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Specific amplification of tumor specific DNA sequences

A tumor-specific and specific technology, applied in the direction of microbial determination/testing, biochemical equipment and methods, etc., can solve the problems of limited amplified loci, unlikely powerful methods for cancer detection, etc.

Inactive Publication Date: 2010-08-25
THE TRUSTEES OF COLUMBIA UNIV IN THE CITY OF NEW YORK
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This constraint severely limits the number of loci that can be amplified
Second, tumors are highly diverse, so detection of only one or a few tumor-specific genomic alterations is unlikely to provide a robust method for cancer detection

Method used

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  • Specific amplification of tumor specific DNA sequences

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1. Identification of tumor-associated hypomethylated regions

[0057] To test whether the CNA methylation profiles of subjects with ovarian tumors differed from those of normal controls, frozen serum samples were obtained from women whose blood was drawn prior to exploratory surgery due to suspicion of ovarian cancer. Similar samples were obtained from cancer-free women. DNA was prepared from 1 ml of cell-free serum by standard methods and all resulting samples were subjected to methylation-sensitive amplification as described above. Such a pair of samples was submitted to NimbleGen for hybridization to arrays that had previously been used to analyze trophoblast methylation.

[0058] The data indicated that both amplifications (cancer and normal) resulted in measurable signal (defined as >3 sd above background) from -5% of array addresses. Also, in ~70% of these cases, the log of the signal 2 - Ratios less than |1.5|, indicating that even though segments we...

Embodiment 2

[0060] Example 2. Development of Comparison Panel (Comparison Panel)

[0061] To facilitate detection and diagnosis using the methods of the invention, a normal population and a specific cancer patient population can be compared to develop a methylation profile associated with a specific type of cancer. The methods of the invention can be used to generate such methylation profiles.

[0062] DNA was isolated from serum or plasma of known cancer patients and normal controls using standard methods (Johnson, K.L. et al., Clin. Chem. 50:516-21 (2004)). Briefly, 10 ml of patient blood was centrifuged twice to remove cells. The resulting plasma was passed over a DNA-binding membrane. DNA was removed from the membrane and the resulting DNA was digested with HpyCh4-IV.

[0063] DNA adapters are annealed and ligated with digested DNA. The linker was designed to form a MluI restriction site when ligated with HpyCh4-IV digested DNA. Linker-mediated PCR was performed as described by...

Embodiment 3

[0068] Example 3. Preparation of microarrays for detection of hypomethylated tumor-associated DNA.

[0069] The genome was broken down into segments bounded by two sites for the relevant methylation-sensitive restriction enzyme (ACGT for HpyCh4-IV) and less than 500 base pairs in length. This provides a range of DNA segments that can be amplified from serum or plasma DNA samples. Algorithms are used to analyze the sequences of these fragments with the goal of finding suitable sequences for display on the microarray. For example, suitable oligonucleotides will have one or more of the following properties: (i) a unique sequence of greater than about 40 nucleotides, or a unique sequence of greater than about 60 nucleotides; (ii) about 40% to about 60% GC, and (iii) should not contain significant repeats or simple sequences, such as runs of greater than about 15 single bases. The array comprises oligonucleotides selected in this manner, wherein each oligonucleotide on the arra...

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Abstract

The present invention provides methods for cancer detection and diagnosis. The present invention provides a method of selectively amplifying hypomethylated tumor DNA sequences derived from a subject for detection of cancer. This method utilizes differential methylation to allow for the selective amplification of tumor specific sequences from DNA mixtures that contain a high proportion of normal host DNA. The invention also provides methods of using the amplified tumor DNA sequences for evaluation of methylation.

Description

technical field [0001] The invention includes methods for cancer detection and diagnosis. Background technique [0002] Existing methods for cancer screening are expensive and largely ineffective, and as a result, most cancers are detected at advanced and difficult-to-treat stages. This is especially true for ovarian cancer. Therefore, the need for new methods for cancer screening has been widely recognized. Numerous recent publications have demonstrated the presence of circulating nucleic acid (CNA) in the body fluids of cancer patients, and various strategies using can for cancer detection, tracking, and prognosis have been considered (reviewed in (Fleischhacker and Schmidt 2007 )). The easiest way is to compare the total amount of CNA between cancer patients and controls. Such studies generally find that cancer patients have more CNAs than cancer-free controls, but have not been able to show a good correlation between tumor size, stage or type, and total CNA concentra...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6855C12Q1/6837C12Q1/6886C12Q1/6827C12Q1/6858C12Q2531/125C12Q2525/191C12Q2521/331C12Q2539/107C12Q1/6818
Inventor 斯蒂芬·A·布朗
Owner THE TRUSTEES OF COLUMBIA UNIV IN THE CITY OF NEW YORK
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