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Oligonucleotide linkers comprising a variable cohesive portion and method for the preparation of polynucleotide libraries by using said linkers

a technology of oligonucleotide and polynucleotide, which is applied in the field of oligonucleotide linkers comprising a variable cohesive portion and the preparation of polynucleotide libraries by using said linkers, can solve the problems of reducing the efficiency of sequencing, affecting and difficult control of the number of g residues added

Inactive Publication Date: 2008-07-31
HAYASHIZAKI YOSHIHIDE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the G-tailing methodology shows several drawbacks, for example, in sequencing efficiency and translation efficiency when cDNAs clones are used for protein expression.
However, the number of G residues added is difficult to control and it is variable, generally between 10 and 30.
A long G-tail has the drawback of impairing a long read sequencing and lowering the sequencing efficiency, whilst a short G-tail has the drawback of providing a low efficient priming, with the consequence of loss of sample, and necessity of repreparing it.
This may be problematic in case of interactions with 5′ UTRs that are typically GC rich.
Similar problems were also observed in cloning vectors having GC rich regions containing a Sfi I or Not I restriction site next to the cloning site.
However, these heavy metals have sometimes caused degradation of cDNAs and decreased rate of production of long strand, full-coding / full-length cDNA.

Method used

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  • Oligonucleotide linkers comprising a variable cohesive portion and method for the preparation of polynucleotide libraries by using said linkers
  • Oligonucleotide linkers comprising a variable cohesive portion and method for the preparation of polynucleotide libraries by using said linkers
  • Oligonucleotide linkers comprising a variable cohesive portion and method for the preparation of polynucleotide libraries by using said linkers

Examples

Experimental program
Comparison scheme
Effect test

example 1

Linker Evaluation Using a Test cDNA

Linkers Preparation

[0199]The population of linker oligonucleotides were purchased from Gibco-BRL Life technologies. The oligonucleotides were distinguished in one single strand (single upper strand) (indicated as A and C, comprising the variable portion) and the other single strand (single lower strand) (indicated as B). Then, one of A and C and B were bound together in order to form two different populations of double strands. The population of linkers A comprises linkers having a fixed portion oligonucleotide (in this case, bases 1-43 of SEQ ID NO:1) and a variable portion (GN5), wherein the first base is G (that is, the base number 44) and the following bases NNNNN (bases from 45 to 49) different for each linker of the population and prepared at random.

[0200]The population of linkers C comprises a constant portion oligonucleotide (bases 1-43 of SEQ ID NO:3) and the variable portion NNNNNN (bases 44 to 49) different for each linker of the populat...

example 2

Full-Length cDNA Library Preparation and cDNA Analysis

Linker Preparation

[0214]Linkers were prepared as above described in Example 1.

Preparation of RNA

[0215]Slices of mouse liver tissue (0.5-1 g) were homogenized in 10 ml of a suspension and extracted with 1 ml of 2M sodium acetate (pH 4.0) and the same amount of a mixture of phenol / chloroform (volume ratio 5:1). After the extraction, the same volume of isopropanol was added to the aqueous layer to precipitate RNA. This sample was incubated on ice for an hour and centrifuged at 4000 rpm for 15 minutes with cooling to collect the precipitates. The resulting precipitates were washed with 70% ethanol and dissolved in 8 ml of water. By adding 2 ml of 5M NaCl and 16 ml of an aqueous solution (pH 7.0) containing 1% CTAB (cetyltrimethylammonium bromide), 4M urea and 50 mM Tris, RNA was precipitated and polysaccharides were removed (CTAB precipitate). After centrifugation at 4000 rpm for 15 minutes at room temperature, the RNA was dissolved ...

example 3

Efficiency of Ligation

[0243]In the ligation of linkers prepared as above to the mouse liver derived cDNAs, 2 μg of mixed linker (N6: GN5=1:4) were ligated to various amounts of cDNAs (1 μg, 0.5 μg and 0.2 μg respectively in Lanes 1, 2 and 3 of FIG. 4). Then, 50 ng of ligated cDNAs were used for the second strand synthesis and analyzed by alkaline gel electrophoresis. All electrophoresis patterns and incorporation rates were the same (Lanes 1-3), suggesting that 2 μg of linker were efficiently ligated to any of the different amount of cDNAs. If the linker amount was not appropriate, excessively expressed cDNA bands would be shifted up to twice size by forming hairpin structures. Instead, the first-strand cDNA and all second-strand cDNAs showed the same pattern (the same size).

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Abstract

The present invention relates to a linker or population of linkers that include an oligonucleotide fixed portion and an oligonucleotide variable portion represented by formula (N)n, wherein N is A, C, G, T or U, or their derivatives, and n is an integer equal to or higher than 1. A linker-polynucleotide or a population of linker-polynucleotides of the invention may be constituted by said linker or population of linkers and a target first strand polynucleotide bound to said linker. The invention also encompasses a method of preparing said linker or population of linkers and a method of preparing a linker-polynucleotide using said linker or population of linkers. The linkers or polynucleotide-linkers of the invention can be used in a method of preparing a cDNA library.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a divisional of U.S. patent application Ser. No. 10 / 398,483, filed Apr. 4, 2003, which is the U.S. National Stage of International Application No. PCT / JP01 / 08805 filed Oct. 5, 2001, which claims priority to Japanese Patent Application No. 2000-306749 filed Oct. 5, 2000, all of which are incorporated herein by reference in their entirety.TECHNICAL FIELD[0002]The present invention relates to a population of linkers comprising an oligonucleotide fixed portion and an oligonucleotide variable portion and to a method for the preparation of polynucleotide libraries using such population of linkers. Further, the invention relates to an improved linker as a marker for specific libraries.BACKGROUND ART[0003]Oligonucleotide linkers and primers have been used in the prior art for priming, binding or annealing single strand polynucleotides and allowing the synthesis of the second polynucleotide complementary strand.[0004]All of Car...

Claims

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Application Information

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IPC IPC(8): C40B80/00C12N15/09C07B61/00C07H21/00C07H21/02C07H21/04C12N15/10C12N15/66C12P19/34
CPCC12N15/1096C40B40/00C12P19/34C12N15/66
Inventor HAYASHIZAKI, YOSHIHIDE
Owner HAYASHIZAKI YOSHIHIDE
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