CA2035471A1 - Techniques for the amplification of nucleic acid - Google Patents

Abstract

Improvement relating to techniques for the amplification of nucleic acids, in particular by polymerization chain reaction (PCR).
Said process for the detection and/or the identification of a nucleic acid sequence or of a mixture of nucleic acid sequences once the bioligical sample has been placed in an appropriate solution in order to extract the nucleic acid or acids, includes the following steps: (1) the destruction of the 5' ends of the nucleic acid sequences with double strand which are present in the sample, by bringing said biological sample into contact with an appropriate reaction agent which is active at a temperature of between 37·
- 42·C; (2) the actual amplification by bringing the resulting sample into contact with (1) appropriate reagents, in particular amplification initiators suitable for the amplification of the target sequence or sequences to be detected, in the presence of a heat-resistant polymerase DNA; (3) detection of the specific amplified target sequences.

Description

~Q3~47~
, ~
WO 91~00, 363 PCT/FR90/~0, 499 IilPROVE~lENTS RE.rlATED TO TECHNIQUES
FOR THE A~IPLIFICATION OF NUCLEIC ACID
The present invention relates to an Lmprovement to the techniques of amplification of nucleic acids, in S particular by the polymerization chain reaction (PCR).
One of the first amplification methods describPd is the polymerization chain reaction (PCR) developed by R. SAIKI et al. (Science, 1985, 230, 1350). This tech-~ nique makes it possible, in particular, to amplify I 10 single-stranded or double-stranded DNA sequences, and is i based on the cyclic operation of a DNA polymerase, which ! enzyme is capable of copying a DN~ strand, used as a template, to a complementary strand by elongation from the free 3'-OH end of an oli~onucleotide primer.
The PCR technique consists in performing n suc-cessive amplification cycles, during which two pr'mers direct the amplification of the double-stranded DNA
¦ sequence enclosed between them. An amplification cycle is composed of three steps, enabling denaturation o~ the DNA
(at a temperature o~ between 90 and 95C), hybridization of the primers (for e~ample at a temperature of between 37 and 75C) and elongation of th~ DNA strands with DNA
polymerase, to ~e carried out successi~ly.
The technologi~al bases of PCR are founded on two ~5 fundamental Points:
the use of two oligonucleotide primers, one ~ compLementary to the DNA~ strand, the other to the DNA-¦ strand, and whose 3' ends are facing one another;
- the repetiti~e use of the activity of a suit-able DNA polymerase.
PCR thus enables, under suitable conditions, all ~he sequence~ neosyn~hesized in the cycle n ~o be us0d as a templat~ for the DNA polymerase in ~he cycle (n ~ 1)O
~ ~ This result~ in an exponential amplification of the 1 35 number of targe~ nucleic acid sequenc~ a~ a function of I : the number of~cycles, in accordance with the ampliflca-i~: tion cur~e which comprises an exponential phase where the i quantity Q ~ t~rget se~uence~ obtained may be linked to ~ `the quantity Qo of inltial target sequenc~st to the r ~! 0 3 ~ 4 7 1 ,j ~
~ amplifica~ion factor x and to the number of cycles n by 3 the following formula: Q = Qo (1 ~ x)n.
PCR thus enables so-called target nucleic acid sequences to be amplified exponentially thousands of times.
This amplification technique is, more especially, described in the CETUS European Patent Application 201,184, which specifies, in particular, that the two ~ oligonucleotide primers employed in the PCR are prefer-i~, 10 ably single-stranded and must be su~ficiently long to ~ prime the synthesis of the extension product in the 3 presence of the DNA pol~merase.
A number of refinements or impro~ements to this ^ technique have ~een proposed, and in particular, in order to avoid the addition of enzymes at each cycle, a DNA
' polymerase capable of withstanding lOO~C~ Taq polymerase, i described in the CETUS European Patent Application ' 258,017, is added from ~he beginning of the process and enables a large nu~ber of con~ecuti~e amplification cycles to be carried out. Taq polym~rase ha~ made it possible, on the one hand to automate ~he amplification process, and on the other hand to~ increase the speci-3 icity of the amplification.
PC~ thus makes it possible to ob~ain, without cloning~ a considerable amplification of a so-called target nucleic acid sequence, and hence a huge gain in sen3itivity. The amplified target sequence is then dlrectly amenable to dLf~erent analytica} processes, such a~ the dot-~lo~, electrophore~is or sequencing process.
! 30 Two patent applications ( CETUS European Patent ~pplica-tion~ No. ~200,362 and No. 229,701) describe, more espeici-ally, the amplification/hybridization combination for the ~ detec~ion of a nucleic acid sequence~ of interest in a `j~ sample.
In the me~an~Lme, variants and/or Lmprovement~
have been proposed, and PCR is currently wid~ely used in both molecular biology and in clinical medicine (diag-i ~ no~is of gene~ic diseases and detection of path~genic microorganisms). Xowever, the rou~ine use of ~his .;
,

2~3~

- 3 -technique has brought ~o light an Lmportant drawback linked to its extreme sensitivity, namely the emergence of false positives which have been shown, after a careful analysis of the results, to be linked to a contamination of the test sample.
A number of papers published during the last two years or so describe such cases o~ contamination and ¦ propose means for counteracting them. These articles il highlight an unexpected type of contamination, namely ¦ 10 contaminatian by sequences identical to those of interest.
- Y.M.D. LO et al., in an articLe published in Lancet (1988, ii, 679), bring to light this drawback of - the polymerization chain reaction, due to its sensi-tivity, and, in order to overcome this important draw-back, recommend taking special care in the preparatlon of the DNA template before amplifîcation. LO et al. des-~1 cribe, more especially, an example of contamination in a ¦ diagnosis of HBV sequences, and show a posi~ive result obtained from nagative samples: HBV sequences were ampli~ie~ from negative samples, a verification of the absence of sequences to be detected in the sample ha~ing ¦ been carrled out by Southern blot~ing.
I LO et al. analysed this result as a contamination ¦ 25 of the prLmers used by plasmids containing the HBV insert I (plasmid pHBV130), inasmuch as, when they get rid of the 3 contamina~ed primers, there ar~ no longer any falsely positive ampliication~ in the negati~e samples.
LO et al. suggest that, inasmuch as PCR is being used increasingly in diaqnosis, the problem of false positives due to con~amination by sequences identical to those to be detected cannot be ignored. LO et al. propose that a so-called negati~e control, consisting only of reagents for PCRr be caxried out syst~ma~ically in order to as~es~ the po~sible plasmid con~amination of ~hese reagents.
LO et al~. also propos~ that the existence of a plasmid contamination he veri~ied by amplification in khe ~ presence o~ primers corresponding to ~aquences placed on 1`
1~ .

e~ , 2 ~ 3 ~
.
,~ each side of the vector-inser~ junction of the said plasmid. This procedure is especially necessary if conflicting results are obtained by conventional methods such as Southern blotting.
It should, howe~er, be noted that the problem of ~j contamination by the presence of a plasmid vector of the target fragment arises only in the context of research la~oratories, whereas diagnos~ic laboratories (medical analytical laboxatories, for example), performing routine identifications, have ready-to-use reagents at their disposal and whexeas, in this case, it is Lmportant to avoid contamination of the template by products other `I than plasmids (products of PCR, free viruses, for exam-ple) for a reliable diagnosis to be carried out, it not being possible, in the opinion of LO et al., for this contamination to be avoided at the present time.
- R.A. GIBBS et al., in a paper published in Genes & Development, 1989, 3, 1095-lO9~, la~ish the following ad~ice: isolate all the reagents used for carrying out PCR from the equipment used for analysing i the products obtained; use screw caps and even go to ~he point Qf freezing the contents befare performing tha final reaction in order to avoid aerosol formation; lLmit the number of amplification cy~les to a minimum, both to reduce the risk of ampLifying small quantities of con-taminants already present and to reduce the total quan-tity of reaction products, and ha~a different sets of pipettes a~ailabl~ for each job.
- G. S ~ et al~, in a paper published in Nature, 1990, 343, 27, propose, in order to avoid this cbntamina`tion, that the sample be treated with W rays be~ore the DNA template tc be~amplified is added.
S. ~W~K et al., in a paper~published in Nature, 98g/ 339, 237-238,propose a n ~ er of ~good laboratory practicasl' for moni~ori.ng and a~oiding con~amination:
physLcally~i~olate the preparations and products for~PCR
t~eparate rooms, W rays, etc. ~; autoclave the bu~fers used; divide the raagents into small~ samplas in order to avoid rapea~ed pipetting; use pratective glo~res during ;~ 3 ~ 4 ~ ~
.~ . .; . I
.
., . manipulations; avoid splashes; use pipettes which do not give risa to aerosols containing samples of DNA; premix reagents where possible in order to decrease the numher .~ o contaminating manipulations; add the DNA last; and ^ 5 choose positive and negative controls carefully.
The contamination described by these di~ferent authors is hence a contamination by sequences identical to those to be detected, the so-called target sequences . derived, in the majority of cases, in the medical ana-lytical laboratory, from previous amplifications and .` which generate a large nu~ber of copies, producing an ' accumulation of the amplified target fragments and i leading, in particular, to the ubiquitous presence of th~
target sequences in the medical analytical laboratory ~l 15 However, the collective suggestions proposed ~y the different authors mentioned abova for a~o.iding this con~amination have the drawback of being especially . restrictinq and difficult to carry out routinely.
! Furthermore~ these different sug~estions do not ! ~ enable the contamination due to the presence of target I sequences derived from pre~ious amplifications to be ll systematically eliminated.
For this reason, the Applicant had as his objec~
~! tive to pro~ide for a process for the ampli~ication of ! 25 target sequences which meets the practical re~uirements better than the previous processes, in particular in that . it mak~s it p~ssible to prevent the exponential amplifi-cation of contaminant double-stranded nucleic acids, that is to say of sequences identical to the sequences to b~
t 30 detected, amplified during previous tests in the labora-tory, and which could give rise to falsely positi~e ' results.
I Contaminant sequence is understood, in the sense of the present in~ention, to mean a short nucleic a id fragment which corre~ponds to a genomic DN~ fragment to `~ be detec~ed ~target sequence) and which possesses an ~! integrated prLmer at its S' end.
; Target sequence i5 undexstood, in the sense of : the present invention, to me~n a sequence defined by the ~ ~ ~ 203~7~
. ~.1 s, . . .
~, amplification pr~mers between which it is enclosed, which . sequence is included in a long-sequence nucleic acid, j genomic DN~ in particular.
j~ The subject of the present invention is a process ~' 5 for the detection and/or identification of a nucleic acid .~ sequence or of a mixture of nucleic acid sequences comprising an enzymatic amplification, characterized in that t aftex the biological sample has been suitably dissol~ed in order to ext.ract the nucleic acid(s), the said process comprises the following steps:
(1) a step of destruction of the 5' ends of the double-stranded nucleic acid sequences present in the j sample, by bringing the said biological sample into } contact with a suitable reagen~ which is active at a lS temperature of between 37 and 42C;
(2) a step of actual amplification, by bringing the sample obtained in (1) into contact with suitable reagents, in particular amplification primers suited to the amplification of the target sequence(s) to be detec-ted, in the presence of a thermostable DN~ polymerase;
and ~
(3) a step of detection of ~he amplified speaific targ~t sequences.
According to an advantageous embodiment o~ the process according to th~ inYention, the reagent of step ~ (1), designated reagent for discrimination between con-; ~ kaminant sequences and target sequences, is chosen from he group which comprises suitable chemical products~and enzyme~ which act in a weakly alkalin~ medium.
According to an ad~antageous arrangement of this ,~
embodiment, the! enzyme is a suitable ~S'-exonuclease, `preferably lambda S'-exonuclease. ~ ~ :
;
~:According to~ an advantageous variant of :this~
~`arrangement, the prLmers employed for the~amplification `~: ~35 o~:step (2)~ are phosphorylated at~their 5' ends. :
.~L ~ da exonucle:ase has the property of destroying~;
;~. the S' ends of double-stranded~nucle1~ acids when the said ends are phosphorylated.~
The action of t:his lambda 5'-exonuclease is more 2~3~ 17~
; - 7 -especially described in the papers by ~.W. LITTLE (J.
Biol. Chem., 1967, 242, 4, 672-686).
Carrying out the abovementioned step 1 is crucial for avoiding contamination by sequences identical to i 5 those to be detected.
I In effect, as pointed out above, the contaminant sequences correspond to relati~ely short sequences containing, at their ends, the amplifîcatian prLmers used during pre~ious amplifications (prLmer extension pro-~' 10 ducts), whereas the target sequences to be detected, defined by the amplLfication prLmers between which they are enclosed (same prLmers as those incorporated into the contaminant sequences), are included in a long-sequence nucleic acid (genomic DNA in particular).
During step (l) of the process according to the j invention, the discrLmination reagent, and more especial-ly the 5'-exonuclease, which has the pxoperty of destroy-ing the nucleotides of a double-stranded nucleic acid . only, stepwise in the 5'~3' direction, leads to th~
production of nucleic acids whose 5' ends are destroyed over a length depending on the tLme of action of the 5'-exonucleas~, whereas it does not destroy the S' ends o~ single-~tranded DNA ~prLmers or denatured DNA, for !~ example).
During step (2) o~ the process according to the invention, the short nucleic acid sequences (contaminant sequences) and the long nucleic acid sequences (sequences containing the target sequence to be detected) will, as a result of the action of the discr ~ination reagent, and more especially the 5'-exonuclease, beha~e differently:
~ as regards the contaminant sequence~, which i comprise, in fact t only tha target sequence and the amplification prLmers incorporated at the ends~ extension ~: will take place only from a single amplification prLmer~
inasmuch as the 5' ends are destroyed; such an e~tension !~ will give rise onl~ to a linear increase in ~hese con-¦ ~aminant sequence~; if it is considered that n successive ¦; amplification cycles ha~e be~n performed during step (2)~
I the contaminant sequences will be amplified only 2n !:
":

tLmes, which may be considered to he a negligible ampli-fication compared with an exponential amplification which gi~es rise to the formation of 2n amplified sequences.
- as regards the target sequences to be detected, in contrast, the destruction of the 5' ends will not a~fect the region of the long nucleic acid sequence in which the target sequences occur; extension will hence take place from bo~h amplification prLmers; such an extension will give rise to an exponential increase in the said target se~uences (production of 2n sequences during n successive amplific~tion cycles) and enable the said sequences to be readily detected.
The time of action of the discrLmination reagent, and more especially the 5'-exonuclease, can vary from a few minutes to more than 1/2 hour, depending on the case.
The process according to the invention hence makes it possible, unexpectedly, by destruction of the 5' ends of the double~stranded nucleic acids present in the sample, to avoid the expon~ntial amplification o~ the contaminant sequences while not affecting the exponential amplification of the target sequences included, in particular, in ~ ~enomic DNA.
It should be noted that, even in the least ~avourable case, that is to say when the target sequence occurs a~ the 5' end of the genomic DNA, this same target sequence on the negative strand is far ~rom the 5' end~, and will hence be the source of an exponentiaL amplifica-tlon. ~ ~ ~
~ Step (1) of the process according to the inven-tion renders the short double-stranded~nucleic aci~
fragments vulnerabLe whereas, in the long nucleic acid fragments, the target sequence will~be protected~ and always ampli~fied~exponentially.
Figure~l~illustrates the con~entional exponential ~mplification p~oces~ob~ained by~PCRr starting with~ a so-called contaminant nucle~ic acid as defined above,~that~
is;~to say one~whose~ends are defined~by the S' ends~of~:
the prLmers A and~B~us~ed.
nuring;~such an amplifica~ion, extension of the ;; 203~47:~
,~P.~,'. .
~.. , 9 .
strands will take place from both primers and the con-taminant sequence will be amplified exponentially, even in the absence of target sequences in the test sample:
after n cycles, 2n copies of the contaminant sequence are ob~ained. In 20 cycles, the initial sequence is, in principle, amplified one million times.
In contrast, when the process according to the invention is carried out, the contaminant sequences, in the presence of 5'-exonuclease, for example, do not lead to the production of false positives, since they are amplified only linearly as shown in Figure 2, where it is seen that extenslon takes place only from a single prLmer and not from bo~h.
~ In effect, as pointed out above, a linear ampli-fication for n cycles leads only to the formation of 2n sequences.
Table I below highlights the number of copies obtained after amplification of a so-called contaminant sequence, without prior treatment wi~h 5'-exonuclease (column A and Figure 1, con~entional exponential amplifi-cation), and wi~h prior treatm2nt with a S'-exonuclease ~column B and Figure 2, linear am~ification).

~ ~ 3 ~3 ~ 7:~

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- 10 - !
TABLE I
, ~ .

Cycles Untreated contaminant Modified contaminant _ . ...... ~ : _ __ 2 4 ~3

4 1 16 5 32 ~6 6 64 7 ~
7 128 ~: 8 ~ :
: 8 : 256 : : ;9 ~ :
9 512 10 .
lS 10 1024 j: l1 : 2048 12 ::
: 12 40~6 :~ 13 :: 13 8192 14 ~
14` : 16384 : 15 : : : :
; ~ ~ 2~ 15 ~2768 : 16 :
: 16 ` 65536 : 17 :
: ~` ` ` ` ~17 131072 ~ ~`. ~ ~ 18 ~ `:;
.~ : 18 .2621~4 ~` ~19 :
~1~ :524288 ~ ~ 20 :: ; ;~
~ ~ 20 : 1048576~ :~ 21 : ~21 ~2097152 : ~ : ~ 22 : ;;~
,~ : : :
~ :~ 22 ~ 4194304 :~ ~ 3~ ~ :
; ~ . ~ . :
23 :~ ~ 8388608 ::~ " : ~ 24 24 :~ ~67772~16 ~ ` :~ :25 ~:: ~
~ ~ 25 ;' ~ 67108~364 ~ `~ 26~ ~ ~ :

. ~:~: 2:7 ~: ~ 1342~177~28 ~ ~3~ ~
28~ ~ 2684~35456;~ ~ 29:: :~ ;:~:
: ~9~ ~ l:~ 5:3,6870912: ~ ~ 30 :~ :-. :: :

~ coordin;g~to another~advantageous~`embodi~ent~:of~
the~ aid~process, ~step~ namely;:the~step:of~destru~
tLon~of;~the:S'~end8~0f:~the~::double~-stranded~nucleic;acid ~ ~ :

2~3 ~7:~

.

.
sequences, is carried out only once whereas step (2) is repeated at least once.
In effect, in the process accoxding to the invention, the discrimination reagent, and more especial-ly the 5'-exonuclease, should act only once, befoxe the first cycle o~ the amplification step, in order to avoid 3 the exponential amplification of the contaminant sequen~
3 ces; it should hence be temperature-sensitiYe in order to be capable of being readily usable in an automated process.
According to another ad~antageous embodLm2nt of the in~ention, step (2) of the said process is carried out, in addition, in the presence o~ a temperature-~ sensitive DNA polymerase.
The combination of a temperature-sensitive polymerase, active at 37C, with the thermcstable poly-;I merase has the advantage of enabling at least two problems linked to carrying out a PC~ to be solved:
- repairing the contaminant sequences obtained fxom a PCR, of which the final amplLfication cycles have poor yields (production of single-stranded contaminants, for example, which would hence no~ be destroyed ~y the process according to the in~ention), - in the case of the application of PCR to RNAo the combination of an RNA-dependent DNA polymerase ~ith the thermostable polymerase enables PCR to be carried aut on RN~ viral particles in a sing~e step.
The process according to the invention has the ad~antage of modifying, at the;beginning of the reacticn, the ~arget fragments amplified during pre~i~us manipula-! 1 ! ~ kion~ (contaminant~j, wi~hout subs~antial modifications of the pla mid or genomic DNA~in which the target , j saquence to be detected occurs. ~fter ~his treatment, the 1` pr~-existin~ amplified target fragment can no longer `~ 35 serve a~ a ~emplzte for an exponential amplification, whereas the plasmid or genomic DNA still remains the s~arti~g template.
Th sub~ect of ~he present in~ention is also an enzyme composition for carrying out a detection process ~ ~.
1 .

~035~ 71 ~. .i~
~ 12 -. ~
according to the in~ention, characterized in that it comprises a reagent which destroys the 5' ends of double-stranded nucleic acid sequences, the so-called reagent for discrLmination between contaminant sequences and target sequences, and a thermostable DN~ polymerase.
According to an advantageous embodLmient of the said composition, the said discrimination reagent is a temperature-sensitive 5'-exonuclease.
According to another advantageous embodiment of : 10the said compos}tion, it comprises, in addition, a DNA
polymerase which ls acti;~e at 37-42C (temperature-sensitive).
The subject of the present invention is, in addition, a ready-to-use outfit, kit o:r coordinated assembly for carrying out the process according to the invention, characterized in that it comprises, apar~ from the appropriate quantities of: buffers and suitable reagents, of at least one sultable pair of primers and, optionally, of at least one suitable.probe, suitable doses of an enzyme composition according to the in~en-tion.
Apart from the foregoln~ arrangements, the in~ention also compris0s othier arrangements, which will become apparen~ ~rom~the description.which followsj which~
refers to examples of implementation of the process which ~: is the subjact of the present invention. ::
~: It should be clearly understood, howe~er, that `~ :these examiples are given only by way of illustxation of ~:~ the sub~ect of the in~ention, ànd in no way constitute a llmitation of the latter.
Generally speaking, t;he process àccording to the ~ in~ention makes it possible to el;Lminate false posi~i~es _ ~ ; due to contamination by seq~enc~s~ identical to the " : ~ sequences to~be detected, pre~iously amplified during : : 3S prQ~ioU3 ~tests`in~:the lab~ratory.: ~
:Example l: Detection of C~ilamydia trachomatis~by the process according to the invention,~in the presence o~ thermos~able DNA polymerase;:comparison with a conven-: :tional (PCR)~amplificatLon.~

203~4~1 , r~~; -- 13 Samples of Chlamydia DNA obtained from the ,; cryptic plasmid of this bacterium are subjected to an amplification in a reaction medium containing, in a total ~olume of 50 ~ M each of the appropriate primers ~i, 5 phosphorylated at the 5~ end, 200 ~M each of the 4 deoxy-ribonucleotide triphosphates (d~TP~ dCTP, dGTP~ dTTP) ~
, 10 mM Tris pH 8; 50 mM KCl; 5 mM MgCl2, 1 unit of thermo-., stable 5'-3' polymerase and one unit of temperature-sensitive lambda 5'-exonucleàse.
The protocol is as follows:
the samples are heated to 37C fox 15 minutes (exonucleas~ aetion) and are then subjected to an ampli-fication comprising 25 cycles, each cycle consisting of:
~ heating to 92C for 15 seconds, cooling to 55C for one minute ~nd then heating to 72C for one minute. The c samples are then stored at 72C for 10 minutes.
~he PCR products are separated by electrophoresis on agarose gel (1.4%) previous~y stained with ethidium bromide.
The DNA is then detected by exposure to W rays.
For comparison, conventional PCRs (without the addition o~ 5'-exonuclease) are carried out according to the same protocol.
The objec~ o~ this comparison is to show the 2S advan~ages of the process according to the in~ention, namely that lambda exonuclease ha~ no effect on the yield of PC~ carried out using genomic DN~, whereas it strongly decreases the yield of PCR carried out using contaminant sequences as defined above, phosphorylated at their S' end.
, ~Figure 3 as a whole illustrate~ this comparison tl and shows the elLmination of false~po~iti~es, ob~ained by mean~ of the process according to the in~ention when PCR
products produced under the~dif~erent condi~ions speci-fied above are separa~ed by electrophoresis on agarose gel (1.4~) s~ained with e~hidium bromidP, as specified aboYe.
~. .
;It comprises:
- in column 1: the a~plification products J
~i~

,!

'` ' r~ 14 - ~03~ 71 ~ obtained by conventional PCR, in the presence o~
3 phosphorylated primers, ~rom a sample containing a cryp~ic plasmid of Chlamydia;
- in column 2: the amplification products obtained by the process according to the in~ention (action of lambda 5'-exonuclease + PCR), in the presence of phosphorylated primers, from a sample containing a cr~ptic plasmid of Chlamydia;
- in column 3: the amplification products obtained by conventional PCR, in the presence of phos-phorylated primers, from a negati~e sample containing contaminants phosphorylated at their 5' end;
- in column 4: the amplifica~ion products ~ obtained by the process according to the invention (action of lambda 5'-exonuclease + PCR~, in the presence of phosphorylated~primers, from a negative sample con-taining contaminant~s phosphorylated a~:their 5' end;
`~ - in column 5: the amplification products obtained by a conventional PCR,;from a negative sample containing contaminants not phosphorylated at their 5' nd;
in column 6: the amplification produc~s obtained by the process according to the invention (lambda 5'-exonuclease + PCR), from a negative sample ~: 25 containing contaminants not phosphorylated at their 5' end.
It emeryes from this figure tha~
` - the~;presence of lambda 5'-exonucl ase does not ~ :
in~luence the ampliication :it~elf (column 1 and coIumn 2);
in~ithè~ absence of l~mbda 5'-exdnuclea~, a negative sample give~ a falsely po~itive result~jcolumn 3],~whereas~this poor rasuLt~disappears in the presence : of l~mbda~5'-ex~nuclease ~col ~ 4); it should be noted~
: that,:in~columns 3 and 4,:a;faint band wh~ch corre~ponds:
to non-speci~ic fra ~ents is observed,: thi~ band usually be~ing se~n in amplifications performed in the absence of a~NA templa~e. ~
` ~ when 1 ambda 5 '-exonuclease: is used, the `- 203~47 L
~, r..
contaminants phosphorylated at their 5~ end are much more sensitive to this 2nzyme (columns 5 and 6.).
Exam~le_2: Detection of Chlamydia ~rachomatis by the process according to the invention.
The reaction is carried out as follows:
Samples of Chlamydia DNA derived from clinical samples are added to a reaction buffer containing: 10 mM
Tris-HCl (pH 8.4); 50 mM KCl; 2 mM MgCl2; deoxyribo-nucleotide triphosphates (dNTP): 200 ~M each; prLmer.
1 ~M each; 0.03 unit of lambda DNA exonuclease per ~1 of reaction; 0.02 unit of thermostable DNA polymexase per ~1 of reaction.
The samples are h~ated to 37C ~or 20 minutes and are subjected to 25 successive cycles as follows: the samples are heated to 90C for 15 seconds, cooled to 55C
for one minute and heated to 72aC for one minute. The samples are then stored at 72C for 10 minutes.
The PCR products obtained with the following prLmers:
primer 1 (+): 5' - TTCCCCTTGTAATTCGTTGC - 3' primer 2 (-) 5' - TAGTAACTGCCACTTCATCA - 3', which are phosphorylated in a conv~ntio~al manner with a polynucleotide kinase, possess 201 bas~ pairs and are s~parated on agarose gel stained with ethidium bromide.
' ~he DNA is then detected by exposure to W rays. The PC~
product3 can also be ~isualized by~auto~adiography after car~ying out Southern blotting on a Hybond N f ilter ~Amersham) with a ~ingle-stranded RNA probe sp~cific for th~ said products, labelled either with 32p ~ Fi ~re 4:
column 1: marker~; col ~ 2: negati~e control; column 3:
pc3sitiYe ;control colllmn 4:` negative clinical! sample;
column~ 5 and 6: positive clinical samples) or with Dig dUTP (Figure ~5: column 1: mark~rs; column 2: positive control; column 3: negative control; column ~: negative clinical sample; columns 5 and 6: positive c}inical samples). The clinical samples containing genomic DNA of Chlamydia trachomatis are weIl detec~ed in the presence o~ lambda exonuclease (Figure 4: columns 3 and 4; Figure ; 5: columns 5 and 6).

~ ~ 3 ~ 4 7 ~
,. ~ .
- lS -Exam~le 3: Role of the contaminant concentration.
So-called contaminant sequences of cytomegalo-~irus tCMV) are prepared.
They are extension product~ of phosphorylated ~ 5 primers ob~ained duxing pre~ious amplifications. Thesei contamiIlant sequences hence contain only target sequences containing the said phosphorylated primers at tneir ends.
The said contaminant sequences are obtained by purification from an agarose gel electrophoresis and then 10by electroelution, ethanol precipitation, suspension in water and quantifica~ion with l'DNA DIPSTRICg from INVITROGEN 1-800-544 4684".
The contaminant sequences thereby obtained are then diluted so as to obtain approximately 105 copies i~
1 1515 ~1 of water. Serial 5-fold dilutions are then per-~1 formed so as to be able to estLmate the number of copies present in each sample before carrying out each PCR.
The different sam~les are subjected either to a con~entional PC~ (absence of lambda S'-exonuclease) or to 20a PCR in the presence of lambda 5'-exonuclease ~process according to the invention).
The roLe of lambda 5'-exon~clease as a function of the concentration of contaminant product is shown in Figure 60 25The PCR products are separated by electrophoresis on agarose gel stained with ethidium bromide, and the DNA
is detected by exposure to W ray~.
Figure 6 ~hows the foll~owing results:
- column 1 contains molecular weight markers 30~Marker VI, Boehringer);
in `column 2: 105 copies of contaminant C~V
sequences amplified in th~ absence of lambda 5'-exo--~~ nucleas~;
`3 - in column 3: 105 copies of contaminant CM~
~ 35sequences ampli~ied after 20 minutes' contact with lambda ;~ exonucleas~ ~0.03 U/Ml); ~
- in col ~ 4: 2 x 104 copies of contaminant C~V
saquences amplified in the absence ~f lambda exonuclease;
- in column 5: 2 x 104 copies of contaminan~ C~V
~ .

~ 0 3 ~ `171 .......
",.. .
i~ - 17 ~

sequences amplified after 20 minutes' contact with lambda exonuclease (0.03 U/~il);
.- in column 6: 4 x 103 copies of contaminant C~V
sequences amplified in the absence of l~m~da exonuclease;
S - in column 7: 4 x 103 copies of contaminant CMV
sequences amplified in the presence of lambda exonuclease (0.03 U/~il, contact tLme: 2~ minutes).
It emerges from this figure:
~ that, in the absence of lambda exonuclease, a band is obtained corresponding to the CMV sequences (columns 2, 4 and 6);
- whereas in the presence of lambda exonuclease, and irrespective of the concentration of contaminant sequences, no band is obtained (columns 3, 5 and 7).
Examiple 4: Role of lambda exonuclease on genomic DN~ containing a target sequence to be detected.
Various genomic DNAs were used to assess the role of Lambda exonuclease on various DNAs. Various herpes-~iruses and papillomavirus were studied more especially.
1) Herpesviru~es:
The DNAs are obtained from cell lines infected by the following human herpesYiruses: ~S~2, CMV, VZ and EBV.
Serial dilutions are carried out before perform-ing PCR in the presence of prLmers suited to the said ~5 Yarious viruses, possessing 20 to~ 30 nucleotides and phosphorylated in a conYentional manner with a poly-nucleotide kinase.
: To carry out PCR, ~he said samples are added toa buffer containing: 10 mM Tris-HCl (pH 8.4); 50 mM ~Cl;
~ mM MgCl2; 0,01% gelatin; dNTP: 200 ~M each; appropriate ! 1~ . ! prLm~rs~ M each; TAQ polymera~e (CETUS PERBIN-ELMER
(i~ic)): 0.02 U/~l of~ reaction;:~ and lambda exonuclease : ~ ~BRL): 0 or 0.03 U/~l of reaction, depending on the pro-~` ~ C~5S carried out ~con~entional PCR or process according :` 35 to the invention, respecti~eIy).
The samples~ are subjec~ed to ~he following di~ferent thermal cycles:~
once: 37-C:for 20 minutes; 92C for 1 minute ~:~ (action of lambda exonuclease where appropriate);

:: :
:

2 ~ 7 1 ,~ .
; - 18 -- 30 times: 92C for 15 seconds; 55C for 1 ~ minute; 72C for 1 minute (actual PCR);
¦ - onca: 72C for lO minutes.
The ampli~ication produc~s are subjected to electrophoresis on 2~ agarose gel stained with ethidium bromide, and photographed on W plates.
The molecular weight markers used (Mar~er VI, Boehringex) have the ~oLlowing sizes: 2176, 1~66, 1230, 1033, 653, 517, 453, 39~, 2g8, 234, 220, 154.
Depending on the virus detec~ed, the following res~lts a~e obtained:
l.a. Varicella Zoster virus (VZV):
Serial 2-fold dilutions of genomi~ DNA of cells infected by Varicella ~oster virus are subjected to a PCR
iS with suitable phosphorylated VZV primers, in the presence and absence of lambda exonuclease, as specified ab~ve.
The PC~ products obtained with the following pri~iers:
primer 1 (~): 5' - A.JJiJiJ~TGC~T~CGT~GG~ TG~C - 3' primer 2 ~ 5' - A~A~G~GC~AATC~CG~C~ CT~C - 3' possess 189 base pairs.
After agarose gel electrophoresis, as specified above, the results illustra~ed in Figure 7 are obtained, in which the odd-numbered column~ show a PCR carried out in the absence of lambda exonuclease and the e~en-numbered columns show a PCR carried out in the presence o~ lambda exonu~lea~e.
It emerges ~rom this figure ~hat a ~detection 3 carried out using a Iong genomic DN~ i.s not in~luenced by ~d 30 the presence of lambda exonuclease.
The s;ame results ar:e obtained on other herpes-viruse~ aY ~pecified above (~icj:
;l l.b. EBV~
`i Serial 10-fold dilutions of genomic DN~ of cells i~fected by Epstein-Barr ~irus are ~ubjected ~o a PCR
with suitable phosphorylated EBV prLmers, in ~he presence and absence of Iambda exonuclease, as specified a~o~e.
The PCR products ob~ained with the ~ollowing prLmers: ~
~: :
3~ ~

2 ~ 3 ~; I r 7 ~

s~
.~ 19 --prLmex 1 (+) : 5' primer ~ 5' - - `.C'`~ C~.~GC'.'G~ .C.`-`C.; -, possess 282 ~ase pairs.
4 After agarose gel electrophoresis as, specified ~, 5 above, the results illustrated in Figure 8 are obtained, in which the odd-numbered columns show a PCR carried out in the absence of l,~m~,bda exonuclease and the even-n~mbered col~mns show a PCR carried out in the presence ~ of lambda exonuclease.
3 lO l.c. HSV2:
Serial 10-foLd dilutions of genomic DNA of cells , infected by herpes sLmplex 2 virus are subjected to a PCR
with suitable phosphorylated HSV prLmers, in the presence and absence of lambda exonuclease.
lS The PCR products obtained with the following primers:
prLm,er 1 (+) : 5' ~ C~ GCC--~GC.~CC CC~C~ 3' ' primer 2 (~) : 5' ~ C~C~_ATCG~C~CG.~G.~CC~ C-~CG - 3' ! possess 185 base pairs.
After agarose gel electrophoresis as specified ¦ above, the results illustra~ed in Figure 9 are obtained, , in which the odd-numbered columns ~how a PCR carried ou~
ti in the absence of Lambda exonuclease and the e~en-numhered columns show a PCR carried out in the presence of lambda exonuclease.
For the detection of cytomegalo~irus r th~ pro-cedure is as above, in the presence of the following prLmers:
~I primer 1 ~ 5' -primer 2 (~) : 5' -and an ampliified fragmen~ of 2S9 base pairs is o~tained.
¦ 2) Papilloma~irus (HPV 11):
Serial 2-fold dilu~ion3 of genomic DNA, obtained ~ from an anal condyloma remo~ed from a patient, are l~` 35 sub~ected to a PCR according to the pro~ocol described abo~e in 1), with suit ble phosphorylated ~PV ll prlmers~
in the presence and ab~ence of lambda exonuclease.
The PC~ products obtained with the following 1~ prLmers:
,;
,, .
.1:
r~

2~3~7~
f''~ ?~

primer 1 (+) : 5' pr~ner 2 (-) : S' - .i~ G~
possess 154 base pairs.
After agarose gel electrophoresis as speciied above, the results illustrated in Figure 10 are obtained, in which the odd-numbered columns show a PCR carried out in the absence of l~mbda exonuclease .and the even-numbered columns show a PCR carried out in the presence of lambda exonuclease.
The results illustrated in Figures 7 ~o 10 show that lambda exonuclease does not modify the yield of an ampliication carried out using a ~enomic DNA template (long ~ragment).
Example S: Role of lambda exonuclease on so-called contaminant sequences.
The contaminant DNAs are isolated by agarose gel electrophoresis and are then subjected to a passi~e diffusion at 4C in water.
The contaminant produc~s thereby obtained are diluted as specified abo~e in Example 4.1), and the PC~
protocol is identical to that described in Example 4.1).
The results obtained with~ C~, EBV and VZV as contaminant sequences are illustrated in Figures 11, 12 and 13) and show that the presence of exonuclease very strongly decreases the amplification yield of these conkaminant sequences.
a~ CMV:
The serial S-fold dilutions of ampli~ication products obtained ~y PCR with suitable phosphorylatad C~V
prLmers are sub jected to a PCR with thesP~ same prLmers~
n the presence and absence of lambda exonuclease.
Aftar agarose gel electrophore~is a~ specified abo~e in Example 4, the results illustrated in Figure 11 are obtainedr în which the odd-n~ered columns show a PC~ ca~ried out in the absence o lar~da exonuclease and the e~ren-numbered columns show a PCR carried ou~ in the presence of lambda exonuclease~
b, EBV:
The serial 5-fold dilution-q of amplification ~ ~ 3 ~ ~5 7~L
.
't~ r.~ '`

~', products obtained by PCR with suitable phosphorylated EBV
prLmers are subjected to a PCR with these s~me primers, l in the presance and absence of lambda exonuclease.
!q ~ter agarose gel electrophoresis as specified above, the results illustrated in ~igure 12 are obtained, in which the odd-numbered columns show a PCR carried out in the absence of la~da exonuclease and the e~en-numbered columns show a PCR carried out in the presence ` of lambda exonuclease.
`110 c. VZV:
The serial 5-fold dilutions of amplification products obtained by PCR with suitable phosphorylated VZ~
primers are subjected to a PCR with these same primers, in the presence and absence o~ lambda exonuclease~
l,15 After agarose gel electrophoresis as speclfied i, above, the results illus~rated in Figure 13 are obtained~
in which the odd-numbered columns show a PCR carried out in the absence of lambda exonuclease and the even-numbered columns show a PCR carxied out in the presence l 20 of lambda exonucLease.
Example 6: Action of increasing concentrations of lambda exonuclease on the amplific~tion yield of genomic DNA.
A constant quantity of genomic DNA obtained from ! 25 cells infected by VZV is subjected to a PCR in the 1 presence of increasing quantities of lambda exonuclease (0; 0.36; 0.18; 0.09; 0.045; 0.022S U/~lj according to ¦ the protocol pre~iously described in ExampL~ 4 above.
~ The re~ults are illustrated in Figure 14, in ;130 which columns 1 to 7 correspond successively to the differen~ con~entrations of lambda'exonuclease specified above. This figure shows that 0.~9 U/~l o~ l~mbda ax~nuclease does not decxease~the yield of the PCR; this shows ~hat a concentrati~n of 0.03 U/yi of reaction is ji~ 3S entirely suitable. ~ ~
As emerges~ ~rom the foregoing, the invention is in no way lLmited to those of its embodiment~ and methods :`~
o~ Lmplementation and application which have ~ust been descri~ed more explicitly; it encompasses, on the con-2 ~ 3 ~ 1 7 1 22 - . !
~, trary, all variants which may occur to the practitioner :j in the field without departing ~rom the scope or range of ' the present invention.

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Claims (11)

WO 91/00,363 - 23 - PCT/FR90/00,499

1. Process for the detection and/or identification of a nucleic acid sequence or of a mixture of nucleic acid sequences comprising an enzymatic amplification, characterized in that, after the biological sample has been suitably dissolved in order to extract the nucleic acid(s), the said process comprises the following steps:
(1) a step of destruction of the 5' ends of the double-stranded nucleic acid sequences present in the sample, by bringing the said biological sample into contact with a suitable reagent which is active at a temperature of between 37° and 42°C;
(2) a step of actual amplification, by bringing the sample obtained in (1) into contact with suitable reagents, in particular amplification primers suited to the amplification of the target sequence(s) to be detec-ted, in the presence of a thermostable DNA polymerase;
and (3) a step of detection of the amplified specific target sequences.

2. Process according to Claim 1, characterized in that the reagent of step (1), designated reagent for discrimination between contaminant sequences and target sequences, is chosen from the group which comprises suitable chemical products and enzymes which act in a weakly alkaline medium.

3. Process according to Claim 2, characterized in that the enzyme is a 5'-exonuclease.

4. Process according to Claim 3, characterized in that the 5'exonuclease is lambda 5'-exonuclease.

5. Process according to Claim 4, characterized in that the primers employed for the amplification of step (2) are phosphorylated at their 5' ends.

6. Process according to any one of Claims 1 to 5, characterized in that step (1), namely the step of destruction of the 5' ends of the double-stranded nucleic acid sequences, is carried out only once whereas step (2) is repeated at least once.

7. Process according to any one of Claims 1 to 6, characterized in that step (2) of the said process is carried out, in addition, in the presence of a tempera-ture-sensitive DNA polymerase.

8. Enzyme composition for carrying out a detection process according to any one of Claims 1 to 7, charac-terized in that it comprises a reagent which destroys the 5' ends of double-stranded nucleic acid sequences, the so-called reagent for discrimination between contaminant sequences and target sequences, and a thermostable DNA
polymerase.

9. Composition according to Claim 8, characterized in that the said discrLminatian reagent is a temperature-sensitive 5'-exonuclease.

10. Composition according to either of Claims 8 and 9, characterized in that it comprises, in addition, a DNA
polymerase which is active at 37°-42°C (temperature-sensitive).

11. Ready-to-use outfit, kit or coordinated assembly for carrying out the process according to any one of Claims 1 to 7, characterized in that it comprises, apart from the appropriate quantities of buffers and suitable reagents, of at least one suitable pair of primers and, optionally, of at least one suitable probe, suitable doses of an enzyme composition according to any one of Claims 8 to 10.