Structured Review

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 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/long range pcr/product/Boehringer Mannheim
Average 92 stars, based on 18 article reviews
Price from $9.99 to $1999.99
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Images

1) Product Images from "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"

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

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

doi:

( 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 .
Figure Legend 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 .

Techniques Used: 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.
Figure Legend 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.

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

2) Product Images from "SMU-2 and SMU-1, Caenorhabditis elegans Homologs of Mammalian Spliceosome-Associated Proteins RED and fSAP57, Work Together To Affect Splice Site Choice"

Article Title: SMU-2 and SMU-1, Caenorhabditis elegans Homologs of Mammalian Spliceosome-Associated Proteins RED and fSAP57, Work Together To Affect Splice Site Choice

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.24.15.6811-6823.2004

Mapping and cloning smu-2 . The scales in panels A and B are identical. (A) Genetic map position of smu-2 with respect to the lin-31 gene and DNA sequence dimorphisms between strains N2 and RC301, which were used in mapping. The left end point of ccDf1 , representing a deficiency that deletes lin-31 and complements smu-2 , was mapped between dimorphisms mnP3 and mnP4 . (B and C) Transformation rescue data for smu-2 with YAC DNA and long-range PCR products. + indicates that at least one line that rescued smu-2 , as assayed by reversal of unc-52 suppression, was obtained; − indicates that several lines that did not rescue smu-2 were obtained. (C) The exon-intron structure of smu-2 is indicated below the restriction map. Arrows, locations of primers used to generate long-range PCR products. Eco01019I, NotI, and HindIII restriction sites used to construct pAS17, pAS19, and pAS21, respectively, are indicated. yk563h8 is a full-length smu-2 cDNA. pAS21 has a frameshift mutation introduced at the HindIII site (light gray box). The placement of the GFP gene for plasmid pAS17 is shown. smu-2 ( mn416 ) has a point mutation located at the 3′ splice site of intron 1. smu-2 ( mn610 ) has a single base pair deletion in the middle of exon 6. LR16F/15R( mn416 ) and LR16F/15R( mn610 ) are PCR products that were generated from genomic DNA made from the respective smu-2 mutants.
Figure Legend Snippet: Mapping and cloning smu-2 . The scales in panels A and B are identical. (A) Genetic map position of smu-2 with respect to the lin-31 gene and DNA sequence dimorphisms between strains N2 and RC301, which were used in mapping. The left end point of ccDf1 , representing a deficiency that deletes lin-31 and complements smu-2 , was mapped between dimorphisms mnP3 and mnP4 . (B and C) Transformation rescue data for smu-2 with YAC DNA and long-range PCR products. + indicates that at least one line that rescued smu-2 , as assayed by reversal of unc-52 suppression, was obtained; − indicates that several lines that did not rescue smu-2 were obtained. (C) The exon-intron structure of smu-2 is indicated below the restriction map. Arrows, locations of primers used to generate long-range PCR products. Eco01019I, NotI, and HindIII restriction sites used to construct pAS17, pAS19, and pAS21, respectively, are indicated. yk563h8 is a full-length smu-2 cDNA. pAS21 has a frameshift mutation introduced at the HindIII site (light gray box). The placement of the GFP gene for plasmid pAS17 is shown. smu-2 ( mn416 ) has a point mutation located at the 3′ splice site of intron 1. smu-2 ( mn610 ) has a single base pair deletion in the middle of exon 6. LR16F/15R( mn416 ) and LR16F/15R( mn610 ) are PCR products that were generated from genomic DNA made from the respective smu-2 mutants.

Techniques Used: Clone Assay, Sequencing, Transformation Assay, Polymerase Chain Reaction, Construct, Mutagenesis, Plasmid Preparation, Generated

Related Articles

Polymerase Chain Reaction:

Article Title: Mutants in the Mouse NuRD/Mi2 Component P66? Are Embryonic Lethal
Article Snippet: .. For the long-range PCR we used the level 2 protocol of the ExpandTM Long Template PCR System (Boehringer Mannheim) but scaled down to a 25 µL reaction volume. .. Gene targeting, ES cell culture, and genotyping We made the targeting construct by cutting a 1.4 kb short homologous arm and a 6.3 kb long homologous arm from the 129/Sv BAC clone 95E12, then used a shuttle vector to place these arms into the targeting vector, ploxPNT.

Article Title: Cytochrome bd Biosynthesis in Bacillus subtilis: Characterization of the cydABCD Operon
Article Snippet: .. Long-range PCR was carried out by using the Expand High Fidelity PCR System (Boehringer Mannheim) according to the manufacturer’s protocol. .. The PCR product was digested with Bam HI and Pst I and was then ligated with the shuttle vector pHP13 , which had previously been digested with Bam HI and Pst I.

Article Title: Biofilm Formation by Staphylococcus epidermidis Depends on Functional RsbU, an Activator of the sigB Operon: Differential Activation Mechanisms Due to Ethanol and Salt Stress
Article Snippet: .. For long-range PCR, the Expand Long Template PCR System (Boehringer, Mannheim, Germany) was used with oligonucleotides specific for the 5′ and 3′ junctions of Tn 917 (5L [5′-CTC ACA ATA GAG AGA TGT CAC CG-3′] and 3R [5′-GGC CTT GAA ACA TTG GTT TAG TGG G-3′]) ( ) as described by the manufacturer for an expected fragment length of 12 to 15 kb. ..

Article Title: Evolution of the Neuropeptide Y Receptor Family: Gene and Chromosome Duplications Deduced from the Cloning and Mapping of the Five Receptor Subtype Genes in Pig
Article Snippet: .. Long-range PCR (EXPAND Long Template PCR System, Boehringer Mannheim) was performed on the NPY1R and NPY5R -containing phage clones to estimate the distance from the phage arms to the coding region of the gene. ..

Article Title: SMU-2 and SMU-1, Caenorhabditis elegans Homologs of Mammalian Spliceosome-Associated Proteins RED and fSAP57, Work Together To Affect Splice Site Choice
Article Snippet: .. YAC DNA was prepared as described previously , and long-range PCR products were made with the Expand long-template PCR system (Boehringer Mannheim). ..

Article Title: Structural Comparison of Three Types of Staphylococcal Cassette Chromosome mec Integrated in the Chromosome in Methicillin-Resistant Staphylococcus aureus
Article Snippet: .. Long-range PCR was performed with Expand Taq (Boehringer Mannheim Biochemica, Mannheim, Germany) according to the procedure recommended by the manufacturer. .. PCR products were purified with High Pure PCR product purification kit (Boehringer Mannheim Biochemica).

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
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). ..

Article Title: A Non-Long Terminal Repeat Retrotransposon Family Is Restricted to the Germ Line Micronucleus of the Ciliated Protozoan Tetrahymena thermophila
Article Snippet: .. Long-range PCR was performed with the Expand Long Template PCR system (Boehringer Mannheim) with conditions as specified by the supplier. .. To obtain 5′ sequence from REP6, we first used inverse PCR with primers targeted within the macronucleus-destined HEH2 gene (Fig. ) to amplify and then sequence the macronuclear locus of the region.

Clone Assay:

Article Title: Evolution of the Neuropeptide Y Receptor Family: Gene and Chromosome Duplications Deduced from the Cloning and Mapping of the Five Receptor Subtype Genes in Pig
Article Snippet: .. Long-range PCR (EXPAND Long Template PCR System, Boehringer Mannheim) was performed on the NPY1R and NPY5R -containing phage clones to estimate the distance from the phage arms to the coding region of the gene. ..

Amplification:

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
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). ..

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    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 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/long range pcr/product/Boehringer Mannheim
    Average 92 stars, based on 20 article reviews
    Price from $9.99 to $1999.99
    long range pcr - by Bioz Stars, 2021-02
    92/100 stars
      Buy from Supplier

    80
    Boehringer Mannheim long range genomic fusion pcr technique
    Disruption of the mouse <t>annexin</t> VI gene locus. Genotyping was done by genomic <t>PCR</t> of DNA extracted from tail snips. Primers MAVI-4 (which lies beyond the end of the targeting construct) and MAVI-5 were used as shown in panel a. Disrupted alleles of annexin VI have a 1.1-kb insert in exon 3 which increases the size of this PCR product from 2.2 to 3.3 kb. The results of PCRs with these primers and template DNA from wild-type (+/+), heterozygous (+/−), and homozygous null (−/−) mice, as shown in panel b, were resolved on a 1% agarose gel and visualized by ethidium bromide staining. The ratios of +/+, +/−, and −/− pups from +/− × +/− matings are shown in panel c.
    Long Range Genomic Fusion Pcr Technique, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/long range genomic fusion pcr technique/product/Boehringer Mannheim
    Average 80 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    long range genomic fusion pcr technique - by Bioz Stars, 2021-02
    80/100 stars
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    ( 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

    Generation of Msh4 mutant mice. ( A ) Gene-targeting strategy. Schematic representation of the Msh4 wild-type gene locus, the p Msh4Ex4 targeting construct and the targeted Msh4 locus. The exons are shown as black boxes. The PCR primers located in the PGK hygromycin cassette and in exon 6 that were used for detecting the gene-targeting events are indicated by arrows connected by a dotted line. The diagnostic Bgl II digestion products for the wild-type Msh4 locus and the modified Msh4 locus that are recognized by the hybridization probe are shown. ( B ) Southern blot hybridization of DNA from mice of the F 2 generation. Tail DNA was digested with Bgl II and hybridized with the probe shown in A . The 5.2-kb band corresponds to the wild type and the 3.6-kb band corresponds to the targeted allele. (+/+) Wild type; (+/−) heterozygous; (−/−) homozygous. ( C ) Detection of Msh4 by Northern blot analysis. Testis poly(A) RNA of the different genotypes was analyzed with a cDNA probe corresponding to the entire Msh4 -coding sequence. A human β-actin-specific probe was used as a control. (+/+) Wild type; (+/−) heterozygous; (−/−) homozygous. ( D ) Detection of MSH4 protein on meiotic chromosomes. Immunofluorescent colocalization of MSH4 (red) and the synaptonemal complex protein COR1 (white) on chromosome spreads of spermatocytes from 20-day-old males. (+/+) Chromosome spread from wild-type spermatocytes; (−/−) chromosome spread from homozygous mutant spermatocytes.

    Journal: Genes & Development

    Article Title: MutS homolog 4 localization to meiotic chromosomes is required for chromosome pairing during meiosis in male and female mice

    doi:

    Figure Lengend Snippet: Generation of Msh4 mutant mice. ( A ) Gene-targeting strategy. Schematic representation of the Msh4 wild-type gene locus, the p Msh4Ex4 targeting construct and the targeted Msh4 locus. The exons are shown as black boxes. The PCR primers located in the PGK hygromycin cassette and in exon 6 that were used for detecting the gene-targeting events are indicated by arrows connected by a dotted line. The diagnostic Bgl II digestion products for the wild-type Msh4 locus and the modified Msh4 locus that are recognized by the hybridization probe are shown. ( B ) Southern blot hybridization of DNA from mice of the F 2 generation. Tail DNA was digested with Bgl II and hybridized with the probe shown in A . The 5.2-kb band corresponds to the wild type and the 3.6-kb band corresponds to the targeted allele. (+/+) Wild type; (+/−) heterozygous; (−/−) homozygous. ( C ) Detection of Msh4 by Northern blot analysis. Testis poly(A) RNA of the different genotypes was analyzed with a cDNA probe corresponding to the entire Msh4 -coding sequence. A human β-actin-specific probe was used as a control. (+/+) Wild type; (+/−) heterozygous; (−/−) homozygous. ( D ) Detection of MSH4 protein on meiotic chromosomes. Immunofluorescent colocalization of MSH4 (red) and the synaptonemal complex protein COR1 (white) on chromosome spreads of spermatocytes from 20-day-old males. (+/+) Chromosome spread from wild-type spermatocytes; (−/−) chromosome spread from homozygous mutant spermatocytes.

    Article Snippet: Genomic DNA from individual colonies was subjected to Long Range PCR analysis (Boehringer Mannheim) and positive ES cell colonies were identified by a 4.3-kb PCR fragment using forward primer 5′-TGGAAGGATTGGAGCTACGG-3′ and reverse primer 5′-GAAAGCAGCTGCTCCGTATC-3′.

    Techniques: Mutagenesis, Mouse Assay, Construct, Polymerase Chain Reaction, Diagnostic Assay, Modification, Hybridization, Southern Blot, Northern Blot, Sequencing

    Amplification of 2.45 kb product of MLL-AF4 rearrangement from RS4;11 cell line DNA and RS4;11 “imitation” Guthrie specimens by PCR. Lanes: M, DNA marker II; 1, no DNA control; 2, 100 ng positive control RS4;11 DNA; 3, 100 ng human placenta negative control DNA; 4, 100 ng normal genomic negative control DNA; 5–9, 10 −1 through 10 −5 dilutions of RS4;11 cells on imitation Guthrie specimens.

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

    Article Title: Backtracking leukemia to birth: Identification of clonotypic gene fusion sequences in neonatal blood spots

    doi:

    Figure Lengend Snippet: Amplification of 2.45 kb product of MLL-AF4 rearrangement from RS4;11 cell line DNA and RS4;11 “imitation” Guthrie specimens by PCR. Lanes: M, DNA marker II; 1, no DNA control; 2, 100 ng positive control RS4;11 DNA; 3, 100 ng human placenta negative control DNA; 4, 100 ng normal genomic negative control DNA; 5–9, 10 −1 through 10 −5 dilutions of RS4;11 cells on imitation Guthrie specimens.

    Article Snippet: Amplification of the MLL-AF4 translocation region in the RS4;11 cell line was performed by using a long-range PCR system (Expand Long Template PCR system, Boehringer Mannheim) under the following conditions: 1× final buffer (50 mM Tris⋅HCl, pH 9.2/16 mM (NH4)2 SO4 /2.25 mM MgCl2 ), 12 pmol each of 27-mer oligonucleotide primers (see below), 500 μM final concentration dNTPS, and 2.5 units of Taq and Pwo enzyme mix, for 10 min at 92°C, 30 sec at 65°C, and 2 min at 68°C × 30 cycles, followed by 7 min of extension at 68°C.

    Techniques: Amplification, Polymerase Chain Reaction, Marker, Positive Control, Negative Control

    Disruption of the mouse annexin VI gene locus. Genotyping was done by genomic PCR of DNA extracted from tail snips. Primers MAVI-4 (which lies beyond the end of the targeting construct) and MAVI-5 were used as shown in panel a. Disrupted alleles of annexin VI have a 1.1-kb insert in exon 3 which increases the size of this PCR product from 2.2 to 3.3 kb. The results of PCRs with these primers and template DNA from wild-type (+/+), heterozygous (+/−), and homozygous null (−/−) mice, as shown in panel b, were resolved on a 1% agarose gel and visualized by ethidium bromide staining. The ratios of +/+, +/−, and −/− pups from +/− × +/− matings are shown in panel c.

    Journal: Molecular and Cellular Biology

    Article Title: Immunological Development and Cardiovascular Function Are Normal in Annexin VI Null Mutant Mice

    doi:

    Figure Lengend Snippet: Disruption of the mouse annexin VI gene locus. Genotyping was done by genomic PCR of DNA extracted from tail snips. Primers MAVI-4 (which lies beyond the end of the targeting construct) and MAVI-5 were used as shown in panel a. Disrupted alleles of annexin VI have a 1.1-kb insert in exon 3 which increases the size of this PCR product from 2.2 to 3.3 kb. The results of PCRs with these primers and template DNA from wild-type (+/+), heterozygous (+/−), and homozygous null (−/−) mice, as shown in panel b, were resolved on a 1% agarose gel and visualized by ethidium bromide staining. The ratios of +/+, +/−, and −/− pups from +/− × +/− matings are shown in panel c.

    Article Snippet: The mouse annexin VI targeting construct was generated by a novel long-range genomic fusion PCR technique with the Expand Long Template or the Expand High Fidelity PCR kit (Boehringer Mannheim).

    Techniques: Polymerase Chain Reaction, Construct, Mouse Assay, Agarose Gel Electrophoresis, Staining