Method for the preparation and amplification of representative and strand- specific libraries of cdna for high throughput sequencing, use thereof, kit and cartridges for automation kit

SCHEDA BREVETTO

Abstract:

The present invention relates to a rapid, simple, effective and economical method for preparing and amplifying representative cDNA libraries for next-generation highthroughput sequencing, kit and cartridges for automation kits which use such method manually or in an automatic manner.

Advantages:

Among next-generation sequencing platforms, the Roche 454 GS FLX pyrosequencer makes it possible to obtain in each run over a million reads, each with a length of about 400 bases, even up to about 800 bases.
This makes it possible to amplify the starting material and in particular the least represented species of RNA which are otherwise hardly detected.
Therefore, information about strand specificity makes it possible to distinguish coding transcripts from antisense ones, to determine from which strand other non-coding RNAs are synthesized, to define more precisely the exact boundaries of adjacent genes, transcribed from opposite strands, and to determine the correct levels of expression of coding and non-coding RNA molecules, transcribed from overlapping sequences.
One of the main problems is represented by the fact that the starting amount of RNA is often very exiguous and that some transcripts, despite having considerable biological significance, are very scarcely abundant.

Application field:

MICRO-ORGANISMS OR ENZYMES COMPOSITIONS THEREOF(biocides, pest repellants or attractants, or plant growth regulators containing micro-organisms, viruses, microbial fungi, enzymes, fermentates, or substances produced by, or extracted from, micro-organisms or animal material
MEASURING OR TESTING PROCESSES INVOLVING ENZYMES OR MICRO-ORGANISMS

Technical board:

The present invention relates to a rapid, simple, effective and economical method for preparing and amplifying representative and strand-specific cDNA libraries for next- generation high throughput sequencing, kit and cartridges for automation kit which use such method manually or in an automatic manner.
Therefore, object of the invention is a method for obtaining a representative and strand-specific cD A library from an RNA sample of at least about 400 ng, comprising the steps of:
In a preferred aspect the method of the invention also comprises a preliminary step of extracting total RNA from a sample, for example a biological, cellular, etc. sample.

itb1[1]

Patent Title

Method for the preparation and amplification of representative and strand-specific libraries of CDNA for high throughput sequencing, use thereof, kit and cartridges for automation kit

Other Title

Metodo per la preparazione e amplificazione di librerie rappresentative di CDNA per il sequenziamento massivo, loro uso, kit e cartucce per kit di automazione

Inventor(s)

MANGIULLI MARINA

PESOLE GRAZIANO

SBISA’ ELISABETTA

TULLO APOLLONIA

Published As

Publ. number Pub. date Appl. number
Appl. date
Publ. Stage
WO2011151777 20111208
EP2576780
20131004
A1 – application with search report

NsRIF

10069 - Catalogo brevetti CNR

Country of validity

PCT

Ownership

 Consiglio Nazionale delle Ricerche (100%) – ITB

Contacts

info@ilo-puglia.cnr.it