Patent Application: US-201013383141-A

Abstract:
the present invention refers to a method for detecting a point mutations of a nucleotide sequence by an improve - ment of the lamp amplification method , as well as to a set of primers and kit there - for . as a non limitative embodiment , the invention refers to the g1849t mutation of the jak2 gene .

Description:
the authors of the current invention have set up a novel method for the detection of point mutations that is selective and rapid . the method departs from the lamp technology , as disclosed in ep 1020534 and depicted here in fig1 . the assay is easy to be performed , since it needs simple instrumentation and produces the results in a single tube reaction . for these reasons it is also less expensive in respect to the other methods described above . the method overcomes the limitations underlined in the other techniques . it is more selective ( down to 0 . 01 % mutant sequences in wt background ); it is isothermal and rapid , completing the diagnosis in a one hour reaction . the method refers to a simultaneous selective amplification and detection of a single base substitution in nucleic acids . therefore it is an object of the invention a method for detecting the presence of a point mutation of a target nucleic acid molecule in a background of nucleic acid wild type molecules , comprising the steps of : b . contacting said nucleic acid sample , in appropriate reaction conditions , with a solution comprising a mixture of oligonucleotides and a dna polymerase with strand displacement activity under hybridization conditions , wherein said mixture of oligonucleotides consists of primers suitable for a loop mediated isothermal amplification of the region of the nucleic acid molecule putatively including the point mutation , said primers comprising : i ) two outer primers f 3 and b 3 ; ii ) two inner primers fip and bip , where fip includes two oligonucleotide sequences , f 2 and f 1 c , and bip includes two oligonucleotide sequences , b 2 and b 1 c , where said inner primers fip and bip are able to recognize and hybridize to two different and opposite regions , f 2 c and b 2 c respectively , of the target nucleic acid molecule , where either the bip primer is designed to hybridize downstream of the point mutation , or the fip primer is designed to hybridize upstream of the point mutation , and in the case that the bip primer is designed to hybridize downstream of the point mutation , then the fip primer is designed to hybridize to the target sequence such that the point mutation is located in or downstream of the f 2 c sequence and upstream of f 1 c , or in the case that the fip primer is designed to hybridize upstream of the point mutation , then the bip primer is designed to hybridize to the target sequence such that the point mutation is located in or upstream of the b 2 c sequence and downstream of b 1 c ; iii ) a self - annealable extensible primer lb or lf respectively comprising :— a central loop sequence able to selectively recognize and hybridize to the region comprising the putative point mutation of the nucleic acid molecule only if the point mutation is present ,— a 5 ′ end sequence and — a 3 ′ end sequence , said 5 ′ end and said 3 ′ end sequences being complementary to each other to form a stem , so that said central loop sequence has an higher hybridization affinity to the region comprising the putative point mutation of the nucleic acid molecule than the hybridization affinity of the 5 ′ end sequence to the 3 ′ end sequence , so that it results in annealing and amplification of the region comprising the putative point mutation of the nucleic acid molecule ; iv ) a non extensible moiety able to selectively recognize and hybridize to the wt sequence of nucleic acid molecule ; d . detecting a signal indicative of amplification of the nucleotide molecule comprising the point mutation . in a preferred embodiment the point mutation is located in the region between f 2 and f 1 c or b 2 and b 1 c ; in an alternative preferred embodiment the point mutation is located in the region probed by b 2 or f 2 . in a preferred embodiment the sequence at the 5 ′ end and the sequence at the 3 ′ end of said self - annealable extensible primer is of at least 3 nucleotides . in a preferred embodiment the non extensible moiety is a peptide nucleic acid ( pna ), preferably having at least 10 nucleotides . dna and rna have a deoxyribose and ribose sugar backbone , respectively , whereas pna &# 39 ; s backbone is composed of repeating n -( 2 - aminoethyl )- glycine units linked by peptide bonds . purine and pyrimidine bases are linked to the backbone by methylene carbonyl bonds . pnas are depicted like peptides , with the n - terminus at the first ( left ) position and the c - terminus at the right . since the backbone of pna contains no charged phosphate groups , the binding between pna / dna strands is stronger than between dna / dna strands due to the lack of electrostatic repulsion . in a most preferred embodiment said peptide nucleic acid ( pna ) comprises a sequence of bases capable of hybridizing with the region including the putative point mutation resulting in double strand structures having respectively a melting temperature ( tm )= x with the wild type sequence and melting temperature ( tm )= y with the mutant sequence , where y & lt ; incubation temperature x and x is at least 5 ° c . higher than y . in a preferred embodiment the non extensible moiety is a self - annealed non extensible primer , comprising a central loop sequence able to selectively recognize and hybrizide to the region comprising the wild type sequence of the nucleic acid molecule , a 5 ′ end sequence and a 3 ′ end sequence , said 5 ′ end and said 3 ′ end sequences being complementary to each other to form a stem , so that said central loop sequence has a higher hybridization affinity to the region comprising the wild type sequence of the nucleic acid molecule than the hybridization affinity of the 5 ′ end sequence to the 3 ′ and sequence , so that it results in annealing and blocking of the wt sequence . in a preferred embodiment the dna polymerase with strand displacement activity is the bst large fragment polymerase , or one of or a combination of : bca ( exo -), vent , vent ( exo -), deep vent , deep vent ( exo -), φ29 phage , ms - 2 phage , z - taq , kod , klenow fragment . in a preferred embodiment the constant temperature is comprised between 62 ° c . and 67 ° c . in a preferred embodiment the signal indicative of amplification of the nucleotide molecule comprising the point mutation is detected by turbidimetry . alternatively the signal indicative of amplification of the nucleotide molecule comprising the point mutation is detected by fluorescence . in a preferred embodiment the nucleic acid molecule comprises the region of the human jak2 gene ( genbank accession no . nm — 004972 ), putatively having the point mutation , guanine - to - thymidine substitution at base 1489 ( g1489t ). preferably f 2 and f 1 c are in position 1730 - 1750 and 1770 - 1795 respectively of the nm — 004972 gene sequence ; b 2 is in position 1862 - 1884 of the nm — 004972 gene sequence and b 1 c is in position 1810 - 1840 of the nm — 004972 gene sequence . most preferably the primers have the following sequences : in a preferred embodiment the non extensible moiety is a pna molecule , preferably having the structure : nh2 gagtatgtgtctgtgga conh2 . the method of the invention is applied also to other genes responsible for a pathology or an alteration , as i . e . kras , egfr , and to snps . the invention will be described with reference to specific not limiting examples , including the following figures : the basic reaction is performed by 4 primers specific for 6 regions of a target genomic sequence . internal primers anneal and extend on the target ; the product is displaced in two steps by external primers ( f 3 , b 3 ) and is shaped as a double stem - loop structure ( starting structure ) ( panel a ). the starting structure is simultaneously amplified from its free 3 ′ and by another internal primer ( panel b ). dna concatamers built by inverted repeats of the initial module are progressively synthesized in an exponential fashion ( panel c ). the primers set is designed with the f 1 c and b 1 c region complementary respectively to one base upstream and one base downstream the nucleotide of interest in position 1849 . furthermore , the 5 ′ end base of fip and bip is specific for the mutated nucleotide of jak2 and both inner primers have a mismatched base at the third base from 3 ′ end . when the dumb - bell structure is formed , if the target in the reaction is wt , the mutant specific f 1 c and b 1 c will not anneal at its 3 ′ end resulting in no amplification . differently , if mutant sequences are present in solution , the mutant specific f 1 c and b 1 c will perfectly anneal , becoming extensible by the polymerase . fig3 . lamp “ allele specific loop primer extension strategy ” principle the only one loop primer in reaction has the last base in the 3 ′ end complementary to the mutant nucleotide t at position 1849 of the jak2 gene . it also presents a mismatched base in the third base from the 3 ′ end . if the target in the reaction is wt , the mutant - specific loop primer will not anneal at its 3 ′ end resulting in no amplification . differently , if mutant sequences are present in solution , the mutant specific loop primer will perfectly anneal , becoming extensible by the polymerase . universal ( mutant insensitive ) set of primers comprising f 3 , b 3 , fip and bip to obtain a dumb - bell presenting the putative mutated nucleotide in the loop region . a particular loop primer designed to recognize the mutated base in the single strand dumb - bell structure is included , together with another modified loop primer complementary to the jak2 wild type sequence and with the 3 ′ end not extensible . when the mutated jak2 sequence is present ( panel a ), the modified mutant loop primer breaks its internal structure to anneal to the target , being consequently extensible by the polymerase : the amplification can proceed . when the wt sequence is present in the sample ( panel b ), the modified wt loop primer anneals to the wt target resulting in no amplification of the wt sequences and avoiding the aspecific binding of mutant loop primer (“ silencing ” effect ). fig5 . lamp “ mutant self - annealed loop primer with pna strategy ” principle universal ( mutant insensitive ) set of primers comprising f 3 , b 3 , fip and bip to obtain a dumb - bell presenting the putative mutated nucleotide in the loop region . a particular self annealed - loop primer designed to recognize the mutated base in the single strand dumb - bell structure is included , together with a pna probe . the pna is designed to be complementary to the loop region comprised between b 2 and b 1 c presenting the wt nucleotide . it forms a stable duplex only with the wt complementary sequence , preventing the annealing and extension of the mut - self - annealed loop primer and therefore suppressing the amplification ( panel b ). it does not anneal to the mut jak2 sequence thanks to the lower affinity ( panel a ). the reaction has been conducted on 7 e3 cps / ul wild type plasmid ( square ), on no - target control and on serial dilutions of mutant plasmid in water ( from 7 e3 to 7 e1 cps / ul , rhomboidal points , and 7 e0 cps / ul , circle point ). each samples has been tested in triplicate . the error bars represent one standard deviation . the method amplify the specific target before the aspecific one until 7 e1 cps / ul concentration . the assay shows linearity between 7 e3 and 7 e1 cps / ul mutant sample . fig7 . sensitivity of lamp “ allele specific mutant loop primer ” the reaction has been conducted on 7 e3 cps / ul wild type plasmid ( square point ), on no - target control and on serial dilutions of mutant plasmid in water ( from 7 e3 to 7 e1 cps / ul , rhomboidal points ) . each sample has been tested in triplicate . the error bars represent one standard deviation . the method amplify the specific target before the aspecific one until 7 e2 cps / ul concentration . the assay shows linearity between 7 e3 and 7 e1 cps / ul mutant sample . the reaction has been conducted on 7 e3 cps / ul wild type plasmid ( square point ), on no - target control and on serial dilutions of mutant plasmid in wild type plasmid , in proportions from 75 % to 1 %, 35000 cps total amount of dna per reaction . each sample has been tested in triplicate . the error bars represent one standard deviation . the method amplify the specific target before the aspecific one until 1 % dose ( 350 cps mut plasmid plus 34650 cps wt plasmid ). the assay shows linearity between 100 % and 1 % mutant sample in wt background . test of the assay in presence and absence of pna on mutated and wt plasmid ( 35000 cps each ), in absence of pna the wt plasmid is aspecifically amplified by the self - annealed mutant loop primer , with a delay of 5 min in respect to the specific mutated target . in presence of pna , the wt plasmid is not amplified within one hour reaction by the self - annealed mutant loop primer . the amplification of the mutant plasmid is delayed of about 5 minutes . the pna forms a stable duplex only with the wt complementary sequence preventing the annealing and extension of the mut - self - annealed loop primer and therefore suppressing the amplification within 1 hour of reaction . fig1 . selectivity of lamp “ self - annealed loop primer with pna ” test of the assay on mutated plasmid ( 350000 cps ), wild type plasmid ( 350000 cps ) and on mutated plasmid serially diluted into wt plasmid in 1 , 0 . 5 , 0 . 1 , 0 . 05 and 0 . 01 % proportion . error bars correspond to 1 standard deviation . the wt sample ( 350000 cps wt plasmid ) is not amplified in one hour reaction . the specific mutant target is detected down to 0 . 01 % mutant sequences in wt ( 35 copies tot mutant plasmid in 349650 copies of wt plasmid ). the assay is linear down to 0 . 1 % mut ( 350 copies tot mutant plasmid in 349650 copies of wt plasmid ). jak2 plasmids were synthesized by the supplier geneart ( regensburg , germany ) to contain the wild type or the mutant jak2 sequence . in details : jak2 plasmid which insert corresponds to the sequence 1689 - 1722 of mn — 004972 , including a g base at nucleotide1849 , referred to as “ wt plasmid ”; jak2 plasmid which insert corresponds to the sequence 1689 - 1722 of mn — 004972 cloned , including a t base at nucleotide1849 , referred to as “ mut plasmid ”. primers : synthesized by the supplier eurofins mwg operon ( ebersberg , germany ) referred to as “ primers ”: reaction buffer : 100 mm tris hcl ph 8 . 8 , 50 mm kcl , 40 mm mgso4 , 50 mm ( nh4 ) 2so4 , 0 . 5 % tween , 5 % dmso “ buffer 5 ×” bst large fragment polymerase 8 u / ul ( new england biolabs ), “ polymerase ” sterile apyrogen water ( salf spa ), “ ddw ” prepare reaction mix as follows : 0 . 2 μm outer primers ( f 3 and b 3 ), 1 . 6 μpm inner primers ( fip and bip ), 0 . 8 um loop primers ( lf and lb ), 1 × buffer solution , 1 . 4 mm dntps mix , 8 u bst polymerase . final volume of the reaction mix must be 4 / 5 of the total reaction volume ( i . e . 20 μl reaction mix + 5 μl sample ). always keep reagents on ice . prepare the mix for at least 17 samples , comprising 3 negative control ( 7e3 cps / ul wild type plasmid ), 12 positive control ( 3 samples 7e3 cps / ul mutant plasmid , 3 samples 7e2 cps / ul mutant plasmid , 3 samples 7e1 cps / ul mutant plasmid , 3 samples 7e0 cps / ul mutant plasmid ) 1 no - target control . prepare serial dilutions of the target (“ target dilutions ”) from shipped solution ( wt plasmid and mut plasmid ). shipped solution is a 7 * 10 10 copies / μl . dilute initially the mutant plasmid to a 7 * 10 4 copies / μl in tris 10 mm , then dilute serially to 7e3 cps / μl , 7e2 cps / μl , 7e1 cps / and 7e0 cps / μl in tris 10 mm . dilute the wt plasmid to 7 * 10 3 copies / μl in tris 10 mm . add 5 μl of target dilutions to the strips , in triplicate . add 5 μl of the target dilutions starting from the less concentrated one to the most concentrated one . close all the tubes . the reaction follows the method scheme of fig1 and 2 . program the turbidimeter ( teramecs ) for incubation at constant temperature and real time monitoring of turbidity , in order to obtain a constant reaction temperature of 66 ° c . for 1 hour . put the strips in the instrument immediately before the beginning of the programs . start the program . analyze the variation of absorbance in terms of a . u . ( arbitrary units of absorbance ) to find the threshold time for each sample analyzed . the threshold time is the minute at which the sample absorbance , after baseline subtraction , reaches the arbitrary unit value representing the threshold ( in this case 0 . 1 a . u .). the threshold time reached by each samples is correlated with its log of dna copies / μl . “ lamp jak2 dumb - bell strategy ” is based on the eiken lamp method for snp detection ( described in ep 1231281 , 20 , 21 , 22 , as well as on http :// loopamp . eiken . co . jp / e / lamp / snps_anim . html ). as shown in fig2 , the primers set is designed with the f 1 c and b 1 c region complementary respectively to one base upstream and one base downstream the nucleotide of interest in position 1849 . furthermore , the 5 ′ end base of fip and bip is specific for the mutated nucleotide of jak2 and both inner primers have a mismatched base at the third base from 3 ′ end . when the dumb - bell structure is formed , if the target in the reaction is wt , the mutant specific f 1 c and b 1 c will not anneal at its 3 ′ end resulting in no amplification because the mismatch at its 3 ′ end should not be extensible . differently , if mutant sequences are present in solution , the mutant specific f 1 c and b 1 c will perfectly anneal , becoming extensible by the polymerase . a shown in fig6 , the assay was able to detect the mutant plasmid from 7e3 cps / μl to 7e0 cps / μl ( 35 copies tot mutant plasmid ). it amplifies aspecifically the wt plasmid 7e3 cps / μl , not distinguishing the lower concentrations of mut plasmid from the aspecific target . the level of selectivity should be less than 1 %, which is the limit shown by the other techniques in literature . with this approach we don &# 39 ; t have any clear advantage . materials , methods and results of the jak2 - modified - lamp “ allele specific loop primer extension strategy ” jak2 plasmids were synthesized by the supplier geneart ( regensburg , germany ) to contain the wild type or the mutant jak2 sequence . in details : which insert corresponds to the sequence 1689 - 1722 of mn — 004972 , including a g base at nucleotide1849 , referred to as “ wt plasmid ” which insert corresponds to the sequence 1689 - 1722 of mn — 004972 cloned , including a t base at nucleotide1849 , referred to as “ mut plasmid ” the underlined base corresponds to a mismatched nucleotide . the bold base corresponds to the mutated nucleotide at position 1849 of the jak2 gene . reaction buffer : 100 mm tris hcl ph 8 . 8 , 50 mm kcl , 40 mm mgso4 , 50 mm ( nh4 ) 2so4 , 0 . 5 % tween , “ buffer 5 ×” stock the primers in aliquots . it is better to store stock solutions at − 20 ° c ., while working dilutions should be stored at 4 ° c . prepare reaction mix as follows : 0 . 2 μm outer primers ( f 3 and b 3 ), 1 . 6 μm inner primers ( fip and bip ), 0 . 8 um loop primer ( lb ), 1 × buffer solution , 1 . 4 mm dntps mix , 8 u bst polymerase . final volume of the reaction mix must be 4 / 5 of the total reaction volume ( i . e . 20 μl reaction mix + 5 μl sample ). always keep reagents on ice . prepare the mix for at least 14 samples , comprising 3 negative controls ( 7e3 cps / ul wild type plasmid ), 9 positive control ( 3 samples 7e3 cps / ul mutant plasmid , 3 samples 7e2 cps / ul mutant plasmid , 3 samples 7e1 cps / ul mutant plasmid ) 1 no - target control . prepare serial dilutions of the target (“ target dilutions ”) from shipped solution ( wt plasmid and mut plasmid ). shipped solution is a 7 * 10 10 copies / μl . dilute initially the mutant plasmid to a 7 * 10 4 copies / μl in tris 10 mm , then dilute serially to 7e3 cps / μl , 7e2 cps / μl , 7e1 cps / μl in tris 10 mm . dilute the wt plasmid to 7 * 10 3 copies / μl in tris 10 mm . add 5 μl of target dilutions to the strips , in triplicate . add 5 μl of the target dilutions starting from the less concentrated one to the most concentrated one . close all the tubes . the reaction follows the method scheme of fig3 . program the turbidimeter ( teramecs ) for incubation at constant temperature and real time monitoring of turbidity in order to obtain a constant reaction temperature of 65 ° c . for 1 hour . put the strips in the instrument immediately before the beginning of the programs . start the program . analyze the variation of absorbance in terms of a . u . to find the threshold time for each sample analyzed . the threshold time is the minute at which the sample absorbance , after baseline subtraction , reaches the arbitrary unit value representing the threshold ( in this case 0 . 1 a . u .). the threshold time reached by each samples is correlated with its log of dna copies / μl . this approach consists of a selective mutant amplification based on a mutant - specific loop primer ( fig3 ). we designed a universal ( mutant insensitive ) set of primers comprising f 3 , b 3 , fip and bip to obtain a dumb - bell presenting the putative mutated nucleotide in the loop region comprised between b 2 and b 1 c . we therefore designed only one loop primer presenting the last base in the 3 ′ end complementary to the mutant nucleotide t at position 1849 of the jak2 gene . it also presents a mismatched base in the third base from the 3 ′ end . if the target in the reaction is wt , the mutant - specific loop primer will not anneal at its 3 ′ end resulting in no amplification . differently , if mutant sequences are present in solution , the mutant specific loop primer will perfectly anneal , becoming extensible by the polymerase . we tested this assay on the mutant plasmid from 7e3 cps / μl to 7e1 cps / μl . ( 35000 and 350 copies tot mutant plasmid ) and on the aspecific wt plasmid ( 7 e 3 cps / μl ), all in triplicate . the assay amplifies aspecifically the wt plasmid 7e3 cps / μl , not distinguishing the 7e1 cps / μl concentrations of mut plasmid from the aspecific target . the level of selectivity should be less than 1 %, which is the limit shown by the other techniques in literature . with this approach we don &# 39 ; t have any clear advantage . ( fig7 ). materials , methods and results of the jak2 - modified - lamp “ self - annealing loop primer strategy ” jak2 plasmids were synthesized by the supplier geneart ( regensburg , germany ) to contain the wild type or the mutant jak2 sequence . in details : which insert corresponds to the sequence 1689 - 1722 of mn — 004972 , including a g base at nucleotide 1849 , referred to as “ wt plasmid ” which insert corresponds to the sequence 1689 - 1722 of mn — 004972 cloned , including a t base at nucleotide 1849 , referred to as “ mut plasmid ” ga231 ( f 3 ) 5 ′ gcatctttattatggcagagag 3 ′ ( seq id no . 16 ) ga232 ( b 3 ) 5 ′ tgctctgagaaaggcatta 3 ′ ( seq id no . 17 ) ga233 ( fip ) 5 ′ gctgcttcaaagaaagactaaggaaatggacaacagtcaaacaac 3 ′ ( seq id no . 18 ) ga234 ( bip ) 5 ′ gctttctcacaagcatttggttttaaattagcctgtagttttacttactctc 3 ′ ( seq id no . 19 ) ga235 ( lf ) 5 ′ gtctccactggagtatgtgtctgtggagaddc 3 ′ ( seq id no . 20 ) the underlined base is the wild type nucleotide in position 1849 of the jak2 gene . ddc stands for not - extensible dideoxy - cytosine . ga236 ( lb ) 5 ′ gtctccactggagtatgtttctgtggagac 3 ′ ( seq id no . 21 ) the underlined base is the mutant nucleotide in position 1849 of the jak2 gene . reaction buffer : 100 mm tris hcl ph 8 . 8 , 50 mm kcl , 40 mm mgso 4 , 50 mm ( nh 4 ) 2 so 4 , 0 . 5 % tween , “ buffer 5 ×” dntps mix 25 mm ( fermentas ), “ dntps ” stock the primers in aliquots . it is better to store stock solutions at − 20 ° c ., while working dilutions should be stored at 4 ° c . prepare reaction mix as follows : 0 . 2 μm outer primers ( f 3 and b 3 ), 1 . 6 μm inner primers ( fip and bip ), 0 . 8 um both self - annealed loop primers ( not - extensible lf and lb ), 1 × buffer solution , 1 . 4 mm dntps mix , 8 u bst polymerase . final volume of the reaction mix must be 4 / 5 of the total reaction volume ( i . e . 20 μl reaction mix + 5 μl sample ). always keep reagents on ice . prepare the mix for at least 26 samples , comprising 3 negative controls ( 100 % wild type plasmid , 7e3 cps / ul , 21 positive control ( 3 samples 100 % ( 7e3 cps / ul ) mutant plasmid , 3 samples 75 % mutant plasmid diluted in wt plasmid , 3 samples 50 % mutant plasmid diluted in wt plasmid , 3 samples 25 % mutant plasmid diluted in wt plasmid , 3 samples 10 % mutant plasmid diluted in wt plasmid , 3 samples 10 % mutant plasmid diluted in wt plasmid , 3 samples 5 % mutant plasmid diluted in wt plasmid , 3 samples 1 % mutant plasmid diluted in wt plasmid , and one no target control . prepare serial dilutions of the target (“ target dilutions ”) from shipped solution ( wt plasmid and mut plasmid ). shipped solution is a 7 * 10 10 copies / μl . dilute initially the mutant plasmid to a 7 * 10 4 copies / μl in tris 10 mm , then dilute serially the mutant plasmid in wt plasmid to obtain the following concentrations of mutant sequences in wild type background : 75 %, 50 %, 25 %, 10 %, 5 %, 1 % ( total amount per tube , 7e3 cps / μl ). add 5 μl of target dilutions to the strips , in triplicate . add 5 ul of the target dilutions starting from the less concentrated one to the most concentrated one . close all the tubes . the reaction follows the method scheme of fig4 . program the turbidimeter ( teramecs ) for incubation at constant temperature and real time monitoring of turbidity , in order to obtain a constant reaction temperature of 65 ° c . for 1 hour . put the strips in the instrument immediately before the beginning of the programs . start the program . analyze the variation of absorbance in terms of a . u . to find the threshold time for each sample analyzed . the threshold time is the minute at which the sample absorbance , after baseline subtraction , reaches the arbitrary unit value representing the threshold ( in this case 0 . 1 a . u .). the threshold time reached by each samples is correlated with its log of dna copies / μl . this approach consists of a selective mutant amplification based on a particular loop primer design resulting in selective hybridization of such loop primer to the dumb - bell formed from the mutant sequence ( fig4 ). we designed a universal ( mutant insensitive ) set of primers comprising f 3 , b 3 , fip and bip to obtain a dumb - bell presenting the putative mutated nucleotide in the loop region comprised between b 1 and b 2 . other experiments were performed presenting the putative mutated nucleotide in the loop region into b 2 or comprised between b 2 and b 1 c , with no relevant differences . we included in the primer set a particular loop primer presenting a 8 - bases sequence region at its 5 ′ end complementary to its own sequence in 3 ′ end . consequently , this special loop primer forms an intra - molecular hairpin structure in equilibrium with its open form at the reaction temperature ( 65 ° c .). when the mutated jak2 sequence is present , this modified loop primer breaks its internal structure to anneal to the target , thanks to the thermodynamic equilibrium ( tm between primer and specific target = 65 ° c .). the loop primer annealed to the specific mutated target is consequently extensible by the polymerase : the amplification can proceed . when the wt sequence is present in the sample , the same loop primer ( specific for the mut jak2 gene ) presents a tm with aspecific target ( 59 ° c .) lower than the intra - molecular hairpin structure ( 65 ° c .). this results in auto - sequestration of the modified loop primer that prefers to fold in the hairpin structure rather than to form a duplex with aspecific target , since the intramolecular forces are higher than the intermolecular ones . to limit the competition of the loop primer previously described for the wild type sequences likely to be present in large excess in the clinical sample , we added another modified loop primer characterized by a structure similar to the one previously described , but whit a sequence complementary to the jak2 wild type sequence ( with g base at position 1489 ). the 3 ′ end of this “ competitor ” loop primer is made not extensible by a modification ( 3 ′ dideoxy ). the task of this competitor is to “ silence ” the wt and allow the specific mutant primer to find its target . when the “ competitor ” recognizes the specific wild type sequence , it breaks its intramolecular structure to anneal to the wt target , thanks to a higher affinity ( tm duplex wt target - wt modified loop primer = 67 ° c . ); the loop primer annealed to the wt target is not extensible , resulting in no amplification of the wt sequences . since the reaction is conducted at constant temperature , the wt - loop primer will remain annealed to the wt sequences preventing aspecific annealing of the mut loop primer . differently , the “ competitor ” presents a tm with its aspecific ( mutant ) target ( 62 ° c .) lower than the intra - molecular hairpin structure that it forms with itself ( 65 ° c .). this results in auto - sequestration of the modified loop primer that prefers to fold in the hairpin structure rather than to form a duplex with the aspecific target , since the intramolecular forces are higher than the intermolecular ones . the selectivity of this assay has been evaluated , performing the reaction on serial dilutions of mutant plasmid in wild type background ( fig8 ). the selectivity achieved is significantly less than 1 % ( 350 copies tot mutant plasmid in 34650 copies of wt plasmid ). this approach has higher selectivity than the assays described in literature . the assay is linear between 100 % mutant ( 35000 cps ) and 1 % mutant in 99 % wild type ( 350 copies tot mutant plasmid in 34650 copies of wt plasmid ). it allows detection and quantification of low percentage of mutant sequences in large amount of wt . it represents an improvement in respect of the other approaches shown in the previous slides and in respect to the methods described in literature . materials , methods and results of the jak2 - modified - lamp “ self - annealing loop primer strategy with pna ” jak2 plasmids were synthesized by the supplier geneart ( regensburg , germany ) to contain the wild type or the mutant jak2 sequence . in details : which insert corresponds to the sequence 1689 - 1722 of mn — 004972 , including a g base at nucleotide 1849 , referred to as “ wt plasmid ” which insert corresponds to the sequence 1689 - 1722 of mn — 004972 cloned , including a t base at nucleotide1849 , referred to as “ mut plasmid ” the underlined base corresponds to the mutated nucleotide at position 1849 of the jak2 gene . pna : eurogentec , referred to as “ pna ” gm43 nh2 gagtatgt g tctgtgga conh2 the underlined base corresponds to the wild type nucleotide at position 1849 of the jak2 gene . reaction buffer : 100 mm tris hcl ph 8 . 8 , 50 mm kcl , 40 mm mgso4 , 50 mm ( nh4 ) 2so4 , 0 . 5 % tween , “ buffer 5 ×” stock the primers in aliquots . it is better to store stock solutions at − 20 ° c ., while working dilutions should be stored at 4 ° c . prepare reaction mix as follows : 0 . 2 μm outer primers ( f 3 and b 3 ), 1 . 6 μm inner primers ( fip and bip ), 0 . 8 um self - annealed loop primer specific for mutant jak2 ( lb ), 0 . 8 um pna , 1 × buffer solution , 1 . 4 mm dntps mix , 8 u bst polymerase . final volume of the reaction mix must be 4 / 5 of the total reaction volume ( i . e . 20 μl reaction mix + 5 μl sample ). always keep reagents on ice . prepare the mix for at least 23 samples , comprising 3 negative controls ( 100 % wild type plasmid , 7e4 cps / μl ), 18 positive control ( 3 samples 100 % mutant plasmid , 3 samples 1 % mutant plasmid diluted in wt plasmid , 3 samples 0 . 5 % mutant plasmid diluted in wt plasmid , 3 samples 0 . 1 % mutant plasmid diluted in wt plasmid , 3 samples 0 . 05 % mutant plasmid diluted in wt plasmid , 3 samples 0 . 01 % mutant plasmid diluted in wt plasmid ( total amount of dna 7e4 cps / μl ), and one no target control . prepare serial dilutions of the target (“ target dilutions ”) from shipped solution ( wt plasmid and mut plasmid ). shipped solution is a 7 * 10 10 copies / μl . dilute initially the mutant plasmid to a 7 * 10 4 copies / μl in tris 10 mm , then dilute serially the mutant plasmid in wt plasmid to obtain the following concentrations of mutant sequences in wild type background : 1 %, 0 . 5 %, 0 . 1 %, 0 . 05 %, 0 . 01 % ( total amount per tube , 7e4 cps / ul ). add 5 μl of target dilutions to the strips , in triplicate . add 5 ul of the target dilutions starting from the less concentrated one to the most concentrated one . close all the tubes . the reaction follows the method scheme of fig5 . program the turbidimeter ( teramecs ) for incubation at constant temperature and real time monitoring of turbidity in order to obtain a constant reaction temperature of 65 ° c . for 1 hour . put the strips in the instrument immediately before the beginning of the programs . start the program . analyze the variation of absorbance in terms of a . u . to find the threshold time for each sample analyzed . the threshold time is the minute at which the sample absorbance , after baseline subtraction , reaches the arbitrary unit value representing the threshold ( in this case 0 . 1 a . u .). the threshold time reached by each samples is correlated with its log of dna copies / μl . this approach consists of a selective mutant amplification based on a particular loop primer design resulting in selective hybridization of such loop primer to the dumb - bell formed from the mutant sequence ( fig5 ). we designed a universal ( mutant insensitive ) set of primers comprising f 3 , b 3 , fip and bip to obtain a dumb - bell presenting the putative mutated nucleotide in the loop region comprised between b 1 and b 2 . other experiments were performed presenting the putative mutated nucleotide in the loop region into b 2 or comprised between b 2 and b 1 c , with no relevant differences . we included in the primer set a particular loop primer presenting a 8 - bases sequence region at its 5 ′ end complementary to its own sequence in 3 ′ end . consequently , this special loop primer forms an intra - molecular hairpin structure in equilibrium with its open form at the reaction temperature ( 65 ° c .). when the mutated jak2 sequence is present , this modified loop primer breaks its internal structure to anneal to the target , thanks to the thermodynamic equilibrium ( tm between primer and specific target = 65 ° c .). the loop primer annealed to the specific mutated target is consequently extensible by the polymerase : the amplification can proceed . when the wt sequence is present in the sample , the same loop primer ( specific for the mut jak2 gene ) presents a tm with aspecific target ( 59 ° c .) lower than the intra - molecular hairpin structure ( 65 ° c .). this results in auto - sequestration of the modified loop primer that prefers to fold in the hairpin structure rather than to form a duplex with aspecific target , since the intramolecular forces are higher than the intermolecular ones . to further increase the discrimination capability of the lamp system based on selective self - annealed loop primer , we added to the reaction mix a peptide nucleic acid ( pna ). pnas are non - extensible and not - displaceable oligonucleotides where the ribose - phosphate backbone is replaced by ( 2 - aminoethyl )- glycine units linked by amide bonds . each base pairing dna / pna contributes to the stability of the duplex structure more than a regular base pairing dna / dna . therefore a single mismatch in a pna / dna duplex results in a significant difference in tm . a pna probe fully complementary to the wt sequence of the jak2 gene prevents annealing and extension of the mutant self - annealed primer , suppressing amplification . in presence of a single mismatch , pna does not inhibit loop primer hybridization , which leads to amplification . therefore pna can be used to selectively block the wt sequence present in the sample . the pna is designed to be complementary to the loop region comprised between b 2 and b 1 c presenting the wt nucleotide . it forms a stable duplex only with the wt complementary sequence ( tm 65 . 7 ° c . ), preventing the annealing and extension of the mut - self - annealed loop primer and therefore suppressing the amplification . it does not anneal to the mut jak2 sequence thanks to the lower affinity ( tm = 56 ° c .). the pna principle has been tested performing the reaction on 7 e3 cps / ul wild type plasmid and on 7 e3 cps / ul mutant plasmid , in parallel in absence and presence of the pna “ wt blocker ” probe ( fig9 ). in absence of pna the wt plasmid is aspecifically amplified by the self - annealed mutant loop primer , with a delay of 5 min in respect to the specific mutated target . in presence of pna , the wt plasmid is not amplified within one hour reaction by the self - annealed mutant loop primer . the amplification of the mutant plasmid is delayed of about 5 minutes . the pna forms a stable duplex only with the wt complementary sequence preventing the annealing and extension of the mut - self - annealed loop primer and therefore suppressing the amplification within 1 hour of reaction . the selectivity of this assay has been evaluated , performing the reaction on serial dilutions of mutant plasmid in wild type background ( fig1 ). the selectivity achieved is less than 0 . 01 % ( 35 copies tot mutant plasmid in 34965 copies of wt plasmid ). this approach has higher selectivity than the assays described in prior art , about 3 logs more in respect to direct sequencing , rflp and pyrosequencing and about 2 logs in respect to arms , real - time techniques and dna - melting curve analysis . as to lamp dumb - bell method , it is intrinsically not useful to detect single point mutations in a high background of wild type sequences . the wt sample ( 35000 cps wt plasmid ) is not amplified in one hour reaction . the specific mutant target is detected down to 0 . 01 % mutant sequences in wt ( 35 copies tot mutant plasmid in 34965 copies of wt plasmid ). this approach has a higher selectivity than the assays described in literature ( about 2 logs ). the assay is linear down to 0 . 1 % mut ( 350 copies tot mutant plasmid in 34650 copies of wt plasmid ). it allows detection and quantification of low percentage of mutant sequences in large amount of wt . it represents a further improvement in respect of the other strategies described in this report and in respect to the methods described in literature . lamp “ self - annealed loop primer with pna ” on clinical samples : comparison with arms 29 samples of dna extracted from patients at ospedali riuniti di bergamo were analyzed using jak2 lamp “ self - annealed loop primer with pna ” strategy , as described in the example 4 . the results obtained have been compared with the ones obtained at the hospital using the arms technology . the arms exploits the fact that oligonucleotide primers must be perfectly annealed at their 3 ′ ends for a dna polymerase to extend these primers during pcr . by designing oligonucleotide primers that match only the specific jak2 point mutation arms can distinguish between wild type and mutant alleles . as shown in table 5 , all the samples diagnosed as positive by arms have been detected as positive by lamp . out of 15 samples resulted negative by arms , 11 have been diagnosed as negative by lamp and 4 as low positive . to exclude that the 4 discordant samples resulted mutated by lamp were false positive and to confirm that the mutation diagnosis was due to an higher selectivity of the modified - lamp method , we tested the samples using a third assay . the assay consists in pcr amplification of the jak2 region of interest in presence of the pna molecule complementary to the wild type target . the purpose is to enrich the mutated base , if present , by suppression of the wild type via pna clamping . if the mutated region is enriched to a level of 20 % of the sample , it can be detected by the direct - sequencing . the primers ( ga231 forward and ga232 reverse ) and the pna are the same described above ( paragraph “ example 4 ”). the amplification was performed in presence of 1 × reaction buffer , 2 . 5 mm mgcl 2 , 200 μm dntps , 500 nm forward and reverse primers , 1 . 5m pna and 0 . 025 u taq gold in a final volume of 45 μl . 5 μl of target 20 ng / μl was added to the reaction mix . the resulting solution was incubated in a thermocycler , following a thermal program consisting in 10 min at 95 ° c . followed by 35 cycles of 30 sec at 94 ° c ., 40 sec at 62 ° c . cycles , 30 sec at 58 ° c . and 30 sec at 72 ° c . and finishing with 10 min at 72 ° c . for the final extension . the four discordant clinical samples , one no - target control sample and a positive and negative plasmid target were tested in duplicate . the resulting amplification products were separated on an agarose gel containing etbr to visualize the amplification bands . the no - target control was not amplified . the negative control containing the wild type plasmid was slightly amplified , and a weak band was visible on the agarose gel . the positive control containing the mutated plasmid was strongly amplified presenting a strong band on the agarose gel . the clinical samples were amplified . the amplification products were consequently analyzed via automatic - sequencing . all the discordant clinical samples show a double peak in position 1849 , corresponding to the guanine ( wild type ) base and the thymine ( mutated ) base . this result confirm that the four discordant samples have been correctly diagnosed mutated by lamp , while they results false negative by arms . which insert corresponds to the sequence 1689 - 1722 of mn — 004972 , including a g base at nucleotide1849 , referred to as “ wt plasmid ” which insert corresponds to the sequence 1689 - 1722 of mn — 004972 cloned , including a t base at nucleotide1849 , referred to as “ mut plasmid ” ga231 ( f 3 ) 5 ′ gcatctttattatggcagagag 3 ′ ( seq id no . 16 ) ga232 ( b 3 ) 5 ′ tgctctgagaaaggcatta 3 ′ ( seq id no . 17 ) ga233 ( fip ) 5 ′ gctgcttcaaagaaagactaaggaaatggacaacagtcaaacaac 3 ′ ( seq id no . 18 ) ga234 ( bip ) 5 ′ gctttctcacaagcatttggttttaaattagcctgtagttttacttactctc 3 ′ ( seq id no . 19 ) ga236 ( lb ) 5 ′ tamra - tgtctccactggagtatgtttctgtggagac 3 ′ ( seq id no . 21 ). the underlined base corresponds to the mutated nucleotide at position 1849 of the jak2 gene . the bold tymine base at 5 ′ end is not complementary to the target sequence . it has been added to separe the fluorophore from the guanine base downstream , which has a quenching effect . ga235 ( lf ) 5 ′- 5 ′ gtctccactggagtatgtgtctgtggagaddc 3 ′( seq id no . 20 ) the underlined base is the wild type nucleotide in position 1849 of the jak2 gene . ddc stands for not - extensible dideoxy - citosine . reaction buffer : 100 mm tris hci ph 8 . 8 , 50 mm kcl , 40 mm mgso4 , 50 mm ( nh4 ) 2so4 , 0 . 5 % tween , “ buffer 5 ×” stock the primers in aliquots . it is better to store stock solutions at − 20 ° c ., while working dilutions should be stored at 4 ° c . prepare reaction mix as follows : 0 . 2 μm outer primers ( f 3 and b 3 ), 1 . 6 μm inner primers ( fip and bip ), 0 . 8 μm fluorescent self - annealed loop primer specific for mutant jak2 ( lb ), 0 . 8 μm self annealed not - extensible loop primer for wild type jak2 ( lf ), 1 × buffer solution , 1 . 4 mm dntps mix , 8 u bst polymerase . final volume of the reaction mix must be 4 / 5 of the total reaction volume ( i . e . 20 μl reaction mix + 5 μl sample ). always keep reagents on ice . prepare the mix for at least 23 samples , comprising 3 negative controls ( 100 % wild type plasmid , 7e3 cps / μl ), 18 positive control ( 3 samples 100 % mutant plasmid , 3 samples 1 % mutant plasmid diluted in wt plasmid , 3 samples 0 . 5 % mutant plasmid diluted in wt plasmid , 3 samples 0 . 1 % mutant plasmid diluted in wt plasmid , 3 samples 0 . 05 % mutant plasmid diluted in wt plasmid , 3 samples 0 . 01 % mutant plasmid diluted in wt plasmid ( total amount of dna 7e3 cps / μl ), and one no target control . prepare serial dilutions of the target (“ target dilutions ”) from shipped solution ( wt plasmid and mut plasmid ). shipped solution is a 7 * 10 10 copies / μl . dilute initially the mutant plasmid to a 7 * 10 4 copies / μl in tris 10 mm , then dilute serially the mutant plasmid in wt plasmid to obtain the following concentrations of mutant sequences in wild type background : 1 %, 0 . 5 %, 0 . 1 %, 0 . 05 %, 0 . 01 % ( total amount per tube , 7e3 cps / ul . add 5 μl of target dilutions to the strips , in triplicate . add 5 μl of the target dilutions starting from the less concentrated one to the most concentrated one . close all the tubes . the reaction follows the method scheme of fig4 . program the real time instrument for incubation at constant temperature in order to obtain a constant reaction temperature of 65 ° c . for 1 hour . program the real time instrument in order to obtain a fluorescence reading per minute . put the strips in the instrument immediately before the beginning of the programs . start the program . the fluorescent self - annealed loop primer in reaction produces a fluorescent signal once it is excited by an appropriated wavelenght - light emission . when the lamp reaction proceeds , the fluorescent self - annealed loop primer is incorporated in the amplification products , being consequently annealed to a complementary nucleotide sequence . the fluorescent self - annealed loop primer is designed to be complementary to a sequence containing at least one guanine nucleotide close to its 5 ′ end . the guanine base can absorbe the wavelengh emitted by the fluorophore ( tamra in our case ), causing a fluorescent signal quenching . the lamp reaction can be consequently detected by analysis of the variation of fluorescence in terms of signal quenching , to find the threshold time relative to each analyzed sample . the threshold time is the minute at which the fluorescence signal in reaction reaches 50 % of quenching . the threshold time reached by each samples is correlated with its log of dna copies / μl . this approach consists of a selective mutant amplification based on a particular loop primer design resulting in selective hybridization of such loop primer to the dumb - bell formed from the mutant sequence ( fig4 ). we designed a universal ( mutant insensitive ) set of primers comprising f 3 , b 3 , fip and bip to obtain a dumb - bell presenting the putative mutated nucleotide in the loop region comprised between b 1 and b 2 . other experiments were performed presenting the putative mutated nucleotide in the loop region into b 2 or comprised between b 2 and b 1 c , with no relevant differences . we included in the primer set a particular loop primer presenting a 8 - bases sequence region at its 5 ′ end complementary to its own sequence in 3 ′ end . consequently , this special loop primer forms an intra - molecular hairpin structure in equilibrium with its open form at the reaction temperature ( 65 ° c .). when the mutated jak2 sequence is present , this modified loop primer breaks its internal structure to anneal to the target , thanks to the thermodynamic equilibrium ( tm between primer and specific target = 65 ° c .). the loop primer annealed to the specific mutated target is consequently extensible by the polymerase : the amplification can proceed . when the wt sequence is present in the sample , the same loop primer ( specific for the mut jak2 gene ) presents a tm with aspecific target ( 59 ° c .) lower than the intra - molecular hairpin structure ( 65 ° c .). this results in auto - sequestration of the modified loop primer that prefers to fold in the hairpin structure rather than to form a duplex with aspecific target , since the intramolecular forces are higher than the intermolecular ones . to limit the competition of the loop primer previously described for the wild type sequences likely to be present in large excess in the clinical sample , we added another modified loop primer characterized by a structure similar to the one previously described , but whit a sequence complementary to the jak2 wild type sequence ( with g base at position 1489 ). the 3 ′ end of this “ competitor ” loop primer is made not extensible by a modification ( 3 ′ dideoxy ). the task of this competitor is to “ silence ” the wt and allow the specific mutant primer to find its target . when the “ competitor ” recognizes the specific wild type sequence , it breaks its intramolecular structure to anneal to the wt target , thanks to a higher affinity ( tm duplex wt target - wt modified loop primer = 67 ° c . ); the loop primer annealed to the wt target is not extensible , resulting in no amplification of the wt sequences . since the reaction is conducted at constant temperature , the wt - loop primer will remain annealed to the wt sequences preventing aspecific annealing of the mut loop primer . differently , the “ competitor ” presents a tm with its aspecific ( mutant ) target ( 62 ° c .) lower than the intra - molecular hairpin structure that it forms with itself ( 65 ° c .). this results in auto - sequestration of the modified loop primer that prefers to fold in the hairpin structure rather than to form a duplex with the aspecific target , since the intramolecular forces are higher than the intermolecular ones . to follow the reaction on a real - time instrument , we labeled the 5 ′ end of the mutant modified loop primer with a fam dye . to avoid the binding of the fluorophore to the guanine base present at the 5 ′ end of the modified loop primer , which has a quenching effect , we added a thymine base to the extremity of the probe . the modified - labeled primer , when present in solution , emits a fluorescent signal if excited by an appropriate - wavelength light . when the lamp reaction starts and proceeds , the fluorescent self - annealed loop primer is incorporated in the amplification products , being consequently annealed to a complementary nucleotide sequence , containing several guanine residues . the guanine bases can absorb the wavelength emitted by the fluorophore , causing a fluorescent signal quenching visible in real time . the lamp reaction can be consequently monitored throughout the analysis of the decreasing of fluorescence signal due to the “ quenching effect ” determined by lamp product generation . 1 . levine , r . l . et al . activating mutation in the tyrosine kinase jak2 in polycythemia vera , essential thrombocythemia , and myeloid metaplasia with myelofibrosis . cancer cell 7 , 387 - 397 ( 2005 ). 2 . james , c . et al . a unique clonal jak2 mutation leading to constitutive signalling causes polycythaemia vera . nature 434 , 1144 - 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