long range pcr  (Boehringer Mannheim)

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

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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

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

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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

Journal: Genes & Development

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

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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

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

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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

Journal: Molecular and Cellular Biology

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

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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