific sequences (black arrows) and are introduced in a single PCR reaction. B : For the two‐step procedure for each individual, the region of interest is first amplified with a pair of gene‐specific primers with M13 forward (green) and reverse (purple) sequence tails. A symmetrical sample barcode, indicated by blue and red for patients 1 and 2, respectively, is introduced in a second PCR using a set of M13 barcode primers. A 5' padding sequence (black) is present on the index primers for both the direct and two‐step barcoding schemes to give all fragment identical end sequences to avoid ligation biases during the SMRT bell library preparation.

    Article Snippet: Long‐Range PCR, SMRT Library Prep, and PacBio Sequencing All work described in this paper is subject to the LUMC Good Research Practice & Integrity guidelines and Ethical requirements.

    Techniques: Polymerase Chain Reaction, Amplification, Sequencing, Ligation

    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

    Deletion breakpoint identification. The chromosome 7p14.3 genomic region is illustrated with tracks for the proband chromosomal microarray (CMA) results, genomic PCR mapping amplicon locations (1–9; green: amplified; red: did not amplify), and copy number variants detected among healthy individuals in the Database of Genomic Variants (DGV; blue: duplication; red: deletion) ( a ). Unambiguous breakpoint mapping was performed by long-read single molecule real-time (SMRT) sequencing (PacBio) of long-range PCR products that amplified across the deleted interval in the proband ( b ). These SMRT sequencing data were also aligned to a modified human genome reference that excluded the identified 72.8 kb deletion (chr7:33130616–33203409) ( c ), confirming that there were no other sequence alterations at the breakpoint locations. The precise deletion breakpoints were subsequently confirmed in the proband and both carrier parents by Sanger sequencing of the long-range PCR amplicons ( d )

    Journal: NPJ Genomic Medicine

    Article Title: Cytogenomic identification and long-read single molecule real-time (SMRT) sequencing of a Bardet–Biedl Syndrome 9 (BBS9) deletion

    doi: 10.1038/s41525-017-0042-3

    Figure Lengend Snippet: Deletion breakpoint identification. The chromosome 7p14.3 genomic region is illustrated with tracks for the proband chromosomal microarray (CMA) results, genomic PCR mapping amplicon locations (1–9; green: amplified; red: did not amplify), and copy number variants detected among healthy individuals in the Database of Genomic Variants (DGV; blue: duplication; red: deletion) ( a ). Unambiguous breakpoint mapping was performed by long-read single molecule real-time (SMRT) sequencing (PacBio) of long-range PCR products that amplified across the deleted interval in the proband ( b ). These SMRT sequencing data were also aligned to a modified human genome reference that excluded the identified 72.8 kb deletion (chr7:33130616–33203409) ( c ), confirming that there were no other sequence alterations at the breakpoint locations. The precise deletion breakpoints were subsequently confirmed in the proband and both carrier parents by Sanger sequencing of the long-range PCR amplicons ( d )

    Article Snippet: Long-read SMRT sequencing : Long-range PCR amplification across the identified breakpoint regions was accomplished using primers targeted to unique DNA sequences flanking the approximated deletion coordinates, and these amplicons were subjected to SMRTbell library construction and long-read SMRT sequencing (Pacific Biosciences, Menlo Park, CA).

    Techniques: Microarray, Polymerase Chain Reaction, Amplification, Sequencing, Modification

    Identification of SMN1 variants. (a) qPCR analysis of four retrotransposon‐free SMN genomic regions in the intron 1 (I1), in the exon 3–intron 3 junction (E3I3), in the intron 5–exon 6 junction (I5E6), and ~1 kb downstream from exon 8 (+1 kb). Compared with four copies of SMN genes that are present in controls, we found that the patient has three copies at the I1, I5E6, and +1 kb loci and two copies at the E3I3 loci. The mother has three copies through the I1 to I5E6 loci and two copies at the +1 kb loci. The father has three copies only at the I5E6 loci. (b) Schematic representation of SMN1/2 exon (E)/intron (I) structure. Positions of sequence differences between SMN1 and SMN2 are represented by black vertical bars. The black triangles denote sequence‐specific variants in exons 7, 8 targeted by MLPA probes in routine testing. Locations of Alus in the breakpoint candidate regions in the intron 1 and 5, including the causal AluSp in the intron 1 and AluSq in the intron 5 indicated by vertical text, and primers binding sites for Alu PCR indicated by black arrowheads are shown below the scheme of the SMN structure. Position of the PCR4 spanning exons 5–8 that showed absence of SMN1 sequence‐specific variants indicating disruption of both SMN1 alleles in the patient is represented by yellow box. Range of the paternal deletion of exons 2a‐5 is represented by red box. (c) DNA sequence trace of the Alu PCR, Alu_259_4A, showing a double sequence caused by presence of AluSq wt in intron 5 together with a sequence originating from the intron 1 AluSp . Red arrows indicate the addition of AluSp ‐specific sequence in an Alu PCR product. (d) PCR genotyping of the SMN1Δ(2a‐5) variant showed presence of the deletion‐spanning amplification product in the patient (P) and father (F), but not in mother (M) and control (C). (e) DNA sequence trace of the breakpoint junction‐specific PCR and detail of the Δ2a‐5 breakpoint junction show the new Alu ‐ Alu chimeric element originating from the recombination between the AluSp in the intron 1 and AluSq in the intron 5. A breakpoint microhomology of the AluSp and AluSq is marked with a black box. (f) Schematic representation of SMN1 and SMN2 in the family members. Pink‐marked boxes represent maternal alleles (M) and blue boxes paternal alleles (F). The red crosses denote identified deletions and the dashed vertical lines denote loci of the qPCR (I1, E3I3, I5E6, and +1 kb) and MLPA (exon 7‐E7, exon 8‐E8) probes used for deletion mapping. The black junctions on the box terminals indicate a cis configuration of SMN1 and SMN2 alleles. The model shows (a) a whole deletion of one SMN1 allele in the patient (P) inherited from her mother and detected by the combination of the qPCR and MLPA; (b) a deletion of the second SMN1 allele in the patient inherited from her father and detected by the E3I3 qPCR and transcript analysis (Figure 1a ); and (c) deletion of one copy of one SMN2 allele in the mother detected by the MLPA and the + 1kb qPCR

    Journal: Molecular Genetics & Genomic Medicine

    Article Title: Spinal muscular atrophy caused by a novel Alu‐mediated deletion of exons 2a‐5 in SMN1 undetectable with routine genetic testing, et al. Spinal muscular atrophy caused by a novel Alu‐mediated deletion of exons 2a‐5 in SMN1 undetectable with routine genetic testing

    doi: 10.1002/mgg3.1238

    Figure Lengend Snippet: Identification of SMN1 variants. (a) qPCR analysis of four retrotransposon‐free SMN genomic regions in the intron 1 (I1), in the exon 3–intron 3 junction (E3I3), in the intron 5–exon 6 junction (I5E6), and ~1 kb downstream from exon 8 (+1 kb). Compared with four copies of SMN genes that are present in controls, we found that the patient has three copies at the I1, I5E6, and +1 kb loci and two copies at the E3I3 loci. The mother has three copies through the I1 to I5E6 loci and two copies at the +1 kb loci. The father has three copies only at the I5E6 loci. (b) Schematic representation of SMN1/2 exon (E)/intron (I) structure. Positions of sequence differences between SMN1 and SMN2 are represented by black vertical bars. The black triangles denote sequence‐specific variants in exons 7, 8 targeted by MLPA probes in routine testing. Locations of Alus in the breakpoint candidate regions in the intron 1 and 5, including the causal AluSp in the intron 1 and AluSq in the intron 5 indicated by vertical text, and primers binding sites for Alu PCR indicated by black arrowheads are shown below the scheme of the SMN structure. Position of the PCR4 spanning exons 5–8 that showed absence of SMN1 sequence‐specific variants indicating disruption of both SMN1 alleles in the patient is represented by yellow box. Range of the paternal deletion of exons 2a‐5 is represented by red box. (c) DNA sequence trace of the Alu PCR, Alu_259_4A, showing a double sequence caused by presence of AluSq wt in intron 5 together with a sequence originating from the intron 1 AluSp . Red arrows indicate the addition of AluSp ‐specific sequence in an Alu PCR product. (d) PCR genotyping of the SMN1Δ(2a‐5) variant showed presence of the deletion‐spanning amplification product in the patient (P) and father (F), but not in mother (M) and control (C). (e) DNA sequence trace of the breakpoint junction‐specific PCR and detail of the Δ2a‐5 breakpoint junction show the new Alu ‐ Alu chimeric element originating from the recombination between the AluSp in the intron 1 and AluSq in the intron 5. A breakpoint microhomology of the AluSp and AluSq is marked with a black box. (f) Schematic representation of SMN1 and SMN2 in the family members. Pink‐marked boxes represent maternal alleles (M) and blue boxes paternal alleles (F). The red crosses denote identified deletions and the dashed vertical lines denote loci of the qPCR (I1, E3I3, I5E6, and +1 kb) and MLPA (exon 7‐E7, exon 8‐E8) probes used for deletion mapping. The black junctions on the box terminals indicate a cis configuration of SMN1 and SMN2 alleles. The model shows (a) a whole deletion of one SMN1 allele in the patient (P) inherited from her mother and detected by the combination of the qPCR and MLPA; (b) a deletion of the second SMN1 allele in the patient inherited from her father and detected by the E3I3 qPCR and transcript analysis (Figure 1a ); and (c) deletion of one copy of one SMN2 allele in the mother detected by the MLPA and the + 1kb qPCR

    Article Snippet: 2.6 Long‐range PCR Long‐range PCR was performed using four primer pairs amplifying both SMN genes (NG_008691.1, NG_008728.1) in four overlapping PCR products (PCR1‐PCR4, Table ).

    Techniques: Real-time Polymerase Chain Reaction, Sequencing, Multiplex Ligation-dependent Probe Amplification, Binding Assay, Polymerase Chain Reaction, Variant Assay, Amplification