Patent Application: US-54892506-A

Abstract:
the invention is based in part on the generation of a double stranded rna molecule substantially covering the whole transcribed region of a gene , and cleaving this using an rna endonuclease to generate small rna molecules which are already or may be subsequently labelled . the invention provides small labelled ribonucleic acid fragments for use as probes to detect potentially small interfering ribonucleic acid fragments produced in vivo . the invention also provides uses of said small labelled rna fragments and kits suitable for preparing said small labelled rna fragments .

Description:
the present invention will now be described further in more detail and by way of example and with reference to the figures which show : fig1 shows a scheme in accordance with one embodiment of the present invention . in summary the scheme shows a method suitable for generating small unlabelled rna fragments from a large unlabelled rna fragment and subsequently labelling said small unlabelled rna fragments . the steps which are carried out , are as follows : a ) a gene / coding region of interest is first amplified using polymerase chain reaction ( pcr ) to generate an amplified dna fragment ; b ) the amplified dna is cloned into an appropriate cloning vector using techniques well known in the art for cloning pcr products ( see for example sambrook et al , 2000 ). for example , ta cloning vectors as known in the art , may be employed ; c ) once cloned , the dna fragment is transcribed using appropriate rna polymerases and their promoters flanking the multiple cloning region into which the gene has been inserted . many vectors are known in the art which are capable of generating sense and anti - sense rna strands such as the topo and gateway vectors supplied by invitrogen , pgem vectors supplied by promega and pbluescript vectors provided by stratagene . all suitable vectors possess two promoters at either end of the cloning region and by using an rna polymerase appropriate for each promoter , it is possible to transcribe either the sense or anti - sense rna using this promoter . the techniques for carrying out such in vitro transcription are well known to those skilled in the art and again are described in sambrook et al ., 2000 or in the protocols provided by the relevant manufacture ; d ) the transcribed sense and antisense rna strands are allowed to anneal , thereby generating said large unlabelled rna fragment ; e ) the large unlabelled rna fragment is thereafter digested using human recombinant dicer ( genetic therapy systems , inc ., san diego , us ), according to manufacturer &# 39 ; s instructions , in order to generate small rna fragments of about 22 - 25 nucleotides in length ; and f ) the small rna fragments are dephosphorylated using alkaline phosphatase and subsequently labelled with [ γ - 32 p ] datp using a polynucleotide kinase ( fig1 a ). the so generated small labelled rna fragments may thereafter be used in northern experiments , known to those skilled in the art , to identify whether or not the same gene / coding sequence is processed in vivo to generate potentially small interfering rnas . fig2 shows a small rna northern blot using radioactively labelled small rna probes prepared according to the present invention . the accumulation of gfp small rnas , a strong band at 21 nucleotides ( nts ) and a fainter band at 24 nts , in the silenced gfp reporter line 8z2 ( glazov et al ., 2003 ) is easily identified . as expected , the wild - type plants col - 0 and the high gfp expressing line 2 ( glazov et al ., 2003 ) did not show any accumulation of gfp smrnas . briefly , 20 μg of smrnas were loaded on a 15 % polyacrylamide / 8m urea gel . the gel was electroblotted on a hybondn + membrane ( amersham ). the membrane was prehybridized for 1 hour in the ultrahyb - oligo buffer ( ambion ). for the preparation of the small rna probe , 5 μg of in vitro transcribed gfp dsrna ( 5 μg of sense gfp rna annealed with 5 μg of antisense gfp rna ) were digested using the human recombinant dicer ( gts ) according to the manufacturer &# 39 ; s instructions . the digestion product was purified with g - 25 spin columns ( amersham ). the small rnas were further dephosphorylated using the shrimp alkaline phosphatase ( sap , roche ) and purified again with the g - 25 spin columns . at this step the concentration of the small rnas is estimated in pmoles / μl and can be kept in − 20 ° c . for future experiments . finally , 20 pmoles of the diced gfp small rnas were labeled at the 5 ′ end with 20 pmoles of [ γ - 32 p ] datp using polynucleotide kinase ( pnk , roche ). the probe was purified with g - 25 spin columns , denatured at 95 ° c . and added to the prehybridization buffer ( ultrahyb - oligo , ambion ). the hybridisation was performed for at least 16 hours and the membrane washed according to the manufacture . the signal was detected either using a phosphoimager screen or using a biomaz mr x - ray film . waterhouse , p . m . & amp ; helliwell , c . a . 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