DNA microarray-based identification and localization of balanced translocation breakpoints

A DNA sequence and microarray technology, applied in the field of identification and location of balanced translocation breakpoints based on DNA microarrays, can solve the problems of routine analysis of inappropriate clinical samples, time-consuming and labor-intensive, limited throughput, etc.

Inactive Publication Date: 2014-10-15
UNIV OF WASHINGTON
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these techniques are time-consuming and labor-intensive, and their throughput is limited, making them unsuitable for routine analysis of clinical samples.

Method used

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  • DNA microarray-based identification and localization of balanced translocation breakpoints
  • DNA microarray-based identification and localization of balanced translocation breakpoints
  • DNA microarray-based identification and localization of balanced translocation breakpoints

Examples

Experimental program
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preparation example Construction

[0122] 4. Preparation of Probes for Detecting Balanced Translocations

[0123] For detection of translocation, any method can be used as long as the method can linearly amplify DNA containing a potential translocation site. An example of a linear amplification method that can be used to practice the present invention includes PCR amplification using a single primer. See Liu, C.L., S.L. Schreiber et al., BMC Genomics, 4: Art. No. 19, 2003 / 5 / 9.

[0124] A typical set of conditions for linear amplification includes a 50 μl volume reaction containing 1 μg of genomic DNA, 200 mM dNTPs and 150 nM linear amplification primers. Amplification can be done using the PCR enzyme system (Advantage 2PCR Enzyme System) of Cloning Technology Co., Ltd. (Clontech) as follows: Denaturation at 95°C for 5 minutes, followed by 12 cycles (95°C / 15 seconds, 60°C / 15 seconds and 68°C / 6 minutes).

[0125] Probes can be labeled during linear amplification or after amplification is complete. In the spec...

Embodiment 1

[0148] Example 1: J H Identification of relevant translocation breakpoints

[0149] Array CGH is designed to be useful for detecting genomic imbalances but not balanced genomic rearrangements. We therefore explored a way for synthetic genome imbalances to represent balanced translocation sites on standard CGH arrays. Using balanced immunoglobulin translocations in lymphoma cell lines as a model, we developed an enzyme-catalyzed linear amplification reaction that allows balanced translocations to also be detected by array-based CGH. Genomic DNA is modified during the amplification step, followed by fluorescein labeling and microarray hybridization. Such as figure 1 Shown, J H - Associated translocation breakpoints available using J H Consensus primers enzymatically amplify (van Dongen, 2003), resulting in linear amplification across breakpoint junctions into translocation partner loci. Able to amplify J using a single primer H - Associated translocations, whether or not ...

Embodiment 2

[0152] Example 2: Identification of IgH Switching (SH)-Associated Translocation Breakpoints

[0153] Linear amplification primers were then designed to identify H ) region translocation. Person S H The region contains multiple tandem repeats of the characteristic repeat unit: the S μ , S α and S ε In the E region, the repeating unit is a degenerated pentameric sequence G(A / G)GCT, in which the S γ The length of the region is 80-90, and the structure is more complex (Max, 1982; Mills, 1990; Mills, 1995). S H The relevant translocation breakpoints are respectively within these repeat regions. To facilitate the detection of these scattered breakpoints, linear amplification primers were designed to recognize these repeat units and μ / S α / S ε District (S 5 Primer) or S γ Repeat region (S γ primers) to initiate synthesis at multiple positions. Use J H S at 5 Primer-performed tCGH was used to identify S in the Burkitt lymphoma cell line Raji μ - MYC translocation (Dy...

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Abstract

The present specification discloses methods for detecting and localizing chromosomal rearrangements associated with various diseases using comparative genomic hybridization. Methods include identifying translocation partners at known loci and determining translocation breakpoints. These methods can be used for the prognosis, diagnosis, and determination of susceptibility to diseases involving chromosomal rearrangements.

Description

[0001] Cross References to Related Applications [0002] none [0003] Statement of Rights Concerning Inventions Resulting from Federally Sponsored Research and Development [0004] This invention was made with government support under NCI Grant No. 5P30 CA015704. The government has certain rights in this invention. [0005] Instructions Regarding Attachments to Sequence Listings, Tables, or Computer Program Listings Submitted on Compact Disk [0006] none Background of the invention [0007] Large-scale genomic aberrations, which include balanced rearrangements (translocations and inversions) and genomic imbalances (deletions, duplications, and amplifications), are common in cancer and play important roles in tumorigenesis. Genomic deletions are often associated with loss of function of tumor suppressor genes, amplifications with overexpression of proto-oncogenes, and translocations with formation of novel oncogene fusions or downregulation of oncogene expression. Histor...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/48C12P19/34
CPCC12Q1/6827C12Q2565/501C12Q2539/101C12Q2531/101C12Q1/6837C12Q2600/118
Inventor H·A·格赖斯曼
Owner UNIV OF WASHINGTON
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