Dual enzymatic amplification

A technology of polymerase and genome, applied in the direction of transferase, biochemical equipment and methods, microbial determination/inspection, etc., can solve problems such as wrongly introduced samples

Inactive Publication Date: 2015-06-24
ZHUHAI LIVZON CYNVENIO DIAGNOSTICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, whole genome amplification introduces errors into the sample, which can prevent interpretable results

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0135] Rare cell analysis without whole-genome amplification by massively parallel sequencing

[0136] Materials and methods

[0137] DNA / cell template construction For amplified genomic experiments, pool purified genomic DNA prior to the amplification reaction. For each WGA reaction, 2.4 ng of genomic DNA (~400 cell equivalent based on ~6 pg / cell) was used [5].

[0138] For direct sequencing libraries (DSL), cell pellets are processed to release DNA, which is then used directly in the library construction process without further purification. For DSL experiments, approximately 400 cells were utilized for all reactions. This number is not arbitrary but is based on the average performance of the Cynvenio Biosystems CTC isolation platform (see, e.g., U.S. Patent Publication Nos. 2011 / 0137018; 2011 / 0127222; 2011 / 0003303; 2010 / 0317093; and 2009 / 0053799, for all purposes hereby incorporated by reference in its entirety). The purity of recovered CTCs depends in part on pati...

Embodiment 2

[0210] Dual Enzymatic Amplification to Validate Genomic Mutations in Rare Cell Populations

[0211] In order to measure mutations in the DNA genome of CTCs isolated from 2 to 4 ml whole blood by any technique, sufficient quantity and quality of DNA is important. Typically, recovery of 2 to 10 CTCs from 2 to 4 ml of whole blood can be expected. This number of cells must be processed with excellent recovery to ensure that chromosomes carrying mutations are not lost during processing. Therefore, special methods are required in order to isolate DNA of sufficient quality and quantity. Conventional methods are not useful because they alter DNA genome representation, produce poor quality DNA and / or result in quantities from such rare samples that are unsuitable for use in various molecular assays (such as, but not limited to, QPCR and DNA sequencing) insufficient.

[0212] Isolation of DNA from rare cell populations (eg, small numbers of blood-derived CTC cells) introduces several...

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Abstract

Provided are methods for validating the presence and character of genomic mutations, particularly single nucleotide polymorphisms (SNPs), by parallel amplification of a portion or the whole genome with at least two different DNA polymerases.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 61 / 674,696, filed July 23, 2012, which is hereby incorporated by reference in its entirety for all purposes. field of invention [0003] The present invention relates to methods for verifying the presence and characterization of genomic mutations, in particular single nucleotide polymorphisms (SNPs), by parallel amplification of parts or the entire genome with at least two different DNA polymerases. Background of the invention [0004] Solid tissue cancer begins to grow at the original site. Metastasis occurs at distant locations as the disease progresses. These metastatic events accelerate disease and ultimately death. Cells or cell debris leave the original site as part of the transfer process. The transfer process is complex. Part of the metastatic process involves rare circulating tumor cells (CTCs). It is becoming...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/48C12Q1/68C12N9/10
CPCC12Q1/6848C12Q1/6869C12Q2521/101C12Q2527/149C12Q2535/122C12Q2545/113
Inventor W.M.斯特劳斯
Owner ZHUHAI LIVZON CYNVENIO DIAGNOSTICS
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