Fragmentation-based methods and systems for sequence variation detection and discovery

A sequence and mutation technology, applied in the field of fragmentation and system for sequence variation detection and discovery, can solve problems such as complex problems

Inactive Publication Date: 2006-05-17
SEQUENOM INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The problem is further complicated when there are multiple base variants or multiple sequence variants rather than just a single base variant or sequence variant

Method used

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  • Fragmentation-based methods and systems for sequence variation detection and discovery
  • Fragmentation-based methods and systems for sequence variation detection and discovery
  • Fragmentation-based methods and systems for sequence variation detection and discovery

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0388] Base-specific cleavage of RNA

[0389] Presented here is a semi-automated protocol for a one-tube reaction involving RNA transcription and G-specific endonucleolytic cleavage using a typical ribonuclease (RNase T1) to analyze sequence variation in the target nucleic acid of interest . Fragments produced by the ribonuclease cleavage methods provided herein can be analyzed according to the methods provided herein. The RNase T1 reaction is performed to cleave approximately 100% of the G nucleotide site on the target nucleic acid. This cleavage produces a characteristic map of fragment masses that represent sequence variation in the target sequence of interest.

[0390] Materials and methods

[0391] Oligonucleotides were purchased from Metabion (Germany). 5-Methylcytidine lithium 5'-triphosphate (Me-CTP) and 5-methyluridine lithium 5'-triphosphate (Me-UTP) were obtained from Trilink (USA).

[0392] PCR amplification

[0393] A 5 μl PCR reaction included 5 ng of genom...

Embodiment 2

[0430] Base-specific cleavage of DNA

[0431] The following examples describe methods for cleaving a target nucleic acid based on the presence of U residues in the nucleic acid by digestion with uracil DNA glycosylase and the use of NH 3 cleavage of the phosphate backbone. The fragmentation methods provided herein can be used to generate base-specific cleaved fragments of target DNA, which can then be analyzed according to the methods provided herein to identify sequence variations in the target DNA relative to a reference DNA.

[0432] The DNA region of interest was amplified by PCR in the presence of dUTP instead of dTTP. The target region was amplified using a 50 μl PCR reaction consisting of 25 ng of genomic DNA, 1 unit of HotStarTaq DNA polymerase (Qiagen), 0.2 mM each of dATP, dCTP, and dGTP, and 0.6 mM each in 1×HotStarTaq PCR buffer. mM dUTP. PCR primers were used in an asymmetric ratio of 5 pmol biotinylated primer and 15 pmol non-biotinylated primer. The temperat...

Embodiment 3

[0451] A. SNP discovery by amplifying base-specific breaks in DNA

[0452] Base-specific cleavage fragments of a target sequence comprising a SNP can be analyzed by the methods provided herein to detect known SNPs or to discover unknown SNPs. High-throughput base-specific fragmentation followed by mass spectrometry can be performed as described by Rodi et al., Bio Techniques, 32:S62-S69 (2002) (incorporated herein by reference), using a system such as that developed by the trademark MassARRAY TM represented system. Mass ARRAY TM Rely on mass spectrometry combined with miniaturized arrays and MALDI-TOF (Matrix Assisted Laser Desorption Ionization-Time-of-Flight) mass spectrometry to deliver results rapidly. Fragment signals generated according to the methods provided herein and Rodi et al., Bio Techniques, 32:S62-S69 (2002) can be analyzed according to the methods provided herein.

[0453] In base-specific fragmentation, a single-stranded copy of the target sequence is creat...

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Abstract

Fragmentation-based methods and systems, particularly mass spectrometric methods and systems, for the analysis of sequence variations are provided.

Description

[0001] related application [0002] This application claims the benefit of priority of US Provisional Application Serial No. 60 / 429,895, filed November 27, 2002, entitled "Fragmentation-based Methods and Systems for Sequence Variation detection and Discocery." [0003] Also related to this application are U.S. Provisional Application No. 60 / 466,006, filed April 25, 2003, entitled "Fragmentation-based Methods and Systems forde novo Sequencing," and U.S. Application Filed November 26, 2003, entitled It is "Fragmentation-based Methods and Systems for SequenceVariation Detection and Discovery", record number (attorney dock) 24736-2073. [0004] Where permitted, the contents of each of the aforementioned applications and provisional applications are incorporated herein by reference. Background technique [0005] The genetic information of all living organisms (eg, animals, plants, and microorganisms) is encoded in deoxyribonucleic acid (DNA). In humans, the complete genome includ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G16B30/00C12NC12N15/00G01N33/53G01N33/68G16B20/20
CPCC12Q1/6872G16B20/00G16B30/00G16B20/20C12Q2521/301C12Q2521/319
Inventor 迪尔克·范登博姆塞巴斯蒂安·伯克尔
Owner SEQUENOM INC
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