Patent Application: US-51320606-A

Abstract:
the osmoprotectants proline , 2 - methyl - 4 - carboxy - 3 , 4 , 5 , 6 - tetrahydropyrimidine ”, and 2 - methyl - 4 - carboxy - 5 - hydroxy - 3 , 4 , 5 , 6 ,- tetrahydropyrimidine ”) are capable of increasing the thermal stability of dna polymerases at elevated temperatures . thp is further effective in lowering the melting temperature of double - stranded dna . proline , thp and thp are thus useful in procedures involving melting of double - stranded dna and / or polymerase - mediated dna synthesis , such as in primer extension , in pcr amplification and in dna sequencing .

Description:
the term “ primer extension ” as used herein in the specification refers to a process of increasing the length of an oligonucleotide complementary to a nucleotide sequence comprised within a template dna . the process consists of repeatedly adding to the oligonucleotide &# 39 ; s 3 ′- end a single nucleotide which is dictated by the nucleotide present at the corresponding position in the complementary template dna strand . the term “ cycled primer extension ” refers to a procedure which involves repeated cycles in which primer extension is alternated with periodic heating whereby separation of the extended primer from the template dna strand occurs . the term “ melting temperature ( tm )” of double - stranded dna ( dsdna ) refers to a temperature at which 50 % of a dsdna sample is separated into its two complementary dna strands . the term “ amplifying ” refers to repeated copying of a specified sequence of nucleotides resulting in an increase in the amount of said specified sequence of nucleotides . the term “ sequencing ” refers to a procedure for determining the order in which nucleotides occur in a target nucleotide sequence . the term “ target nucleotide sequence ” refers to a nucleotide sequence which is intended to be duplicated , amplified or sequenced . the term “ template dna ” refers to dna molecules or fragments thereof of any source or nucleotide composition , that comprise a target nucleotide sequence as defined above . according to the present invention , thp ( b ) or proline or mixtures thereof can significantly lower the melting temperature of dsdna , and proline , thp ( b ) or thp ( a ), alone or in combination , increase the stability of dna polymerases incubated at elevated temperatures . thp ( b ) and thp ( a ) for use in the invention can be isolated from natural sources such as , for example , from actinomycin d - producing microorganisms of the streptomyces species , e . g . s . parvulus , s . chrismomalus , or s . antibioticus , and separated in purified form as described in il patent no . 100810 and corresponding u . s . pat . no . 5 , 789 , 414 and ep 0553884 . thp ( b ) alone can be isolated and purified from halophilic and halotolerant bacteria such as bacteria of the genus ectothiorhodospira , e . g . e . halochloris , e . halophila and mutants thereof or from heterotrophic halophilic eubacteria of the family halomonadaceae grown in high salinity conditions . thp ( a ) alone can be isolated and purified from soil microorganisms of the streptomyces species , e . g . s . clavuligerus , s . griseus and mutants thereof , under low salt stress such as 0 . 25 - 0 . 5m nacl . thp ( b ) can also be synthetically produced as described in japanese patent application no . 63 - 259827 . l - proline is a common amino acid that is commercially available or can be synthetically produced and obtained in highly purified form . according to the invention , thp ( b ) was found to decrease the tm of oligonucleotides as short as 6 - 8 mers and of dsdnas being either genomic dnas , cdnas or recombinant dna molecules , in a concentration dependent manner in concentrations ranging from 0 . 5 to 4m . the melting temperature of short oligonucleotides ( 6 or 8 mers ) were reduced by 3 to 6 ° c . in the presence of 0 . 5m and 1 . 0 m thp ( b ). the magnitude of the tm decrease depends on the gc content of the particular oligonucleotide or dsdna , being more pronounced with high gc content dnas . for example , the tm decrease of calf thymus dna ( 42 % gc ) and of micrococcus lysodeikticus dna ( 72 % gc content ) in the presence of thp ( b ) was significantly higher than that of clostridium perfringens dna ( 26 % gc content ), while no change in the tm of the synthetic oligonucleotide poly ( da - dt ) could be observed in the presence of thp ( b ) at concentrations as high as 4m . at 4 . 0 m concentration of thp ( b ), dnas with different gc content melt in a very narrow temperature range ( 40 - 43 ° c . ), while in the absence of thp ( b ) the melting temperature ranges from 39 to 75 ° c . isostabilization of the dna molecule by thp ( b ) may be explained as a result of greater destabilization of gc - rich than at - rich dnas . thp ( b ) eliminates the dnas base pair composition - dependence on dna melting . proline , known to decrease dna melting temperature ( rajendrakumar et al ., 1997 ), was found according to the invention to only slightly decrease klenow polymerase enzymatic activity and to be a better stabilizer of klenow polymerase than glycerol , with a half - life of the enzyme of 21 min at 65 ° c . in 5 m l - proline . these findings have enabled a successful design of a pcr protocol for a rather gc - rich genomic dna template . the amount of klenow polymerase in the herein presented protocol ( 10 - 15 units ) can be further reduced when 7 - deaza - dgtp is used instead of dgtp , due to the expected decrease of denaturation temperature . the results herein reveal that proline concentration in the range of 3 - 5 . 5 m is sufficient to confer stability to klenow polymerase . proline can be used as a sole additive in the protocol or in combination with glycerol or any other dna - destabilizing agent which the polymerase tolerates , such as thp ( b ) or thp ( a ). proline ( up to 5 . 0 m ) decreases the melting temperature ( tm ) of various dnas and leads to dna partial “ isostabilization ” ( a decrease of tm difference between gc and at pairs , manifested by an apparent linear decrease of dtm / dgc factor ( melchior et al ., 1973 ; rees et al ., 1993 ), while at higher concentrations , proline destabilizes gc and at pairs evenly . a complete “ isostabilation ” of dna , as in the case of betane ( rees et al ., 1993 ) thp ( b ) ( equal stability of at and gc pairs , dtm / dgc = 0 ), was not reached for proline . the tm values of the tested natural dnas ( 57 - 78 ° c .) decreased to a narrow range of 28 - 32 ° c . in the presence of 6 . 2 m prolione . the partial “ isostabilization ” of dna by proline at high concentration may cause low specificity of pcr , when 20 - 25 b . p . primers are used . primers of 30 - 35 b . p . length , used in the herein presented pcr protocol , were found to be effective to remedy the decreased priming specificity at high concentrations of proline , and to achieve a good selectivity of amplification . besides standard pcr and dna sequencing , the protocol with proline can be interesting for the following methods : a ) use of klenow polymerase in combination with contiguous hexamer primers and single - stranded dna binding protein for a specific primer formation ( kieleczawa et al ., 1992 ) utilizing a rather low amount of a source dna ; b ) low denaturation temperature cycling might enable usage of less thermostable labels for dna sequencing or pcr . this approach might be useful for other thermolabile polymerases in pcr and other dna amplification methods . for example , t7 dna polymerase and its modifications , able to amplify gc - rich dna and regions with stable secondary structures , could provide solutions to the cases still remaining beyond today &# 39 ; s practical pcr and dna sequencing capabilities , such as amplification of long cgg triplet repeat sequences . introduction of t4 polymerase to cycled pcr might be of interest for the cases requiring high fidelity , e . g . for amplification of sequences present at a very low frequency requiring many cycles of amplification to be detected . according to the invention it was further found that proline , thp ( b ) and thp ( a ), alone or in combination , can stabilize both thermostable and non - thermostable dna polymerases incubated at elevated temperatures , the stabilizing effect being more pronounced when the enzyme is incubated for prolonged periods of time and at a higher temperature than the temperature of their optimal activities . the thermostable taq polymerase , after 30 - 35 min incubation under typical dna denaturation temperature at 95 ° c ., is only 50 % active , and after 30 min incubation at 97 ° c ., only 10 % active in comparison to 40 % in the presence of 1m thp ( b ) and even higher , 55 %, in the presence of thp ( a ). a much more dramatic effect is obtained at longer incubation time ( 60 min ), where the remaining activity is less than 5 % without additives and is 55 % in the presence of thp ( a ) ( by 10 - fold higher ). the non - thermostable polymerases are much more sensitive to thermal inactivation , for example , the half life of klenow dna polymerase is around 30 - 50 seconds at 65 ° c ., whereas in the presence of 5m proline it is 25 min , about 30 - 50 fold longer . in preferred embodiments , cycled primer extension of any template dna is conducted with the thermostable taq polymerase at 60 - 80 ° c . in the presence of 0 . 5 - 3 . 5m thp ( b ), optionally with 0 . 5 - 3 . 0m thp ( a ), or 1 - 5m proline , or with a non - thermostable polymerase at 30 - 65 ° c . in the presence of 1 - 3 m thp ( b ), optionally with 0 . 5 - 3 . 0m thp ( a ), or 1 - 5m proline . lowering the tm of dsdna by proline and / or thp ( b ) and stabilization of dna polymerases by proline , thp ( b ) and / or thp ( a ) are beneficial for cycled primer extension procedures that comprise steps of dna melting and of polymerase - mediated dna synthesis , such as dna sequencing and pcr procedures , leading to high yields of dsdna denaturation , namely separation of dsdna into its two complementary strands at a lower temperature , and high performance of dna polymerases . the concentration of the osmoprotectant to be used in a particular cycled primer extension reaction depends on the specific template dna , the primer ( s ), the dna polymerase and the reaction conditions employed . low concentrations of thp ( b ) or proline , typically around 0 . 5 - 1 . 5m , are preferred for lowering tm of an average gc - content dna , while higher concentrations , typically 1 - 3m , are preferred for high gc - content dna , so to further lower the tm and hence the denaturing temperature employed . to avoid major dissociation of primer / template dna , when high concentrations of thp ( b ) ( 3 - 4m ) and proline ( 4 - 5m ) are used to lower dna tm to the range of 40 - 55 ° c ., primers of at least 30 nucleotides are used . these modifications improve annealing and yield of the reaction . the use of non - thermostable dna polymerases such as t7 dna polymerase or klenow is of major importance in cases where accuracy of dna amplification is crucial such as in detection of subtle changes in a dna sequence and in processes of pcr typing and diagnosis of some genetic diseases and cancer caused by minor mutations , due to their high fidelity in dna replication and proofreading ability . performing primer extension reactions at reduced temperatures also permits the use of thermosensitive fluorescent and other labile compounds for labeling newly synthesized dna strands for use as probes in the detection of complementary target sequences of nucleotides by sensitive assays such as , chemiluminescence detection . reaction conditions used in pcr are variable depending on the nature of the template dna and primers , and optimal ph and salt and magnesium ions concentrations are usually determined empirically for each particular reaction . a typical pcr procedure involves temperature cycling to provide adequate conditions for accomplishing three steps in each pcr cycle : ( i ) dna denaturation ; ( ii ) primer annealing ; and ( iii ) primer extension . a standard denaturation incubation step ( i ) at 94 - 95 ° c . for 0 . 5 - 2 min is usually sufficient for separating dna strands of an average gc content from the original and newly synthesized dna . the primer annealing step ( ii ) is performed usually around 5 ° c . lower than the melting temperature of the primer - template dna duplex . however , if non - specific pcr products are obtained in addition to the expected product , the annealing temperature should be increased . the extension ( step iii ) of the annealed primer at its 3 ′ end to synthesize a new dna strand , complementary to the template strand , is usually carried out by the thermostable enzyme taq polymerase at 70 - 75 ° c ., which is the optimal temperature range for the enzyme activity (˜ 2 - 4 kb / min .). the complete denaturation of the dna template , especially at the first amplification cycles , is of most importance in pcr procedures , otherwise its use as a template for the following reaction steps decreases and results in poor yield of the pcr product . this is especially relevant when an amplified dna duplex has a very high gc content , rendering it difficult in strand separation , or when a target nucleotide sequence is present in a minute amount in the initial reaction mixture . thus , pcr buffers containing solutes leading to significant lower tms of the dna templates are most important in pcr procedures . the addition of proline , thp ( b ), thp ( a ) or mixtures thereof to pcr procedures is beneficial in three levels : ( i ) increased yield of the amplified dna products ; ( ii ) increased sensitivity ; and ( iii ) increased specificity of the reaction . the effect of proline and thp ( b ) in decreasing tms of oligonucleotide primers and of template dnas , and the effect of proline , thp ( b ) and thp ( a ) in stabilizing dna polymerases , result in more efficient use of the template dnas , primers and enzymes of the reaction , leading to high yield of pcr - amplified dna product . moreover , the increased sensitivity of pcr assays in the presence of the additive enables detection of target dna sequences that are not detectable in its absence . this is especially significant in cases where very rare or long target sequences are to be amplified . in addition , the additives also improve the quality of pcr amplification by reducing significantly or eliminating nonspecific products . the improved accuracy of pcr in the presence of proline , thp ( b ) and / or thp ( a ) enables performing pcr protocols with increased number of cycles and longer cycle times , without impairing the quality of the reaction products . in another embodiment , the invention provides a method for cycle dna sequencing comprising contacting a template dna with a primer homologous to a specific sequence on a target dna in the presence of a dna polymerase and an effective amount of proline , thp ( b ) and / or thp ( a ) under conditions allowing dna sequencing . a commonly used cycle dna sequencing protocol known as sanger or dideoxy sequencing method , typically includes isolating double stranded template dna , separating it into its component single strands , adding a sequencing primer homologous to a sequence of nucleotides on the target dna and performing a cycled primer extension of said primer on the target dna . the cycled primer extension is performed in four paralleled reactions , each including a small amount of a dideoxynucleotide triphosphate , either ddatp , ddctp , ddgtp or ddttp , along with a molar excess of the four deoxynucleotide triphosphates ( dntps ) normally required for dna synthesis , i . e . datp , dctp , dgtp or dttp . the growth of the extended dna chain is stopped once a ddntp molecule is incorporated into it , thus generating series of extension products of various lengths . when these extension products of the four extension reactions are separated side by side , for example on a polyacrylamide gel , a pattern is obtained . by using a labeled primer or labeled ddntp , typically radioactive or fluorescent , this pattern can be monitored , for example , by autoradiography , fluorescence detectors etc , and the dna sequence can be determined . cycle dna sequencing also involves cycle primer extension , thus the sequencing outcome is influenced by similar criteria as mentioned above for pcr . the degree of template dna and primer denaturation , as well as the polymerase performance , are of crucial importance for the sensitivity and accuracy of a sequencing reaction . the exact reaction conditions for performing a cycle dna sequencing method and the effective concentrations of the added osmoprotectant vary depending on the template dna , primers , target dna to be sequenced and the dna polymerase used in a particular reaction . cycle sequencing performed in accordance with the invention in the presence of proline , thp ( b ) and / or thp ( a ), is a beneficial and sensitive tool . the osmoprotectant additive permits obtaining a sequence of a longer stretch of nucleotides in a single reaction , as well as to sequence minute amounts of dna present , for example , in limited samples of blood or tissue used in forensic pathology and in evolutionary biology . in addition , some gc - rich dnas or other dnas with complex or rigid secondary structure that are very difficult to sequence using conventional reaction mixtures , can thus be successfully sequenced . since in the presence of the additives the specificity of primer annealing is increased and non - specific extended products are mostly eliminated , detection of rare mutations becomes feasible . this is especially important in diagnosis of diseases characterized by a small mutation in a gene nucleotide sequence or in identification of high cgg repeats that are indicative of many human disorders , such as huntington &# 39 ; s disease ( han et al ., 1994 ). the kits for performing dna amplification by pcr or for dna cycle sequencing of the invention include , respectively , the reagents necessary for pcr or dna sequencing ( e . g . appropriate buffers , dntps , either taq or a non - thermostable polymerase , etc .) and , in separate containers , thp ( b ) optionally with thp ( a ) or proline . for examples 1 - 4 , thp ( a ) and thp ( b ) were prepared according to malin and lapidot ( 1996 ) and their water solutions were passed through a chelex column to remove divalent cations before use . betaine ( sigma ) was dissolved in water and passed through a chelex column before use . taq dna polymerase ( recombinant ) and klenow fragment of dna polymerase i ( 10 units / μl ) were purchased from mbi fermentas , calf thymus dna ( used in the dna melting examples ) and activated calf thymus dna ( used in the polymerase activity assays ), micrococcus lysodeikticus dna , colstridium perfringens dna and poly ( da - dt ) from sigma . the oligonucleotides [ d ( atgcat )] 2 ( seq id no : 1 ) and [ d ( gcttaagc )] 2 ( seq id no : 2 ) and the following 28 - mer primers 1 and 2 were prepared by solid - phase phosphouramidate synthesis : 1 . 5 ′& gt ; cgg gat cca tgg aat acg tat acg ctg c & lt ; 3 ′ 2 . 5 ′& gt ; cgg aat tct tag ccg aag agt tcg ccg a & lt ; 3 ′ for examples 5 - 9 , l - proline 99 +% and 99 . 5 +% were purchased from sigma and from fluka , respectively , and glycerol was from bdh . activated calf thymus dna and calf thymus dna was from sigma . taq dna polymerase ( recombinant ) and klenow fragment of dna polymerase i ( 10 units / μl ) were purchased from mbi fermentas , klentaql dna polymerase from ab peptides and pfu dna polymerase ( cloned ) from stratagene . halobacterium marismortui genomic dna template was a generous gift of dr . shulamith weinstein ( kimmelman laboratory of biocrystallization , weizmann institute of science ). two pairs of primers were used in examples 7 - 9 : two 28 - mer primers 3 and 4 with 22 of complementary nucleotides each and with end restriction site : primer 3 containing banihi restriction site and primer 4 containing ecori restriction site , and two 30 - mer primers 5 and 6 with all 30 complementary nucleotides : ( seq id no : 3 ) 3 . 5 ′& gt ; cgg gat cca tgg aat acg tat acg ctg c & lt ; 3 ′ ( seq id no : 4 ) 4 . 5 ′& gt ; cgg aat tct tag ccg aag agt tcg ccg a & lt ; 3 ′ ( seq id no : 5 ) 5 . 5 ′& gt ; atg gaa tac gta tac gct gca ctc atc ctg & lt ; 3 ′ ( seq id no : 6 ) 6 . 5 ′& gt ; tta gcc gaa gag ttc gcc gag gcc ctc acc & lt ; 3 ′ all oligonucleotides and primers for examples 1 - 9 were prepared by the chemical service unit of the weizmann institute of science , rehovot , israel , and their solution concentrations were determined by uv absorbance at 260 mn . dna melting studies were conducted in a buffer ( 1 ml ) containing 5 . 0 mm k 2 hpo 4 and 0 . 1 mm na 2 edta at ph 7 . 5 . the buffer and thp ( b ) or proline solutions were filtered through 0 . 22 μm millipore membrane filter , prior to addition of the dna , and then degassed with helium at room temperature . dna samples were adjusted to o . d 260 = 0 . 2 and incubated overnight at 37 ° c . before use , as previously described ( rees et al ., 1993 ). dnas in the above buffer with and without thp ( b ) or proline were measured in 1 - cm path teflon - stoppered quartz cell and incubated at the initial assay temperature for 5 min . the increase in absorbance at 260 nm was monitored in hewlett packard 9450a diode array spectrophotometer attached to a temperature programmer and controller . both the sample and the reference cells were heated together at a rate of 0 . 5 ° c ./ min and the net absorbance was recorded after every 0 . 5 ° c . increase . the tms were determined graphically from the midpoints of the absorbance versus temperature profile . 1 h nmr measurements were carried out on a bruker amx 400 nmr mhz spectrometer at 400 . 13 mhz ( equipped with an aspect 300 control ). for the 1 h nmr measurements , 1 . 0 mm dna oligonucleotides were dissolved in 0 . 5 ml phosphate buffer solution ( ph 7 . 2 ) in d 2 o ( 20 mm , for [ d ( atgcat )] 2 ( seq id no : 1 ) and 40 mm for [ d ( gcttaagc )] 2 ( seq id no : 2 ) containing 50 mm nacl and 0 . 1 mm edta . the solutions were lyophilized and then redissolved in 0 . 5 ml d 2 o ( 99 . 96 %), heated to 65 ec and gradually cooled to 5 ec , and then degassed with argon at room temperature . the assay was performed at 37 ° c . in 15 μl reaction mixture containing 67 mm tris - hcl ( ph 7 . 4 ), 1 . 0 mm β - mercaptoethanol , 5 . 2 nm [ α - 32 p ] datp , 6 . 4 μm datp and 320 μm of each dctp , dgtp , dttp , 0 . 6 mm activated calf thymus dna , and 6 . 7 mm mgcl 2 for thp or 6 . 7 , 10 . 0 and 15 mm mgcl 2 for proline . klenow fragment ( 0 . 1 units ) was added to the microtubes with reaction mixture pre - heated to 37 ° c ., and following 7 . 5 minutes incubation at 37 ° c . ( a time - point within the region of linear kinetics determined in a separate experiment , not shown ), the reaction microtubes were placed on ice , and the reaction was stopped by addition of 12 μl of 50 mm edta and then applied on strips of chromatographic paper ( whatman no . 3 ). strips were washed three times by cold tca 10 %, dried and the radioactivity was counted . ( v ) determination of remaining activities of taq polymerase after incubation with thp ( a ) or thp ( b ) at elevated temperatures taq polymerase ( 0 . 5 units ) was added to 50 μl buffer containing : 10 mm tris - hcl ( ph 8 . 8 at 25 ° c . ), 2 . 5 ng halobacterium marismortui genomic dna template , 2 μm of each of the dntps : datp , dctp , dgtp and dttp , 0 . 12 nm of each of the two 28 - mer oligonucleotide primers 1 and 2 described in section ( i ) above , 50 mm kcl , 0 . 08 % nonidet p40 and 1 . 0 mm mgcl 2 . thp ( b ), thp ( a ) or glycerol were added from 3m stock solutions ( ph 8 . 8 at 25 ° c .). the reaction mixtures were overlaid with paraffin oil and incubated at 95 ° c . or 97 ° c . aliquotes ( 7 . 5 μl ) were taken for polymerase activity assay at different periods of time . ( vi ) determination of remaining activity of klenow dna polymerase after incubation at 65 ° c . with proline klenow dna polymerase ( 0 . 5 unit ) was incubated at 65 ° c . in 50 μl buffer containing : 67mm tris - hcl ( ph 7 . 4 at 25 ° c . ), 2 . 5 ng halobacterium marismortui genomic dna template , 4 μm of each of the dntps : datp , dctp , dgtp and dttp , 0 . 12 nm each of the two 28 - mer oligonucleotide primers 3 and 4 described in section ( i ) above , 6 . 7 mm mgcl 2 and either without or in the presence of 5 . 0m glycerol or proline . tris - hcl buffer , template dna , dntp , primers and mgcl 2 were added to pcr microtubes , evaporated to dryness by speed - vacuum and respective volumes of water , proline ( from a 5 . 5m stock solution ) or glycerol ( from a 5 . 5m stock solution ) were added . the microtubes were vortexed and klenow enzyme was added to the samples . aliquots ( 5 μl ) were taken for polymerase activity assay at different periods of time as indicated in fig1 . the klenow dna polymerase activity assay was performed as described in section ( iv ) above at 6 . 7 mm concentration of mgcl 2 . to each aliquot ( 5 μl ) 20 μl of stock solution containing all other components of the assay were added , making a total reaction volume of 25 μl and a 5 - fold dilution of the aliquots . thus , proline and glycerol concentrations in the polymerase assay were 1 . 0m , shown to be stimulative for klenow polymerase activity in a separate experiment ( data not shown ). pcr was performed in 25 μl reaction mixture containing 3 ng template dna , 0 . 12 nm of each 28 - mer oligonucleotide primer 1 and 2 described in section ( i ) above , 0 . 5 units taq dna polymerase , 200 μm of each dntp , in pcr buffer containing : 10 mm tris - hcl ( ph 8 . 8 at 25 ° c . ), 50 mm kcl , 0 . 08 % nonidet p40 . mgcl 2 concentrations of 1 . 0 mm and 1 . 75 mm were used in the absence and presence of thp ( b ), respectively , added from a 3m stock solution ( ph 8 . 8 at 25 ° c .). reaction mixtures were overlaid with paraffin oil and preheated for 3 min at their respective denaturing temperatures , except for mixtures of reactions performed at td 95 ° c ., that were preheated for 3 min at 94 ° c ., and then subjected to 35 thermal cycles as follows : ( i ) 30 sec incubation at 89 - 95 ° c ., as indicated in each experiment ( denaturation step ); ( ii ) 90 sec incubation at 55 ° c . ( annealing step ); and ( iii ) 60 sec incubation at 72 ° c . ( primer extension ). ( viii ) pcr in the presence of proline , using klenow dna polymerase . pcr was performed in a 25 μl reaction mixture containing 100 ng halobacterium marismortui genomic dna template , 0 . 12 nm of each 30 - mer oligonucleotide primers 5 and 6 described in section ( i ) above , 10 or 15 units of klenow dna polymerase , 0 . 9 mm of each dntp , in pcr buffer containing : 10 mm tris - hcl ( ph 7 . 4 at 25 ° c .) and 15 mm mg ( oac ) 2 . tris - hcl buffer , template dna , dntp , primers and mg ( oac ) 2 were added to pcr microtubes , evaporated to dryness by speed - vacuum and dissolved in 22 μl of a proline - glycerol solution ( 5 . 5m of l - proline in 12 . 5 % w / v solution of glycerol in water ). reaction mixtures were preheated for 3 min at 75 ° c ., and then subjected to 35 thermal cycles as follows : ( i ) 20 sec incubation at 70 ° c . ( denaturation step ); ( ii ) 4 min incubation at 37 ° c . ( primer annealing and primer extension steps ). klenow dna polymerase ( 10 or 15 units ) diluted up to 3 μl volume , containing 50 % w / v glycerol , was added during the first primer annealing step at 37 ° c . ( ix ) pcr in the presence of proline , using tag dna polymerase . pcr was performed in 25 μl reaction mixture containing 3 ng of halobacterium marismortui genomic dna template , 0 . 12 nm of each 28 - mer oligonucleotide primers 3 and 4 described in section ( i ) above , 0 . 5 units of taq dna polymerase , 200 μm of each dntp , in pcr buffer containing : 10 mm tris - hcl ( ph 8 . 8 at 25 ° c . ), 50 mm kcl , 0 . 08 % nonidet p40 . mgcl2 concentrations of 1 . 0 mm and 1 . 8 mm were used in the absence and in the presence of 1 . 0m l - proline , respectively . l - proline was added from 5 . 5 m stock solution adjusted to ph 8 . 8 at 25 ° c . reaction mixtures were preheated for 3 min at their respective denaturation temperatures , except for reactions performed at td 95 ° c ., that were preheated for 3 min at 94 ° c ., and then subjected to 35 thermal cycles as follows : ( i ) 30 sec incubation at 91 - 95 ° c ., as indicated in each experiment ( denaturation step ); ( ii ) 90 sec incubation at 55 ° c . ( primer annealing step ); and ( iii ) 60 sec incubation at 72 ° c . ( primer extension ). ( x ) pcr in the presence of proline , using a mixture of klentaq1 and pfu ( or vent ) dna polymerases . pcr was performed in 25 μl reaction mixture containing 250 ng of halobacterium marismortui genomic dna template , 0 . 12 nm of each 30 - mer oligonucleotide primers 5 and 6 described in section ( i ) above , 0 . 3 μl of klentaq1 and pfu ( or vent ) enzymes mixture , prepared as described ( barnes , 1994 ), 200 μm of each dntp , in pcr buffer containing : 10 mm tris - hcl ( ph 8 . 3 at 25 ° c .) and 50 mm kcl . mg ( 0ac ) 2 concentrations of 1 . 0 mm and 14 . 5 mm were used in the absence and in the presence of 4 . 0m l - proline , respectively . l - proline was added from 5 . 5 m stock solution adjusted to ph 8 . 3 at 25 ° c . reaction mixtures were preheated for 1 min at their respective denaturation temperatures , except for reactions performed at td 95 ° c ., that were preheated for 1 min at 94 ° c ., and then subjected to 35 thermal cycles as follows : ( i ) 30 sec incubation at 72 - 95 ° c ., as indicated in each experiment ( denaturation step ); ( ii ) 90 sec incubation at 37 - 55 ° c ., as indicated in each experiment ( primer annealing step ); and ( iii ) 7 min incubation at 63 - 69 ° c ., as indicated in each experiment ( primer extension ). the effect of different concentrations of thp ( b ) on the melting profile of calf thymus dna ( 42 % gc ) was studied . melting experiments were conducted as described in materials and methods , section ( ii ), in the absence or presence of 0 . 8m , 2m , 3m and 4m thp ( b ). as shown in fig2 , the addition of thp ( b ) significantly lowered the dna melting temperature and sharpened its transition profile . the dna melting temperature in aqueous solution , 62 ° c ., was lowered to 41 ° c . in the presence of 3 or 4m thp ( b ). the effect of thp ( b ) on dna melting temperatures was examined on other dnas with different base compositions , such as micrococcus lysodeikticus and clostridium perfringens dnas ( 72 % and 26 % gc , respectively ) and on the synthetic poly ( da - dt ). as shown in fig3 , the melting temperatures ( tm ) of the different dnas decreased with the increase of thp ( b ) concentration in the incubation mixture . this effect is more pronounced for gc - rich dnas . while the oligonucleotide poly ( da - dt ) did not exhibit any change in the melting temperature in the presence of 1 - 4m thp ( b ), 3 - 4m thp ( b ) eliminated the base - pair composition dependence of dna thermal melting . as shown in fig3 , in the presence of 4m thp ( b ), all dnas with a wide range of gc content melt in a very narrow temperature range ( 40 - 43 ° c . ), while in the absence of thp ( b ), the melting temperatures ranged from 39 to 75 ° c . this isostabilization effect by thp ( b ) may be explained as a result of greater destabilization of gc - rich than at - rich dnas . the thermal transitions of the short oligonucleotides [ d ( atgcat )] 2 ( seq id no : 1 ) ( fig4 a ) and [ d ( gcttaagc )] 2 ( seq id no : 2 ) ( fig4 b ) were studied in the absence ( open squares ) and presence of 0 . 5m ( open circles ) and 1 . 0m ( filled squares ) thp ( b ). nmr chemical shift changes of the c4h5 proton of [ d ( atgcat )] 2 ( seq id no : 1 ) and of the g1h8 proton of [ d ( gcttaagc )] 2 ( seq id no : 2 ) were measured as a function of increasing temperatures as described in materials and methods section ( iii ). the results of these experiments are depicted in fig4 a - b and summarized in table 1 . as shown in table 1 , the melting temperatures of [ d ( atgcat )] 2 ( seq id no : 1 ) and of [ d ( gcttaagc )] 2 ( seq id no : 2 ) decreased by 2 ec and by 3 ec , respectively , in the presence of 0 . 5m thp ( b ), and by 3 . 5 ec and 6 ec , respectively , in the presence of 1 . 0m thp ( b ). data were compared to the melting temperature of [ d ( gcttaagc )] 2 ( seq id no : 2 ) in the presence of betaine . the decrease in tm by betaine was only − 2 ec at 1 . 0m concentration , about two - fold higher concentration of betaine is needed for exerting the same tm decline caused by thp ( b ). as shown in table 1 , the melting temperatures of [ d ( atgcat )] 2 ( seq id no : 1 ) and of [ d ( gcttaagc )] 2 ( seq id no : 2 ) decreased by 2 ec and by 3 ec , respectively , in the presence of 0 . 5m thp ( b ), and by 3 . 5 ec and 6 ec , respectively , in the presence of 1 . 0m thp ( b ). data were compared to the melting temperature of [ d ( atgcat )] 2 ( seq id no : 1 ) in the presence of betaine . the decrease in tm by betaine was only − 2 ec at 1 . 0m concentration , about two - fold higher concentration of betaine is needed for exerting the same tm decline caused by thp ( b ). thp ( b ) and thp ( a ) effects on taq dna polymerase stability at elevated temperatures the effects of thp ( b ) and thp ( a ) on the remaining activity of taq dna polymerase incubated at elevated temperatures for different periods of time were studied . after 90 min incubation at 95 ° c ., taq dna polymerase was only 30 % active . the enzyme was remarkably stabilized upon addition of either thp ( b ) or thp ( a ). after incubation at 95 ° c . in the presence of 0 . 5m thp ( b ) or 0 . 5m thp ( a ), the half life of taq polymerase was 70 min and 60 - 90 min , respectively , in comparison to the half life of 30 - 40 min observed in the absence of additive ( not shown ). comparable protective effects were obtained when taq dna polymerase was incubated at 95 ° c . in the presence of a combination of thp ( a ) and thp ( b ) ( results not shown ). thus , thp ( b ) and / or thp ( a ) present in the reaction mixture enable doubling pcr cycles without increased loss of enzyme activity . in fig5 are shown results of similar experiments measuring the thermal inactivation of taq polymerase at 97 ° c . in the absence ( filled circles ) or presence of 1m thp ( b ) ( filled squares ), 1m thp ( a ) ( open squares ) in comparison to 1m glycerol ( open diamonds ). the thermal inactivation of the enzyme at the elevated temperature 97 ° c . was , as expected , more rapid than the inactivation at 95 ° c . ; almost a complete loss (& gt ; 95 %) of enzyme activity was observed following 60 min incubation at 97 ° c . with no additives . however , also the protective effects by thp ( b ) and thp ( a ) were more dramatic : the remaining taq polymerase activities , following 30 min incubation at 97 ° c . were 40 % and 50 % in the presence of 1m thp ( b ) or thp ( a ), respectively , in comparison to 10 % remaining activity in the absence of additives . as a result of 60 min incubation at 97 ° c ., the remaining taq polymerase activity in the absence of additive was 5 %, whereas in the presence of 1m thp ( b ) or thp ( a ) the remaining activities were 20 % and 45 %, respectively . the results shown in fig5 indicate that thp ( a ) is more effective than thp ( b ) or glycerol in stabilizing taq dna polymerase . the combined effect of thp ( b ) on dna melting temperatures and on taq dna polymerase activity and stability at elevated temperatures was followed under pcr conditions . pcr reaction was performed by taq dna polymerase as described in materials and methods , section ( vii ), using as a template whole genomic dna of halobacterium marismortui ( 66 . 5 % gc ) and the 28 - mer primers 1 and 2 described in section ( i ). in fig6 are depicted the amplified dna sequences produced by pcr performed at 95 ° c . and 92 ° c ., in the absence and presence of 0 . 5m thp ( b ), respectively , showing that yield and specificity of the dna amplification was improved by the presence of thp ( b ). at 92 ° c ., amplified sequences were produced only in the presence of thp ( b ), but not in its absence . the effect of 1 . 0m thp ( b ) in the pcr buffer mixture is presented in fig7 a - c . a “ control ” assay was performed at td 95 ° c . in the absence and presence of 1 . 0m thp ( b ) with two concentrations of taq dna polymerase , 0 . 5 and 0 . 75 units in 25 μl pcr reaction mixture . the presence of 1 . 0m thp ( b ) improved pcr specific amplification at td 95 ° c . ( fig7 a ). however , the most significant results were obtained when denaturation temperatures of the dna were reduced from 95 ° c . to 90 ° c . ( fig7 b ) in the presence of 1 . 0m thp ( b ) ( either with 0 . 5 or 0 . 75 units of taq dna polymerase in 25 μl reaction mixture ). under these conditions , specific amplified sequence was generated only in the presence of thp ( b ), while no trace of amplified dna could be detected in the absence of this additive . when td was further lowered to 89 ° c . in the presence of 0 . 5 units taq dna polymerase in 25 μl reaction mixture , amplified dna sequence was markedly lower , even in the presence of 1 . 0m thp ( b ) but no trace of amplified dna was detected in the absence of thp ( b ) ( fig7 c ). the effect of different concentrations of proline on the melting profile of calf thymus dna ( 42 % gc ) was studied . melting experiments were conducted as described in materials and methods , section ( ii ), in the absence or presence of 2m , 3 . 5m 5m and 6 . 2m proline . as shown in fig8 a , the addition of proline significantly lowered the dna melting temperature and sharpened its transition profile . the dna melting temperature in aqueous solution , 62 ° c ., was lowered to 27 ° c . in the presence of 6 . 2m proline . the effect of proline on dna melting temperatures was examined on different dnas with different base compositions , such as micrococcus lysodeikticus and clostridium perfringens dnas ( 72 % and 26 % gc , respectively ), calf thymus dna ( 42 % gc ) and on the synthetic poly ( da - dt ). as shown in fig8 b , the melting temperatures ( tm ) of the different dnas decreased in the presence of 6 . 2m proline concentration in the incubation mixture . the range of melting dna with gc content of 72 % is about 5 ° c . higher than that of gc content of 42 % and 26 %, while poly ( da - dt ) melts about 15 ° c . lower . the effect of increasing concentration of proline as depicted in fig9 a on the four dnas reveals that the effect was pronounced for gc - rich dnas . while the oligonucleotide poly ( da - dt ) did not exhibit any change in the melting temperature in the presence of 1 - 5m proline , a small effect occurred in the range of 5 - 6 . 2m proline . proline at 6 . 2m almost eliminated the base - pair composition dependence of dna thermal melting . as shown in fig9 a , in the presence of 6 . 2m proline all dnas with a wide range of gc content melt in a very narrow temperature range ( 25 - 32 ° c . ), while in the absence of proline the melting temperatures ranged from 38 to 78 ° c . fig9 b depicts changes in dtm / dgc as a function of proline concentration . a linear correlation is presented for proline concentration of up to 5m . to study the effect of 5 . 0m l - proline on the klenow dna polymerase activity , experiments were conducted as described in materials and methods , section ( iv ), in the presence of different concentrations of mgcl 2 : 6 . 7 mm , 10 . 0 mm and 15 . 0 mm mgcl 2 . as shown in fig1 , l - proline only slightly decreased klenow dna polymerase activity . the activity of the enzyme remained high enough , particularly at 10 . 0 ( hatched bar , middle ) and 15 . 0 mm mgcl 2 ( hatched bar , right ). the effect of 5 . 0m proline on the stability of klenow dna polymerase at 65 ° c . the remaining activity of klenow dna polymerase incubated at 65 ° c . in the presence of 5 . 0m proline , 5 . 0m glycerol or without any additives , was measured as described in materials and methods , section ( vi ). as shown in fig1 , klenow dna polymerase at 65 ° c . has a half - life of less than one minute with no additives ( filled circles ), 3 minutes in the presence of 5 . 0m glycerol ( filled triangles ) and 21 minutes in the presence of 5 . 0m proline ( open diamonds ). the combined effects of proline on klenow dna polymerase stability at elevated temperatures and on dna denaturation temperature step , permitted a successful design of cycled pcr conditions for this enzyme . pcr was performed by klenow dna polymerase as described in materials and methods , section ( viii ). pcr amplification of a 349 b . p . fragment ( 66 . 5 % gc ) of halobacterium marismortui genomic dna ( from position 2546 to 2843 ) was performed in a 251 μl reaction mixture containing 100 ng of the dna template , 0 . 12 nm of each 30 - mer oligonucleotide primers 5 and 6 described in section ( i ) above , 0 . 9 mm of each dntp , 10 mm tris - hcl ( ph 7 . 4 at 25 ° c .) and 15 mm of magnesium acetate . tris - hcl buffer , template dna , dntp , primers and magnesium acetate were added to pcr microtubes from stock solutions , evaporated to dryness by speed - vacuum and dissolved in 22 μl of a proline - glycerol solution , containing 5 . 5m of l - proline in a 12 . 5 % w / v solution of glycerol in water . klenow polymerase ( 10 units / ul , storage buffer contains 50 % w / v glycerol ) and , in order to keep constant glycerol concentration in the pcr mixtures , aliquots of glycerol solution in water ( 50 % w / v glycerol ) were added during the first primer annealing step . as shown in fig1 , in lanes 1 . and 2 . 1 . 0 μl of klenow polymerase ( 10 units ) and 2 . 0 μl of the glycerol solution were added , and in lanes 3 . and 4 . 1 . 5 μl of klenow polymerase ( 15 units ) and 1 . 5 μl of the glycerol were added . the final concentration of l - proline in all pcr mixtures was 4 . 85m and of glycerol was 17 % w / v . all pcr reactions were run on a mj research ptc - 100 machine equipped with a normal block ( ramping rate is 1 ° c . per second ). reaction mixtures were preheated for 3 min at 75 ° c ., and then subjected to 35 thermal cycles as follows : a ) 20 sec incubation at 70 ° c . ; b ) 4 min incubation at 37 ° c . reaction products were run on a 2 % agarose gel and stained by ethidium bromide . the results shown in fig1 reveal that proline concentration in the range of 3 - 5 . 5m is sufficient to confer stability to klenow dna polymerase and to conduct a successful pcr protocol . fig1 shows pcr , using taq dna polymerase , performed in the absence and in the presence of 1 . 0m proline , as described in materials and methods , section ( ix ). addition of 1 . 0m proline to the reaction mixture did not impair pcr performance at denaturation temperature 95 ° c . and enabled successful pcr at decreased denaturation temperature , namely 91 ° c . pcr in the presence of proline , using mixture of klentaq1 and pfu dna polymerases pcr was performed in the presence of 4 . 0m proline , using a mixture of klentaq1 and pfu dna polymerases , as described in materials and methods , section ( x ). reaction mixtures were preheated for 1 min at their respective denaturation temperatures ( 77 ° c . and 75 ° c . ), and then subjected to 35 thermal cycles as follows : ( i ) 30 sec incubation at 77 ° c . or 75 ° c . ( denaturation step ); ( ii ) 90 sec incubation at 44 ° c . ( primer annealing step ); and ( iii ) 7 min incubation at 65 ° c . ( primer extension ). as shown in fig1 , there is a clear correlation between the concentration of proline in the mixture and the minimal denaturation temperature . thus , true for above mentioned conditions , 4 . 0m concentration of proline was enough for successful pcr at the 77 ° c . denaturation temperature , but not at 75 ° c . 1 . barnes , w . m . 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