High throughput detection of molecular markers based on AFLP and high throughput sequencing

A molecular marker, high-throughput technology, applied in the field of molecular biology and biology, nucleic acid detection and identification

Inactive Publication Date: 2009-05-13
KEYGENE NV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Occasionally, AFLP markers may include inserti

Method used

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  • High throughput detection of molecular markers based on AFLP and high throughput sequencing
  • High throughput detection of molecular markers based on AFLP and high throughput sequencing
  • High throughput detection of molecular markers based on AFLP and high throughput sequencing

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Embodiment 1

[0098] For each DNA sample, a restriction ligation step was performed with EcoRI and MseI. The linker is based on the hybridization sequence located on the surface of the Slexa high-throughput sequencing system. More specifically, the EcoRI linker contains the P5 sequence (sequence primer part), and the MseI linker contains the P7 sequence (bridge PCR primer sequence). EcoRI adapters also contain sample identity tags. 96 different EcoRI adapters and 1 MseI adapter were used. A degenerate EcoRI linker can be used. Template preparation included a size selection step: After the restriction digestion (EcoRI+MseI) step but before the adapter ligation step, the mixture was incubated at 80°C for 10 minutes. Fragments smaller than 130 nt were removed (in one corn sample).

[0099] The complexity of the mixture is reduced by a selective preamplification: using a +1 primer (i.e., containing a random selective nucleotide at the 3' end), using 96 EcoRI+1 primers and 1 MseI+1 primer (o...

Embodiment 2

[0104]Sequence-based detection of AFLP fragments was performed with Solexa's Clonal SingleMolecule Array (CSMATM) technology, a sequencing-by-synthesis platform capable of analyzing up to 40,000,000 individual fragments in a single sequence run. The experimental procedure included: AFLP template preparation, selective (AFLP) amplification, single-molecule bridge amplification, and sequencing of thousands of sequence tags from one restriction enzyme end of the AFLP fragment. Maize parental lines B73 and Mo17 and 87 recombinant inbred lines (RILs) were used and sequenced, and more than 8,900,000 EcoRI AFLP fragment ends were sequenced to provide proof-of-principle for sequence-based AFLP detection. The parental lines B73 and Mo17 and 87 RILs were selected. AFLP templates were performed with the restriction enzyme combination EcoRI / MseI. Selective amplification was performed with +2 / +3 AFLP primers. Solexa CSMA bridge amplified template fragments were prepared with a second res...

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Abstract

The present invention relates to a high throughput method for the identification and detection of molecular markers wherein restriction fragments are generated and suitable adaptors comprising (sample-specific) identifiers are ligated. The adapter- ligated restriction fragments may be selectively amplified with adaptor compatible primers carrying selective nucleotides at their 3' end. The amplified adapter-ligated restriction fragments are, at least partly, sequenced using high throughput sequencing methods and the sequence parts of the restriction fragments together with the sample-specific identifiers serve as molecular markers.

Description

technical field [0001] The present invention relates to the fields of molecular biology and biotechnology. In particular, the present invention relates to the field of nucleic acid detection and identification. More particularly, the present invention relates to methods of detecting and identifying markers, especially molecular markers. The present invention relates to the provision of high throughput methods for the detection and identification of molecular markers. The invention further relates to the use of this method for identifying and / or detecting nucleotide sequences associated with a range of genetic traits, genes, haplotypes and combinations thereof. The present invention can be used in the field of high-throughput detection and identification of molecular markers from any source, whether plant, animal, human, human body or other. Background technique [0002] The scientific community, especially the medical community, has long wanted to exploit genomic DNA. Ge...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor M·J·T·范艾吉克R·C·J·赫格思
Owner KEYGENE NV
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