CA2205392A1 - Quantitative method for early detection of mutant alleles and diagnostic kits for carrying out the method - Google Patents

Abstract

There is disclosed a quantitative sensitive method to enable the detection of point mutations at a known site and a diagnostic kit which uses a multi step (for example, four steps) or a single step reaction. The method uses selective polymerase chain reaction (PCR) amplification of mutant test gene sequences involving first stage amplification of both mutant and wild-type sequences, first stage restriction enzyme digestion of only wild-type sequences, second stage amplification of undigested amplified fragments enriched in mutant sequences and second stage digestion of previously undigested wild-type sequences. Long and short tail primers are used in the first and second stages of amplification respectively to enable selective amplification (in the second stage) of only previously amplified material and none of the original test genomic DNA. The short tail primers are labelled with biotin and fluorescence at their respective 5' and 3' ends to enable easy detection and quantitation of mutations in the test gene via automated fluorescence readers. The use of multi steps as well as a single step reaction is disclosed. The process is exemplified with respect to its use in detecting mutations in the human K-<u>ras</u> gene, yet it is applicable for any given mutation in a defined site.

Description

, W O 96/15139 . PCT~US95/1475s " ~ 1 1 O~ANTITATIVE h~l~O~ FOR EARLY DETECTION
OF MUTANT ~T~T~r~S AND DIAGNOSTIC ~ITS FOR
CAk~Yl~ O~T T~E M~l~O~
Field o~ the Invention The in~ention relates to methods and respective kits ~or the early detection of genetic mutations. The methods and kits make use of quantitative sensitive reactions to enable detection of point mutations at known sites.
R~ll,,,.,,~ d of the Invention The development of metho~Jogies for the early detection of genetic mutations is an important issue in the ~ ~v~tion and treatment of m~ n~n~y, ~ t knowledge of the association of specific genetic alterations with the development of certain types of tumors enables an d~ oach to the early detection of cancer long before histologic or pa~ho~ogic evidence indicates the development of neoplastic tumor y owth. The identification of genetic alterations aQ
biological markers allows for early ~i~gnQsis, which in turn may di`ctate a particular regimen of treatment to ~ e~t subsequent tumor development.
The multistep process of transformation is thought to be directed by an accumulation of specific ~m;n~nt and receQsi~e genetic lesions. - -For G~- .L~le, one fre~uent ~ n~nt event in SUBSTITUTE SHEET (RULE 26) WO96/1513g PCT~sg5/l~7ss somatic cell cancers has been found to be an acti~ating mutat~on in a me_ber of the ras gene family. Strong ~oY~ exiRts for the concept that ras oncogenes are causati~e players in the multistep process of Lu~o,igenesis. Ras gene mutations occur in ~Lo~m~tely 15% of ~l~m~
Lu~o~, ho.r~vF~ their ;nC~nce appears to ~ary according to tumor type. Specifically, mutations in the hl~m~n R-ras gene ha~re been le~G~ Led to be as high as 90% in carc;nnm~Q of the pancreas, 60%
in ~A~nOC~ 'nn~~~ of the lung, and 50% ~n enocarc~n~m~R o~ the colon. In certain tumor types, R-ras gene mutations present themsel~eR as - early e~ents in the tumorigenic pathway, and e~;~n~e suggests that they are more pre~alent during the later stages of tumor development.
The most fre~uently acti~ated position of the R-ras gene in hl~m~n ~u~o,~ has been found to be co~n 12. Therefore, by way of example, the detection of mutations at thi~ ~osition could be of critical i~ Lance in many aspects of research and ~;~gno~tic application. Furth~n~e, the rl; n; cal application of the assay for detection of mutations in biological samples could be of great ~ oy~ostic ~alue, and could assist in e~aluating early courses of patient intervention.
Presently, the detection of mutant genes has been accomp~ h~ through the use of the polymerase rh~n reaction (PCR), a guick and simple in-~itro reaction through which adequate ~ounts of a specific gene region ca~ be generated ~or subsequent analys~s. Ampl~ied DNA fr~m~nts ha~e been analyzed for the presence of point mutations employing one of several technical ~ o~ch~R briefly out~ ;n~ below:
1. Single-Rtrand conformation poly~o hism analysis is a method for analyzing SUBSlTl UTE SHEET (RULE 26) CA 0220~392 1997-07-10 WO 96/15139 PCT/~JS95/14755 DNA for nucleotide substitutions. In this method, amplified m~terial is denatured to create single-str~n~ DNA and separated on a native polyacryl ~m; ~e gel under conditions that enable distinction between single strands of n~ and mutant alleles, each migrating at a different rate. This t~chn;que can be used to identify alterations in any given gene without reguiring knowledge of the specific site where a mutation has OC~ul ed.

2. Se~n~;ng an amplified product of a specific gene is an approach that leads to the direct identification of a mutated site. This .~ d~ oach is the most labor-intensive, yet it provides c~plete information with respect to the type of mutation and its precise location.

3. Restriction fr~m~nt length poly~hism where PCR amplified products are digested with specific restriction enzymes which can selectively digest either a no~-l or a mutated allele or a particular gene; To obtain higher sensitivity, the RF~P has been modified through the ;n~o~poration of a liquid hybr;~ tion step in wh;r~ ~mpl;fied material i~
hybridized with a labelled oligonucleotide sequence which is specific for a mutated region prior to separation on PAGE. This approach, also known as high resolution RF~P analysis, ~l;~;n~tes the need for se~enc~ng, but it is l;~;ted to the 3~ analysis of mutations at a precise location that involves a naturally occurring restriction enzyme site. To overcome this l;~;tation, one can artific;~lly introduce restriction enzyme sites to permit a distinction to be made between no~-l and mutant alleles where the position of the point mutation does not h~hor a naturally occurring site. In this approach, base-pair substitutionR

SUBSTITUTE SHEET (RULE 26) WO96/15139 PCT~S95/1475s are introduced into the primers used for the PCR, - yiel~;n~ a restriction enzyme site only when the primer flanks a specific point mutation. This approach enables the Relecti~e identification of a point mutation at a known site of pres~ hly any gene.

4. Enriched PCR is a modification introduced by the in~entor (coll~ho~ating with others) into the RF~P 'analysis which permits the detection of a mutant gene e~en when the mutation -is present at ~ery low frequency (i.e. 1 in 104 ~nrm~l alleles). T_e pr;ncirle of this a~Loach is to create a restriction enzyme site only with norm~l seguences, thus enabling selecti~e digestion of norm~l but not of mutant alleles amplified in a first amplification step. This e~c~ts the non-mutant'DN~ from further amplification in a second amplification step while, upon subsequent amplification, the mutated alleles are enriched.
' 5; ''Mismatched 3' end amplifications is a PCR techn;gue ~-h;rh utilizes 5' primers that ha~e been modified at the 3' end to match only one specific point'mutation. This method relies on ron~;tions under which primers with 3' ends c~mrlementary to specific m R~~tches are Amrl ~fied, whereas wild-type seguences preclude primer elongation. Thi~ procedure requires a specific primer for each suspected alteration and must be carried out under rigorou~ c~n~itions.
The in~entor, in rol~ho ation with others, has pre~iously shown that PCR
~ fication of human X-ras gene first exon sequences can be ~cQmrl~he~ using an upstream primer (R5') ~nao~n~ a G ~ C substitution at the first posit~on of ~o~n 11 (Jiang et al, Qn~o~ene, 4,923 - 928 (1989)). The sequence of R5' thus SUBSTtTUTE SHEET(RULE 26) WO 9~i/15139 P~ /14755 m~A; Ate8 a B~tNI restriction enzyme site (CCTGG) overlapping the first ~wo nucleotide8 of wild-type coA~n 12. Since this site is ab8ent from mutuant coAn~ 12 frA~m~nts, RF~P analysis of the ampiified ~Lod~cts can be used to detect X-ras oncogenes activated at coAQn 12. I~G Lantly, a 8~c~nA
BstNI site may be strategically inco ~o~ted into the downstream primer (R3') as an int~n~ control for enzyme fidelity.
The principle behind the 'enriched' amplification procedure of the prior art is described in an article co-authored by the inventor (Rahn et al, (1991). Oncogene, 6, 1079 -1083) and shown in the schematic flow diagram of Figure 1. R-ra~ first exon sequences are PCR
amplified using the upstream primer, R5', and a new downstream primer R3' wt which lacks an -int~n~l control BstNI restriction site. The 157 nt long frA~m~nt is digested with BstNI, thereby clea~ing wild type fr~m~nts and rendering them inaccessible for subsequent amplification. The ~ od~cts of the dige~tion, enriched in full length mutated ~oAon 12 sequences, are then used in a 8~CQ~ round of PCR amplification with primers R5 and R3'. These samples are subject to RFLP
analysis by digestion with BstNI, followed by polyacryl ~m~ Ae gel electrophoresis of the ~cts.
Referring to Figure 1, in a first round of amplification (A), primers R5' and R3' wt (wild-type) are utilized for the synthesis of a 157 nt fragment inclùding codon 12 sequencès. R5' contains a nucleotide substitution at the first position o~ codon 11, creating a BstNI restriction s~te (~lW) o~erlapping the first two nucleotides of wild type codon 12 (hatched box). Digestio~ of - -PCR amplified ~equences rom the first round with SUBSTITUTE SHEET (RULE 26) WO 96115139. , PCT/US9S/147!i5 r BstNI lea~es uncleaved products enriched in mutant roAnn 12 sequences (black box). These unclea~ed oducts are suhject to a second round of ampli~ication (8) using primers R5' and R3' (Cont~;ntng a control BstNI site; crogg-ha~ch~A
box). ~pon RF~P analysis with BstNI, sequences deri~ed ~rom a mutated codon 12 allele show h~n~R
of 143 and 14 nt, while amplified wild type allele r~mn~nts are clea~ed to generate fragments of 114, 29, and 14 nt.
While the enriched PCR and other ~chn;ques discussed above pro~ide appro~rhe~ for the possible early detection of mutant alleles, - there are drawh~kR in their use for the identification of a mutant allele in a pre-neoplastic lesion. So, for example~, in the enriched PCR technique, although it may be desirable to amplify in a second ~m~l; f;c~tion step only duplexes which were form~ in a first amplification step, no procedure has been pro~ided to ~ev~t amplification in a second amplification step of genomic DNA which was present in a test ~rle originally. Another drawback of the enriched PCR ~chn;que and t~e other prior art techn;~ues discussed above is that they are c~mhersome and are not easily adapted for use in ~ no~tic kits. For example, the pre~iously used method for detection of mutant alleles in the enriched PCR ~echn;que in~ol~es a gel separation of selecti~ely ~plified mutant alleles from others. What has been ~eeAe~ is a more sensiti~e and less cumbersome method of detection that can be Qa8ily C~vel Led into a A; ~n~tic k;t. What has also been n~eA~ iB a quantitati~e procedure to enable quantification of the results of a genetic scr~en; n~ . What has furt_er been needed is a simplification of the t_ree stage procedure SUBSTITUTE SHEET (RULE 26) CA 0220~392 1997-07-10 W096/15139 PCT~S95114755 of the prior art (involving ampl~fication, digestion, re-amplification and final digestion followed by PAGE analysis).
SummarY of the Invention 0 5 It is an ob;ect of the invention to provide a ~uantitative assay for the detection of a mutant allele in a pre-neoplastic lesion.
It is another object of the invention to provide a method for ~rlifying a ~ucleic-acid duplex present in a test sample in a two step amplification process which enables amplification in a s~r~n~ amplification step only of duplexes formed in a first amplification step and thereby prevents ~mplification in the s~ro~ amplification ~tep of duplexes which were present in the test sample prior to the first amplification step (e.g., genomic DNA).
It is a further object of the invention to provide a more sensitive assay for the detection of a mutant allele in a test sample.
A still further object of the invention is to provide a quantitati~e assay for the detection of mutations in a test gene, such as codon 12 of a R-ras gene.
Yet ~other object of the invention is to provide reagent mi~.es-which can be used in a ~;~gnostic assay to detect and quantify the presence of mutations in a gene.
It is a still urther object of the invention to provide ~;~nostic kits which may be used easily to detect point mutations in a test gene.
It is also an object of the invention to provide a process whi rh simplifies and affords time savings over the multi-stage processes of the prior art.
To achieve the above and other objects SUBS 11 l UTE SHEET (RULE 26) ~096/15139 , PCT~S95/14755 of the in~ention, there is pro~ided a process for analyzing a nucleic acid test sample taken $rom the gen~ of an organism for the detect~on of a mutant nucleotide sequence in a spe~ f; c region of the g~n~?, wherein the region can contain the mutant nucleotide.sequence e~en at a frequency'of 10 5. The process comprises:
(i) a first ~mpl; f; cation step comprising amplifying,material in first and sec genomic duplexes present in the test ~^mrle ~n a $irst polymerase rh~;n reaction in which u,pstre~
,and downstream long tail primers, comprising upstream primer and downstream primer nucleotide ",. sequences respecti~ely, DNA polymerase, four different nucleotide triphosphates and a buf$er are used in a repetitive series of reaction step~
in~ol~ing template denaturation,'primer ~nn~l ;ng and extenR;Qn o$ ~nn~led,primers to form first and s~cQn~ synthesized nucleic acid duplexes.
Each of the first syn~he~;~ed nucleic acid duplexes has an upstream end and a downstream end and consists of a first synthesized ~trand and a first complementary synthesized stra,nd. Each of the 8~con~ synthesized nucleic acid duplexes has an ,upstream end and a downstream end and consi~tR
of, a 8~c~n~ syn~h~R;~ed strand and a 8~n~
c~mrlementary synthesized strand. Each of the first and sec~n~ synthe~;~ed strands has a first end portion co~rising the upstream primer nucleotide sequences and a second end port~on comprising nucleotide se~e~c~R'sufficiently complementary to the downstream primer n~leotide sequences to nnne~l therewith. The first synthesized duplexes ha~e the region with a mutant 35 nucleotide se~uence, and the second synthesized duplexes ha~e the reg~on w~th a wild-type nucleotide seguence. The upstream and downstream SUBSTITUTE SHEET (RULE 26) CA 0220~392 1997-07-10 W096115139 PCT~S9~/147~

_ g long tail primers are selected such that nucleic acid strands formed in the first polymerase rh~n reaction using the upstream and downstream long - tail primers can ~nn~l with short tail primers which do not ~nn~l with any nucleic acid strands in the (first or seconA) genomic duplexes. The long tail upstream primers are also selected such that the s~ronA synthesized duplexes have a restriction site which is not present in the first synthesized duplexes due to the presence in the first synt-h~n~ed duplexes of the region with the mutant nucleotide seguence. The restriction site is cleavable with a first restriction enzyme.
ii) a digestion step comprising treating at least a portion of the test sample with the first restriction enzyme whereby selecti~ely to cleave the seconA synthesized duplexes while lea~ing the first synthesized duplexes unclea~ed, iii) a second amplification step comprising amplifying material that was subjected to restriction enzyme digestion in step (ii) and r~m~:n~A unclea~ed. In this amplification step, upstream and downstream short tail primers are used in a s~conA polymerase chain reaction , 25 selecti~ely to ream,plify material which was synth~R;~ed in the first,amplification step and was not affected by the restriction enzyme in step ~ii) since it h~ho. 8 a mutation in the specific region of the genome. The upstream and downstream 30 ' short tail primers are selected such that they e~l with the first synth~ ed complementary and first syn~h~R~èd strands respecti~ely but do not ~nne~l with strands of the first or second genomic duplexes whereby the upstream and downstream short tail primers can be used in the 8~cQnA ~r~ cation step selecti~ely to amplify material in duplexes fo~meA in the first SUBSlTTUTE SHEET (RULE 26) . CA 0220~392 1997-07-10 ~ , .

, WO96/15139 . PCT~S95114755 - 10 _ amplification step but c~nnot amplify material in the first or second genomic duplexe8. Each of the upstream short tail primers are labelled with a first substance that bindB tightly with a second substance such that upstream ends of the further synthesized duplexes bind to a ~u~o ~ ng surface coated with the second 8UbstanCe. Each of the downstream short tail primers are labelled with a downstream label such that downstrQam ends of the further synthesized duplexes ha~e the downstream label. The second amplification step is perfo,~7 in a ~essel (e.g. microwell plate: Eppendorf tube) ha~ing the ~u~o Ling surface coated with the second substance such that further synthesized duplexes labelled with the first substance contact and bind to the ~u~v~Ling surface, or the process includes a b;n~in~ step comprising contacting the test sample with the ~o Ling surface coated with the second substance whereby further synthesized duplexe~ labelled with the ~irst substance bind thereto. The b~ n~; ng step can be per$ormed, for example, after second stage amplification or after a subsequent digestion with the restricti~n enzyme.
i~) a second digestion step wherein the test sample is again treated with the first restriction enzyme selectively to clea~e synthesized duplexes cont~in~ng regions having the wild-type sequence;
~) a w~nh~ng step to ~ve at least downstream portions of clea~ed duplexe~ from the ~u~G Ling surfacef and vi) a detection step comprising assaying for the presence of the downstream label on the ~u~L Ling surface.
In accordance with the invention, there is also pro~ided a ~ nostic kit for use in an SUBSTITUTE SHEET (RULE ;!6) . .

assay for detecting the presence or absençe on a first gen~m;c nucleic acid strand of a gPn~;c ~ region conta~n;n~ a mutant nucleotide sQquence, wherein the genomic region ca,n contain'the mutant nucleotide sequence or a wild-type sequence, wherein the f~rst genomic nucleic acid strand is present in a test sample in the form of a first gPnom~c duplex consisting of the first genomic -nucleic acid strand and a first complementary nucleic acid ~trand, and wherein the assay romr~ises at least a first and a seco amplification step. The kit compr~ses:
a) a first reagent mixture for use in , the first amplification step wherein material in the first nucleic acid dupl OEes is amplified in a - polymerase ch~;n reaction with synthesis of a first sy~thesized duplex ha~ing a first synthesized nucleic acid strand and a first complementary syn~hP~i~ed strand. The first reagent mi~L~ a comprises upstream a~d downstream long tail primers. Each of the upstream and downstream long tail primers comprises a complementary primer portion and a non-c~plementary primer portion. The complementary primer portion of the upstream long tail primerR
is sufficiently r~rlementary to a first end portion of the first complementary nucle~c acid strand to enable the upstream long tail primers,to ~nn~l therewith and'thereby to initiate synthesis of a nucleic acid ex~en~ product using the ~irst c~ ementary nucleic acid strand as a template. The compl~mentary primer portion of the~
downstream long tail primers is sufficiently c~mrlementary to a first end portion of the first gPnnm;c strand to enable the downstream primers to ~nne~ 1 therewith and thereby to initiate synthesis of a nucleic acid extension product using the SUBSTITUTE SHEET (RULE 26) WO 96115139 PCI~/~JS95/14755 first genomic strand as a template. The no~-complementary primer portion8 of the upstream and ~ downstream long tail primer8 are not su~ficiently - complementary to either the first genomic strand or the first complementary nucleic acid strand to ~nn~l with either. The non-complementary primer portions of the respective upstream and downstream long tail primers are positioned on the respective upstream and downstr~am long tail primers such that a first end portion of the first synthesized strand has nucleotide sequences that are identical to the nucleotide seguences of the non-complementary primer portion of the upstream long tail primers and such that a first end portion of the first complementary synth~ ed strand has nucleotide sequences that are identical to the nucleotide sequences of the non-complementary primer portion of the downstream primers; and b) a second reagent mixture ~or use in the second amplification step c~ ising upstream and downstream short tail primers. Each of the upstream short tail primers has nucleotide seguences which are sufficiently complementary to the nucleotide seguences in the non-complementary primer portion of the upstream long tail primers to ~nne~l therewith but which are not sufficiently complementary to nucleotide Qequences in either the first g~n~m; C strand or the first complementary genomic strand to ~nn~l therewith.
Each of the downstream short tail primers has nucleotide sequences which are sufficiently complementary to the ~ucleotide sequences in the non-complementary primer portion o~ the downstream long tail primers to ~nn~l therewith but which are not sufficiently comrl~entary to nucleotide sequences in either the first gen~m;c strand or the first complementary gen~m~c strand to ~nn~l SUBSTITUTE SHEET (RULE 26) -` W O 96/lS139 PCTrUS95/14755 .

therewith, whereby the upstream and downstream short tail primers can be used in the second - ampliication step selecti~ely to amplify material - in dupl OE es synth~;red in the fir8t ~rl~fication u 5 step and none o~ the first genomic duplexes.
Each of the first and seq~n~ reagent mixtures can also contain four different nucleotide triphosphates, an agent _or nucleic acid polymerization under hybridizing conditions 10 and a buffer. The upstream short tail primers of the 8~0~ reagent mixture can be labelled with a first r~r~und, such as biotin, which binds tightly with a 8~ro~ compound, such as avidin or strepta~idin, and the downstream short tail 15 primers of the second reagent mi~L~e can be labelled with a r~;o~rti~e or fluorescent label.
The kit may be provided with a first microtiter plate which contains the first reagent m~xture such that the first amplification step can 20 be perfsrme~ in the fir~t plate simply by ~;ng the test ~rle thereto. The kit can also contain a second microtiter plate which is coated with the s~r~n~ c~ d. The seco~ plate can contain a restriction enzyme ~-h;rh selecti~rely digests 25 synthesized nucleic acid duplexes if a wild-type nucleotide seguence is present in a genomic region of one of the nucleic acid strands of the duplexes. The reaction mi~Lu~e of the first ampl;f;r~tion step, or a portion thereof, can thus 30 be ~eA to the s~rQn~ plate whe~e~ synthesized duplexes cont~;n;n~ a genomic region with a wild-type seguence will be selecti~ely digested. The s~ron~ reagent mi..L..Le can be s~9-g to the 8e90 plate after the digestion to perform the secon~
- 35 amplification step. The second reagent mi~L~e can contain the upstre~m short tail primers labelled with biotin such that nucleic acid SUBSTITUTE SHEET (RULE 26) W096/15139 PCT~sgS/147~s duplexes formed in the s~ron~ amplification step will bind to the second plate. The kit can contain additional amounts of the restriction enzyme and its buffer 80 that the duplexes bound to the second plate can be further digested.
After such further digestion, the s~cQn~ plate can be w~hP~ to ~ve ~"hound duplexes such that only uncleaved duplexes ha~ing regions with the mutant nucleotide se~en~e will r~-; n in the second plate. These unclea~ed duplexes will ha~e fluorescent or r~;o~cti~e labels at their downstream ends and can be rs~ y assayed and ~uantified.
Brief Description of the Drawin~
Figure 1 is a flow chart depicting the - enriched PCR procedure of the prior art;
Figure 2 is a flow chart depicting the steps of the quantitati~e process of the present in~ention.
Figure 3 is a dia~ d~atic exemplifica-tion of lo~g and short tail primers of the~
invention for use with roA~n 12 of the h--m~n R-raB
gene.
Detailed DescriPtion The i~l~val process of the present in~ention will now be discussed with reference to the figures of the drawing. As will be understood in this discussion, the process can be readily adapted for use in a ~ nostic kit. For e~d~ple, the procedure can be performed in microtiter plates pro~ided in the kit. A set of two plates can be used to ~nmplete the process. Each plate can come with its own set of reagents to r~n;m~e the need for addition of reagents. Additional steps can be perfo~m~ using pro~ided solutions (i.e. pre~ously prepared m~L~las in separate cont~:n~s) all on the same plate or plates. With SUBSTlTtJTE SHEET(RULE 26) . ~ , the exception of the last step, which is guantitation on a microtiter plate using, for e, an auto_ated ~r;T,c~ reader, all steps can be performed in a PCR m; c~ycler.
Figure 2 is a f}ow chart showing the ~arious stages of a preferred ~mhoA~m~nt of the in~enti~e process for detecting a _utation in a target coAnn of a genomic duplex fr~m~nt. In a first stage of the process (see Figure 2: ~First Stage Amplificationn), _ateriai in the genomic dupl OE is ~rl~fied in a polymerase ~ n reaction using upstream and downstream long tail primers, each of which comprises a complementary portion which is complementary to one of the nucleic acid strands in the genom;c duplex and a non-complementary portion which is not complementary to either of the strands in the duplex. In a preferred embo~;ment of the invention, a test ~mple cont~;~; ng genomic DNA duplexes suspected of cont~: n; ng a mutation in a spec~ f; C codon of one of the strands iB ~ d to a first microtiter plate that has a plurality of wells each of which contains a first reagent mix with all reagents neces~ary ~or first stage amplif~cation of the duplexes in the test sample. The original test or genomic DNA can be taken for eY~m~le from a hllm~n or other mammal. The source of the DNA can be tisQue, biopsy, or body fluids (e.g., hlooA, effluents, pancreatic juice). ThQ reagent mix includes long tailed primers, dNTPs, a DNA
poly~elase and its respectiYe buffer. The plate is then placed on a PCR mic o~yeler for first stage Ampl; fication of material in the genomic duplexes in a repetiti~e series of reaction steps in~ol~ing template denaturation, primer ~nn~ n~
and exten~;Qn of ~n~l ed ~oducts to form 8yn~h~ ed DNA duplexes.

SUBSTITUTE SHEET (RULE 26) .
~096/15139 PCT~S95/1475s After the first stage of amplification, the reaction mix will contain amplified _aterial and genomic DMA. The amplified material consists of the portion of genomic DNA fl~nke~ by primers (e.g., 196 base pairs of ~-ras roA~n 12- see Example I, infra), which was multiplied (~ 106 times within the first 20 cycles) during first stage Ampl; fication. The amplified material -- includes material which h~ hQ~8 mutation and that which does not. The long tail primers simply add tails to the amplified region. The tails have no effect at this stage of reaction. ~.~ev~ , after digestion, the tails will allow amplification of previously amplified material and none of the original genomic DNA.
As will readily be apparent to those of skill in the art, the upstream end portions of the duplexes synthesized in the first amplification stage contain the nucleotide sequences of the upstream long tail primers. The upstream pr~mers can be synthesized or selected 80 as to m~ te a restriction site in a specific co~n of a synthesized strand if and only if the co~on is present in the strand with the wild-type nucleotide sequence. Products of the first stage - A~rl;fication _ay be treated with a specific restriction enzyme which will clea~e at the me~; ~ted restriction site, and synthesized duplexes con~; n; ng reagents with the wild-type nucleotide sequences will be selecti~ely cleaved while lea~ing synthesized duplexes ha~ing the co~nn with a mutant nùcleotide sequence uncleaved.
Of course, it is important that the synthesized duplexes contain one and only one restriction site for the ~pecific restriction enzyme.
The ~,od~cts of the firnt stage amplification can be kept as a reference for SU8STITUTE SHEET (RULE 26) WO 96115139 PCI~/US9~/147~;5 . ~ .

future needs, with the exception of a portion, for G~ ~ .L.le 5 ~1, that can be taken from each of the ~- wells and transferred to a 8econd microtiter plate ha~ing a plurality of wells, each of which is provided with a second reagent mix cont~; n; n~ the specific restriction enzyme and its respecti~e buffer. The 8~cnn~ plate can be placed in the PCR
microcycler for a time and at a temperature sufficient for the restriction enzyme to clea~e 10 the synth~ ed duplexes ha~ing wild-type ~equences in the gennm;c region (for example, 1 hour at 60C.). Total ~olume can be controlled 80 as not to exceed, for example, 10 ~l. This plate can be already coated with avidin, to be used in subse~uent steps.
The duplexes synthesized in the first stage amplification are then treated with the specific restriction enzyme in a digestion step (see Figure 2: "First Stage Digestion"). If the ~o~ contains a wild-type sequence such that the upstream long tail primer m~ tes a restriction site in the synthesized duplexes, the duplexes will be clea~ed by the restriction enzyme during the digestion ~tep, a~ shown in the duplex labelled nNormaln in Figure 2. If the co~n of the synthesized duplexes contains a mutated nucleoti~e se~uence such that there is no restriction site for the enzyme present in the ~yn~h~;7ed duplex, the duplex will not be cleaved by the restriction enzyme during the digestion step and will appear as shown in the duplex labelled nMutant" in Figure 2.
A second amplification step is then performed selectively to amplify material in synthesized duplexes which were not digested in the first stage dige~tion (see Figure 2: "Second Stage Amplificationn). For example, to each well SUBSTITUTE SHEET (RULE Z6) W,096115139 of the s~cQn~ microtiter plate a third mix of reagents can be ~ . This mix consists of the reagents necessary to carry out the second stage amplification, including the short tailed primers which are labelled with biotin B on the upstream or 5' primer and fluorescence F on the downstream or 3' primer. Addit~ nn~l polymerase, buffer, and water to adjust total buf$er to a desired ~olume (e.g. lO0 ~l) can also be part of this mix. The plate will be r~ ~c~ in the PCR mi~ ~ ycler under suitable conditions and for sufficient cycles in~ol~ing template denaturation, primer ~nn~l ;n~
and extension of ~nn~led products (e.g., 30 cycles) to effect further ~mpl;~ication.
The upstream and downstream short tail primers used in the second stage ~plification can ~nn~ 1 to the respecti~e strands of the duplexes fo~m~ in the first stage amplification but cannot ~nn~l to the gtrands of the gennmic duplexes.
The upstream short tail primers are synthesized such that they contain biotin B which can bind to a ~OL Ling surface coated, for example, with a~idin or streptavidin. Downstream short tail primers are labelled with a fluorescent material F
(preferably fluorescein) such that strands formed with these primers can be eas~ly detected and guantified in subsequent steps. Technigues for in~Lo~ ;n~ biotin at the 5~ or 3~ t~m~n-ln o~
olig~n--~l eotides are known in the art, as are ~rhn;ques ~or repln~;n~ dT re~ with biotin-dT residues within the oligonucleotide seguence.
For ~ le, the litèrature describes the coupling of a biotin phosphoramidite during olig~n-~leotide sy~thesis. A.J. Cocuzza, Tetrahedron ~ett., 1989, 30, 6287 - 6290. The literature also describe~
p ~l~cts capable of br~n~h;n~ to alIow multiple biotin additions at the 3' or 5~ ~m; n~

SUBSTITUTE SHEET(RULE 26) CA 02205392 1997-07-10 . -WO 96115139 . PCI~/US95/14755 P.S. Nelson, M. Kent, and S. Muthini, Nucleic Acids Res. 1992, 20, 6253 - 6259. S~m;larly, ~~techniques for the synthesis of fluorescein labelled se~enc~n~ primers are known. E.g., F.

- 5 Schubert, R. Ahlert, D. Cech, and A. Rosenthal, Nucleic Ac;~ Res. 1990, 18, 3427.
Further synthesized strands formed in the 8~con~ amplification stage comprise a first end portion with the nucleotide sequences-of the upstream short tail primer and a ser~n~ end portion c~is~ng nucleotide sequences comp}ementary to the nucleotide sequences in the downstream short tail primers. The further synthesized duplexes are labelled with biotin B at their upstream ends and with fluorescence F at their downstream ends.
Following the second stage amplifica-tion, the unbound material can be washed away from the plate, thus lea~ing only the a~idin biotin coupled material intact. The further synthesized duplexes are then subjected to a second digestion step (see Figure 2: "Second Stage Digestion") selectively to cleave duplexes ha~ing the region comprising wild-type sequences. To this end, a fourth mix can be ~e~ to the plate. This mix can contain new portions of the restriction enzyme ~long with a suitable buffer. Digestion can be performed for sufficient time to cleave any r~m~ ~ n ~ ng unclea~ed duplexes cont~ n ~ ng a wild-type region (e.g. 1 hour at 60C.). Followingthis step, the plate can be~washed and subjected to quantitation ~ia known techniques. Preferably, direct quantitation of the fluorescein signal is made ~ia a fluorescence reader (available comm~cially). Alternative quantitation procedures include (a) using antibodies against fluorescein which are then detected ~ia known SUBSTITUTE SHEET (RULE 26) CA 0220~392 lgg7-07-lo . ~r 96/15139 PCT~S95/14755 enzyme-l;nk~ ;mm~nosorbent assay (~T-T,~) techn;ques (see, e.g. ~andgraf et al, Anal Biochem 198:86 - 91 (1991), Alard et al, Biotechn;ques- -15:730 - 7 (1993); Rostyn et al, Hum T~nol 38:148 - 58 (1993) and Taniguchi et al, J. Tmmnnol -Methods 169: 101 - 9 (1994)); (b) using a r~A;s~ti~e labelled 3' primer and quantitation in a suitable counter, or (c) use of non-labelled 3' primer but, following the second ampl;f;~tion, denaturing the fragment and perfQ~ ;n~
hybr;~7~tion with a probe which corresponds to the ~oAnn 12 mutant allele (or other test region).
For example, the denatured _ragment can be hybridized with 32P-labelled or fluorescent probes specific for each possible mutation in coA~ 12 o_ the h~m~n K-ras oncogene (probes a~ailable, e.g., from Clontech, Palo Alto, CA). Specific hybridization will add further assurance for the specific quantitation of the mutant alleles a~d enables a further tenfold increase in sensitivity.
The in~entor has used the latter approach successfully with r~;o~cti~e probes for ~oAn~ 12 of R-ras when the syn~h~Ri7ed fr~gm~nt was immobilized on a nitrocellulose m~L due, and ha~
achieved a sensiti~$ty of 1/10-5.
The polymerase chain reaction in each o_ the ~mrl~f;cation steps ~iRcl~Rsed abo~e can be performed by incubating the test sample at three temperatures corresron~; n~ to the three steps in a cycle of amplification - denaturation, ~nn~ ng and extension. This c~ycling can be perfo~m~A
automatically, for example, in a PCR mi~ G~ycler such as the 1605 AirThermo-Cycler (Idaho Technology), or with a DNA Therm~l Cycler (Perk~nelm~ Cetus Instruments). It can also be performed ~nl-~lly~ for ~Y~rle, with pre-set water baths. Typically, the nucleic acid duplexes SUBSTITUTE SHEET (RULE 26) .
~ .
WO 96/15139 PCI~/US95114755 .

in the test sample may be denatured by briefly heating the sample to about 90 - 95C., the primers may be allowed to ~nne~l to their complementary seguences by briefly cooling to 40 -- 5 60C., followed by heating to about 70 - 75C. to extend the ~nn~led primers with a suitable agent for polymerization. Suitable agents for polymerization may be any c~yO~d or system which will function to ~cr~mrl~h the synthesis of primer extension products, including enzymes.
Suitable enzymes for this purpose include, for ~"'L'l e, E.-coli DNA polymerase I. ~lenow fragment of E. coli DNA polymerase I, T4 DNA polymerase, other a~ hl e DNA polymerases, incl~; ng heat-stable enzymes, which will facilitate combinationof the nucleotides in the proper m~nne~ to form the primer extension products which are complementary to each nucleic acid strand.
Generally, the synthesis will be initiated at the 3' end of each primer and proceed in the 5' direction along the template strand, until .8ynth~ te~m; n~ tes, pro~a; ng molecules of different lengths. There may be polymerization agents, ho~_v~L, which initiate synthesis at the 5' end and proceed in the other direction, using the s~e process as described abo~e. A
partic~ ly preferred polymerization agent ~or use in the in~ention is a therm~stable DNA
polymera~e isolated from ~herm~ aauaticus (known as Ta~ poly~e ~se) ~ince it can withstand repeated exposure to the high temperatures (90 - 95C.) required for strand separation.
Although conditions for achieving opt;m~l success in polymerase chain reactions can ~ary, ron~tions for ron~rting suitable polymerase ~ n reactions to amplify nucleic acid (preferably DNA) duplexes in any gi~en test sample SUBSTITUTE SHEET (RULE 26) CA 0220~392 1997-07-10 W09~/15139 PCT~S95/1475s of the invention can be routinely deri~ed from --' st~n~d parameters. By way of example, a st~n~d PCR of the invention _ay be perform~-in the first microtiter plate in a buf$ered solution ha~ing a ~olume of between about 25 - 150 ~1. In addition to a sample DNA, the solution can contain the DN~ polymerase'(preferably Taq polymerase) ~n an amount of between about 1.5 - 5 units (preferably 2.5 units)','each deoxynucleotide triphosphate (dATPi dCTPj dGTP and dTTP) in an amount of between'about .1 - .25 mM (preferably 0.2 mM), and a buffer in an amount of between about 2 - 15 ~1 (pen~ ng final ~olume). The buffer will preferably contain 10 mM Tris HCl, pH
8.3, and 50 mM ~Cl and will be present in an amount sufficient to maintain the solution at a pH
of about 7 - 9. The''amount of primer can be carefully quantitated for each amplification step.
For example, in the first amplification step, not less than 5 ng and not more than 70 ng (preferably 20 ng) can be used to ~l~v~t excess o$ primers that could be transferred to the subsequent steps (such an excess creates artificial h~qhJ G~d which should be a~oided). The second amplifica--25 tion step can contain a st~n~d excess ha~ing, for example, not less than 50 ng'and not more than 200 ng (preferably 150 ng) of each primer.
As can be appreciated, in one embodiment of the in~ention the nucleotide sequences of the respective oligonucleotide primers in the first and second ~mpl~f cation steps are selected such that they are capable of selecti~ely amplifying in the sec~n~ amplification step PCR material that was synth~Q~ed in the first amplification step PCR and none of the genomic DNA. Thus, the step amplification is selecti~e $or the mutant allelQs a~d el~m~n~tes bac~y ~u~d material that SUBSTITUTE SHEET (RULE 26) WO 96/15139 PCI~/US95/14755 could, l~ntil now, be synthe~ized from the origina g~nnm~ C DN~. Thig is i~o L~ut in applications where, for example, sequences not complementary to a g~n~m; C template are ~ to the 5' end of the primers to provide a means of intro~c~n~, for G-- lle, restriction sites into the PCR product.
This has been achieved using sequence8 taken from polyo_a ~irus, not present in the human gPn~m~.
Once the sequence of a specific region of a target gene is known and the position of a mutation within the region defined, the design of suitable long tail and short tail primers in ~dance with the above principles will be routine for those of skill in the art using known t~hn;~ues. Tn~ to ~LGlùce specific upstream~
and downstream long tail oligonucleotide primers each having a portion which is complementary to different strands of a nucleic acid duplex cont~n~n~ desired nucleotide sequences, it is only nece~sary that a sufficient number of bases at both ends of the seguence be known in su ~icient detail 80 that c plementary portions of the up~tream and downstream oligonucleotide primers can be prepared which will ~nn~l to di~ferent strands of the desired sequence and at relati~e positions along the sequence such that an exten~lon ~ ~uct syn~h~s ~ed from one primer, when it i8 separated from its template ~ ement), can serve as a template for extension of the other primer into a nucleic acid o~-defined length. The long tail primers are fo-meA such that seqùences not complementary to the t~rl~te are present at the 5' ends of the upstream and downstream long tail primers respectively. These e~G~ OU8 non-complementary se~uences ~ecome inco~ ated into the first stage PCR amplification products and the amplification , SUBSmUTE SHEET (RULE 26) CA 0220~392 1997-07-10 ~096/15139 , PCT~S95/14755 ducts thus co_prise ~y~ous end portions on both their upstream and downstream ends. These portions are not present in the original g~nnm;c duplexes. The upstream and downstream short tail primers are selected or synth~R;~ed 80 as to only to ~Gy~OU8 end portions whereby they can be used to selecti~ely ~mrlify only mat'erial in the PCR amplification ~ oducts and none of the geno_ic strands. The long tail primers will preferabiy be between a,bout 40 and 60~base pairs in len,gth and the short tail primers will preferably be between about 15 and,25 base pairs in length and will o~erlap the 5' ends of the long tail primers.
The selection of suitable primers will - now be ~Y~mplified with respect to Figure 3 which shows long and short tail primers of the invention for use in syn~h~ ;n~ a 196 nucleotide fr~m~nt, including coAon 12, of the h-lm~n R-ras gene. As ' 20 can be seen in Figures 3 and Example 1 (infra), a preferred upstream long tail primer can consist of 44,nucleotides of which 20 nucleotides overlap the exon sequences of the K-ras gene ~m~ tely preceding codon 12. (The upstrQam long tail primer shown in Figure 3 is A~ neA to o~erlap the exon seguences up to, but not including, the nucleotide seguences of coAnn 12). The upstream long tail primer encoA~ a G ~ C substitution at the first position of codon 11. The upstream long tail primer in Figure 3 thus meA;~tes a BstNI
restriction enzyme site (CCTGG) overlapping the first two nucleotides of wild type ~oAnn 12 (GG).
The upstrQam short tail primer shown in Figure 3 consists of 24 nucleotides which o~erlap a 24 nucleotide region at the 5' end of the upstream long tail primer. The short tail primer ,seguences will preferably be derived from polyoma SUBSllTUTE SHEET (RULE 26) CA 0220~392 1997-07-10 W096115139 pcT~sssll47ss .. ' :

virus DN~, a virus whose nucleotide sequences are not present in the human genome.
~ The downstream long tail primer shown in Figure 3 con~;Rts of 45 nucleotides which o~erlap 45 nucleotides at the downstream end of the l96 nt ~ragment. This primer neither contains nor m~A;~tes a BstNI restriction site 8uch that duplexes synthesized using the upstream and downstream long tail primers will be digested by BstNI restriction enzyme only at the rero~n;tion site m~A~nted by the upstream long tail primer and only if the test DNA contains wild type sequence~
at the first and second positions of coAn~ 12.
The downstream short tail primer shown in Figure 3 consists of 26 nucleotides which overlap a 26 nucleotide region at the 5' end of the downstream long tail primer.
Although the in~ention has heretofore been discussed with reference to the use Or separate plates and/or reaction mixes for the respective steps of amplification digestion and amplification, the invention also ~nrnmr~ses a method wherein the~ respective steps of amplification, digestion, further amplification, further digestioni etc., are perfo~m~A in a single reaction mix. In accordance~with this aspect of the inventioni the in~ento~ has discovered a method for combining, for example, amplification, digestion (selection) and amplification in a single reaction. The prior art reaction was a three step procedure since the reagent used in the digestion step (the restriction enzyme) was not thought to be sufficiently heat stable to withstand the high temperatures which are an integral part of the first and third cycles of PCR
~mrl~rication (i.e. denaturation and extension).
The inventor has now found that, by short~n;ng the SUBSTITUTE SHEET (RULE 26) W1096115139 , PCT~Sg~/14755 cycle times in the polymerase rh~; n reaction and by moderating the high temperatures of the first ~ cycle of the PCR (duplex denaturation), it is possible to preserve the activity o$ certain restriction enzymes whereby they can be included in the PCR reaction mix without being i~ev~ ~ibly denatured (inactivated). Thi8 el;m;~tes the need for a separate reaction _ix for each separate stage of amplification and digestion.
Whereas, for a great majority of restriction enzymes, th temperature for optimal ac~tivity for a restriction enzyme to cleave DNA is about 37C., a restriction enzyme for use in t~he single reaction mix process of the invention will display optimal activity at between about 50C.
and 70C. and preferably at about 60C. and above.
In any event, a restriction enzyme suitable $or .
use in the one-step process of the invention will be sufficiently thermostable to maintain its activity in cleaving DNA duplexes at the m;n;m~lm temperatures and cycle t~mes n~e~e~ to effect amplification of the duplexes in the polymerase rh~; n reaction of the one-step process.
S;m;l~ly, a polymerase suitable $or use in the one-step process of the invention will be sufficiently ~herm~stable to maintain its activity at the temperatures and cycle times used in the process. The restriction enzymes and polymerases that will work in the one-step process of the invention include those restriction enzymes and polymerases that are extracted $r G ~h~mostable bacteria including The~ favus, TherTnlR ruber, ~he~m--R ~h~r~P_ilus, Bacillus stearoth~ J~hilus, (which has a somewhat lower temperature op~;m-m than the others listed), Therm~ aauaticus, ~h~rmllR lactus, ~h~ rubens, and Methano~h~m-~ -~
fervidus. In addit~on, th~-m~stable polymerases SUBSTITUTE SHEET (RULE 26) -CA 0220~392 lss7-07-lo W096tl5139 PcT~sg5ll47ss and.restriction enzymes for use in the invention include those isolated from the ~h~mophilic -- arc~eh~cteria, such a~ Sulfolobus solfataricus, Sulfolobus acidocaldarius, Th~m~Plasma acido~hilum, Meth~n~hacterium thermoautotroPhicum, and Desulfurococcus mobilis.
Although as previously noted, temper-atures used to denature nucleic acid duplexes in the denaturation cycle of a polymerage ~h~n reaction will typically be between about 90 -95C., denaturation by heat will occur at temperatures.as low as about 88C. Tn~e~A the inventor has found that, even at the m;n;m~l temperatures.needed to effect denaturation of nucleic acid duplexes, duplexes can be denatured by heating.to such m;n;m~l dupiex denaturation temperatures and then ;~m~A;~ tely cool; n~ to the temperatures required for primer ~nn~l;ng. In other.words, the duplexes need not be kept at the m~n;m~l duplex denaturation temperatures (i.e.
they can r~;n 0 8~co~A~ at such temperatures) and denaturation will ~till occur. Moreover, the inventor has found that, if the duplexes are . heated to the m~n;~-l temperatures ne~A for template denaturation sufficiently rapidly (preferably at.a rate.of between about 5.-20C./secQnA, and more preferably at about 10C./sec~nA), and are then ;~mediately cooled to primer ~nn~l;ng temperatures sufficiently rapidly (preferably at a rate Of at least about 10C./8econA), restriction enzymes in the reaction mix having optim~l acti~ity at the aforementioned temperatures will not be inactivated during at least the first 10 cycles of the amplification.
With respect to the heating of the duplexes to the afo ~cnt-;oneA denaturation temperatures, the inventor has found that the upper l;~t on the SUBSmUTE SHEET (RULE 26) W~96115139 .PCT~S9Sl14755 rate of heating is limited only by instability which may result between the polymerase and the DNA if the rate is too fast (i.e. it appears that the enzyme dissociates from the DNA when the temperature rises too rapidly).
. S;m; 1 A~1Y, the inventor has found that the nucleic acid ext~nR;Qn cycle8 of a polymerase ch~; n reaction can be con~cted at the temperatures re~uired for f~m~ns ext~n~Qn ~ Gdùcts (about 70 - 75C.) without inacti~ating the restriction enzymes of the inve~tion if the extension cycles are sufficiently rapid. So, for example, such cycles should be-c~nA--~ted for le than 30 secQn~R at the temperatures required for forming extension products, and will preferably be ~onA-~ted for less than 15 8~conA~ at such temperatures. The ~;n;mllm times for the extension cycles will be limited only by the time required for the synthesis of the desired primer extension products. Although this will vary in accordance, for example, with the length of the extension ~ducts being synthesized, an ext~nR~Q~ cycle for the one-step method of the invention may typically last between about 3 to I5 seco~AR.
To effect the rapid temperature cycles needed to preserve the activity of the the~stable restriction enzymes of the invention during PCR amplification, use may be made of a rapid temperature cycler which operates based on heat transfer by hot air to s~rles contAi~A in thin capillary tubes. Such cyclers include the 1605 Air Th~mo-Cycler c~mm~cially avA;1A~le from Idaho Technology of Idaho Falls, T~nho. This thermocycler uses capillary tubes and air heating to enable transition rates of 5 - lOC./second.
Complete 30-cycle reactions can be ~inished using such cyclers in as little as lO minutes (see "The SUBSTITUTE SHEET (RULE Z6) Wo96115139 - PCT~S95/147s5 1605 Air ~h~mo-Cycler ~ser's Guide~ r~hl~nhe~ by T~h.~ T~hnt~ y) .
To optimize the acti~ity of the poly-merase and restriction enzyme when included in the same reaction mix, it is necessary to satisfy the buffer requirements of the respective enzymes.
Changes to the PCR reaction buffer may affect the f; ~ ty of the polymerase; that is, the poly-merase has a certain error rate (in~o ~o ating a wrong base pair during DN~ syntheR~) that _ay increase if an optimal polymerase buffer is not used. S;m51~ ly~ the restriction enzymes for use in the in~ention ha~e buf~ers that will opti~ize their acti~ity in clea~ing DN~ duplexes at or near specific nucleotide sequences. Since optimal buffers for the polymerase and restriction enzymes will differ, c~mh~ n~ tion of the respecti~e enzymes in a one-step reaction requires selection of a buffer that can suitably maintain the acti~ity of each enzyme. Since the f;~ ty of the polymerase is of prime concern, a suitable buffer for use in the one-step process of the in~ention will be one that enables the restriction enzyme to clea~e duplexes at or near a ~p~c; f; C nucleotide sequence without substantially decr~ n~ the degree of f;~l;ty of the polymerase. In this respect, an e~ o~ rate of 5 x 10-4 or;less is preferred for ~u~o~es of the in~ention. More preferably, the polymQrase e~o~ rate will not exceed 1 x 10-4.
In this respect, some polymerases have h~ ~h~
f; ~1; ty than do others, and in certain applica-tions, if a particul`ar poly~e ~se is 1Q88 than optimal, it may be desirable to U8Q a polymerase with a higher f i ~ ty and a rQspecti~e suitable buffer.
The pre~erred polymerases for use with the one-step process o~ the in~ention is Ta~

SUBSTITUTE SHEET (RULE 26) CA 0220~392 1997-07-10 WQ96~15139 PCT~S95/14755 polymerase. Other suitable polymerases include Vent DNA polymerase (a~ailable from New England Biolabs, Be~erly, MA); Pfu DNA polymerase (a~ailable from Stratagene, La Jolla, CA) and Altima DNA polymerase (a~ailable from Perkin ~1 m~
Cetus, Norwalk, CT).
The preferred restriction enzyme for use with the one-step process of the invention is BstNI. Other suitable restriction enzymes which could be n ~; 1 ~ed~ to other target sequences designed for a given point mutation include Ban I, Bcl I, BsmI, BssH II, BstXI, and Sfi I. These enzymes are all ro~o~cially a~ailable (from, for - example, Stratagene) and ha~e 50 - 60C. as optimal digestion temperature and their recognition sites are known.
Suitable buffers for use in the one-step reaction of the in~ention can be prepared, for example, by modifying a buffer known to be suitable for the respecti~e polymerase of restriction enzyme used in the reaction. For example, the in~entor has found in a preferred embodiment of the one-step process of the in~ention wherein Taq polymerase is used in the amplification of 196 nucleotide frA~nt cont~;n;n~ coA~n 12 of the h~ n R-raQ gene and the amplification products are digested with BstNI
(see Example II, infra), a suitable reaction mix can contain a buffer conQ;Qting of between about 60 - 80% (preferably 80%) of BstNI buffer. (BstNI
buffer, as used herein, consists of 50 mM NaCl, l0 mM Tris-HCl, l0 mM MgC12, 1 mM DTT (p~7.0) and BSA
(100 ~g/ml); 80% of BstNI buffer is B~tNI buffer diluted with water to 8b% of its original concentration). The reaction mix in the preferred emboA;mont can also ~Lise between about 5 - 30 ng of long tail primers (preferably about 20 ng), SUBSTITUTE SHEET(RULE 26) WO 96/15139 rCT/US9~/14755 .. .

between about 50 and 150 ng of short tail primers (preferably about 100 ng); between about 7.5 and 10 units of Taq polymerase (preferably 7.5 units);
between about 2.5 - 7.5 un$ts (preferably 5 units) of BstNI enzyme; about 1 ~g of test DNA and about O.2 mM dNTPs. The ratio of long tail primers to short tail primers will preferably be between 3-1 and 6:1, and will more preferably be about 5:1.
If the actiYity of the restrict~on enzyme with a certain buffer is not sufficient to cleave nucleic acid duplexes at or near specific nucleotide se~enc~ with a desired efficiency (for ~le, about 90 - 95%), additional digestion steps can be ~ to the reaction.
Foll o.wing ~mnl; ~; catin - n in t h~ ~ are ~t~p reaction, material iB subjected to -analysis Yia b;n~ ng to a~idin coated matrix followed by flubrescent mea~u ~t. The short 3' (tailed) primer doe~ not contain a restriction enzyme site for BstNI (since such a site would not enable further amplification). To control h~r~lJ ~u~d of no~m~l ampli$iable material, a threshold value is established in each reaction using no~m~l or no~m~ diluted with mutant DNa in different ratios (i.e. a c~l;hration cur~e can be made). The latter also serves as a reference for quantitation of mutant allele ; n~ n~e .
The present processes and kits can be used for the detection of a mutant allele in any gi~en gene as long as: a) the sequence of the target gene is known and b) the position of the mutation is defined. While a differen~ set of primers has to be ~e~;~ne~ for each target site, and a different restriction enzyme may haYe to be used, the principle and the me~ho~ology will r~; n the same. For eY~rl e, this inYention can be used for the analysis of specific point SUBSTI I UTE SHEET (RULE 26) -~096115139 PCT~S95/147ss mutations in the p53 tumor ~u~.e880r gene, and in particular, in so-called "hot spots" in the p53 tumor suppressor gene. These hot spots include (l) coAQn 249 which is a hot spot for the potent carcinogen aflatoxin (2) coAnn 248 which is a hot spot for N-ethyl-N-nitrosourea and (3) codon 3g3 which is a hot spot of UV in s~nl:ght.
As another example, the present invention can be used for the analysi8 of specific point mutations in the ras oncogene fam~ly ~n each of the know~ sites for mutations, which include ~-ras, R-ras and N-ras genes at ~oAnn~ 12, 13, and 61. Table l exemplifies restriction enzymes that may be used in the process of the in~ention with respect to each o these genes. The process of the in~ention is belie~ed to be espe~ y important for assaying codon 12 of R-ras, since it is the most frequent mutation in hllm~n colon and pancreatic LU~OL ~ .
EXAMPLE I
Amplification and Digestion of Fr~$r-nt of ~llm~n R-ras Gene Using ~ong and Short Tail Primers The methods and kits of the invention will now be ex~m~rlified with respect to their use in the ~mrl~fication and digestion of test DNA
including sequences of coAnn 12 of the hllm~n R-ras gene:
A. Sequence of primers used for PCR ~mplification o~ ae~uences of coAon l2 of the h~ n R-ras gene:
Downstream long tail primer (45 mer) 5' CTG CTC GTA TCT ATC ACT TCA GGT CTC
GAA GAA TGG TCC TGC ACC 3' Downstream short tail primer (26 mer) 5' CTG CTC GTA TCT ATC ACT TCA GGT CT 3~ -~pstream long tail primer (44 mer) 5' GCG GTT GGG GCT TAA TTG CAT ATA AAC
TTG TGG TAG TTG GAC CT 3' SUBSTITUTE SHEET (RULE 26) WO 9611S139 Pcr/us9s/l47~5 Upstream short tail pr~mer (24 mer) 5' GCG GTT GGG GCT TAA TTG CAT ATA 3' -- Figure 3 shows the relati~re posit;Qn;nr of the respecti~re long and Rhort tail primers with respect to ~oA~-n 12 of the test fr~r~nt.
B. ConA; tions for amplification and digestion reactions.
First step amp~f;~tion is performed ~Q; n~ 1 ~g of test DNa., 2.5 u~its of Tag enzyme (Perkin Elmer-Cetus, Norwalk, CT), 5 ~ul of Tag buffer (50 mM ~CCl, lO Mm Tris-HCl, pH 8.3, 0.01%
gelation) 0.2 mM dNTPs, 20 ng of long tail primers (downstream and upstream) 3 mM MgCl2 in a total ~rolume of 50 ~l. 20 cycles of amplification are perf~ A using 3 steps for each cycle, composed of l' ~ 94C., l' ~ 53C., l' ~ 72C.
~ 5 ~1 al~uot of the first step amplification reaction is taken into a restriction enzyme reaction in which 5 units of the restric-tion ~nA~nu~lease BstNI (purchased from New England Biolabs, Be~rerly, MA)) i8 ~A~ 9 and supplemented with a 1 f~l of the BstNI buffer (50 mM NaCl, lO mM Tris-~Cl, lO mM MgCl2, 1 mM DTT (p~
7.9) and BSA (lO0 ~g/ml) to a total ~rolume of lO
~l. This step is perfo~ at 60C. for l hour.
2 ~l of digested m~terial are ::-AA~A to a lO0 ~l of reaction ~olume which cor~ t of: 150 ng of short tailed primers, 2.5 units of Taq enzyme, lO ~l of Taq buffer (lOx), 0.2 mM dNTPs.
Secr~nA step amplification is perfo~^~ for 35 cycles, consisting of 3 steps each l' ~ 94C., l' 59C., l' ~ 72C. ~
Short tailed primers ~AeA to this reaction are labelled with biotin (5' or upstream tail) and fluorescein (3' or downstream tail).
Thi~3 lab~ll ;ng is performed according to chemistry av~ hle ~ ^cially (from, for example, .

SUBSTITUTE SHEET (RU~E 26) WO 96/15139 PCTI~S9S/147~5 r ~ 34 ~
Milligen Biosearch, Bedford, MA and Glen Research Sterling, VA) during the synthesis of the ~- oligonucleotide in a DNA synthesizer. These modified oligonucleotides can also be purchased.
Following the second step ~plification, the material is ~e~ to an avidin coated matrix, and digested again with BstNI.
Fluorescein mea~u-~~nt is then t~k~n using a fluorescence reader.
C. D;~sn~stic ~it ~or Reactions For k'it purposes two microplates and 3 sets of pre-made solutions are utilized.
(1) Microplate A is a rl ~;n PCR
compatible microplate.
(2) Microplate B is as in (1), but pre-coated with avidin.
(3) Solution A -- consists of all reagents required to perform the first step amplification with the exception of test DN~ (or control DN~). It therefore contains Taq enzyme, buffer, dNTP, and the long tail primers.
Solution B -- consists of reagents required to per$orm the in~e~me~;~te digestion step. It contains both the restriction enzyme and its respecti~e buffer.
Solution C -- consists of all reagents required to perform ~econd step ~mpl; f; cation. It therefore contains short tailed primers labelled with biotin or fluorescein, respectively, enzyme, buffer, and dNTPs.
D. Robotic PCR
The reaction mixes developed for the processes of the in~ention, such a~ Solutions A, B
and C (above), are easily adapted for use in robotic PCR processes. For Example, a portion of the first stage amplification product of the invention can be transferred by an i~strument SUBST1TUTE SHEET(RULE 26) . . CA 0220~392 1997-07-10 W O 96/15139 PCTrUS95/14755 (robot) to a ser~n~ ~essel which contains the restriction enzyme reaction for first stage -- digestion. A portion o~ the product of first - stage digestion can be transferred by the instrument to a third ~essel cont~;n;n~ the reagents for 8~0~ stage amplification. A
portion of the product of second ~tage amplification can be transferred by the instrument to a fluorescence reader, which can be a part of the instrument. Thus, in this Example, first stage ampli~ication can be ron~ ted in Microplate A by intr~A~c: n~ the test DNA and Solution A into the microplate. Then, Solution B can be ~e~ to the microplate for first stage dige~tion. The test s~rle, or a portion thereof, can then be ~oved and reamplified in Microplate B with Solution C (cont~;n;~g the short tail primers).
Further digestion can be performed in Microplate 8 with Solution B following which the instrument can carry out the ~uantitation step in the f luorescence reader.
EXAMPBE II
Multiple States of Ampli~ication and Digestion in Single Reaction Mix A. There is now ~Yemrl; ~ied a process in which the repetiti~e cycles-of amplification and digestion of ~Y~mrle I are performed in a single reaction. The following have been modified to perform such a reaction.
(1) Reagents (a) buffer is adjusted to enable both digestion and amplification and consists of 80% of BstNI buffer (i.e. BstNI buffer diluted with water to 80%). (b) Primers - 20 ng of long primers are m; Y~ with 100 ng of short tailed primer~. (c) 7.5 units of the Ta~ enzyme is used (d) 5 units of BstNI enzyme. (e) all other reagents - DNA; dNTPs; - remain the same as SUBSTITUTE SHEET (RULE 26) W~96/15139 PCT~S9S/147~5 in F~ ~ ~le I.
(2) Total ~olume reduced to lO ~l and reaction is performed in capillary glass tubes.
(3) Reaction is per~ormed in a Hot Air mi~ ~ler (the 1605 Air Th~rmo-Cycler a~ailable from T~ho Technology) under the following ~n~; tions-a) hot start - heat test DNA at 88C. for lO se~nn~.
b) 5 cycles consisting of a) 0 sec~n~R ~ 88C., b) 5 se~o~ 53C., c) 15 se~n~ Q 72C.
c) l cycle of 60C. for 20 minutes.
d) 5 cycles as in b.
e) l cycle of 60C. ror 20 minutes.
f) steps b-c are repeated once more.
g) 20 cycles consisting of 0 seco~ at 88C.; 5 secon~R at 33C.; and 15 secQn~ at 72C.
h) material is taken ~rom capillary glass to microplate coated with a~idin.
i) ~uantitation is then performed as set forth in Example I.
Total time for single step reaction is less than 90 minutes.
B. D;~gnostic ~it for Single Step Reaction It may be appreciated that, for use with a single step reaction, a ~;~gnostic kit of the i~vention (including a suitable ~he~mnstable restriction enzyme, DNA polymerase and buffer) need contain only a single PCR compatible glass tube which is pre-coated with a~idin. The kit may contain a single pre-made ~olution cont~ n ng the reagents of Section A(l) of this ~ le.
In summary, the present in~ention is seen to pro~ide processea and ~ nostic kits for SUBSTmJTE SHEET (RULE 26) = ~

analyzing a nucleic acid te6t sample taken from the g~n~vme of an org~n;Rm. The processes ~ described herein in~olve less cumbersome techn;ques and/or reguire ~ewer Rteps to carry out amplification and digestion than do the procedures pre~iously deRcribed.
Other moA~ tiQns of the abo~e-described ~mho~;m~ts of the in~ent~on that are ob~ious to those of skill in the area of molecular 1~ biology and related discipl;n~Q are inten~ to be within the scope of the following cl ~;m~ .

SUBSTITUTE SHEET (RULE 26)

Claims (28)

C L A I M S

1. A process for the detection in a nucleic acid test sample taken from the genome of an organism of a mutant nucleotide sequence in a specific region of the genome, wherein said region can contain said mutant nucleotide sequence or a wild-type nucleotide sequence, wherein the test sample is suspected of containing a first genomic strand of nucleic acid having said region with the mutant nucleotide sequence together with a second genomic strand of nucleic acid having said region with the wild-type nucleotide sequence, wherein the first genomic strand, if present in the test sample, is present or is caused to be present in the form of a first genomic duplex consisting of the first genomic strand and a first complementary strand, and the second genomic strand is present or is caused to be present in the test sample in the form of a second genomic duplex consisting of the second genomic strand and a second complementary strand, the process comprising:
(i) a first amplification step comprising amplifying material in the first and second genomic duplexes present in the test sample in a first polymerase chain reaction in which upstream and downstream long tail primers, DNA polymerase, four different nucleotide triphosphates and a buffer are used in a repetitive series of reaction steps involving template denaturation, primer annealing and extension of annealed primers to form first and second synthesized nucleic acid duplexes, said first synthesized nucleic acid duplexes having said region with the mutant nucleotide sequence, and said second synthesized nucleic acid duplexes having said region with the wild-type nucleotide sequence, said upstream and downstream long tail primers being selected such that synthesized strands formed in the first polymerase chain reaction using the upstream and downstream long tail primers can anneal with upstream and downstream short tail primers which do not anneal with any nucleic acid strands in the first or second genomic duplexes, said long tail upstream primers also being selected such that the second synthesized duplexes have a restriction site which is not present in said first synthesized duplexes due to the presence in said first synthesized duplexes of said region with the mutant nucleotide sequence, said restriction site being cleavable with a first restriction enzyme, ii) a first digestion step comprising treating at least a portion of the test sample containing the first and second synthesized duplexes with said first restriction enzyme whereby selectively to cleave said second synthesized duplexes while leaving said first synthesized duplexes uncleaved, iii) a second amplification step comprising amplifying material in the uncleaved first synthesized duplexes in a second polymerase chain reaction in which the upstream and downstream short tail primers are used selectively further to amplify nucleic acid strands not cleaved in step (ii) whereby to form further synthesized duplexes, said upstream and downstream short tail primers being selected such that they anneal with nucleic acid strands synthesized in said first amplification step but do not anneal with any strands of the first or second genomic duplexes whereby the upstream and downstream short tail primers can be used in the second amplification step selectively to amplify material in duplexes synthesized in the first amplification step but cannot amplify material in the first or second genomic duplexes, each of said upstream short tail primers being labelled with a first substance that binds tightly with a second substance such that upstream ends of the further synthesized duplexes bind to a supporting surface coated with the second substance, each of said downstream short tail primers being labelled with a detectable marker;
iv) a binding step comprising causing contact between the test sample and the supporting surface coated with said second substance whereby further synthesized duplexes labelled with the first substance bind thereto;
v) a second digestion step wherein the test sample is again treated with the first restriction enzyme selectively to cleave synthesized duplexes containing nucleic acid strands having said region with the wild type sequence; and vi) a detection step comprising washing to remove unbound duplexes and assaying for the detectable marker to detect the presence of the mutant nucleotide sequence on uncleaved duplexes bound to the supporting surface.

2. A process as claimed in claim 1, wherein the first substance comprises biotin and the second substance comprises avidin or strepavidin, wherein each of said downstream short tail primers is labelled with a fluorescent label such that downstream ends of the further synthesized duplexes have the fluorescent label, and wherein the detection step comprises assaying for the presence of the fluorescent label.

3. A process as claimed in claim 2, further comprising a second digestion step following the binding step wherein the test sample is again treated with the first restriction enzyme selectively to cleave synthesized duplexes containing nucleic acid strands with regions having the wild-type sequence.

4. A process as claimed in claim 2, wherein the test sample is placed on a first microtiter plate for the first amplification step following which the test sample, or a portion thereof, is transferred to a second microtiter plate which is coated with the avidin or strepavidin, the test sample being left in said second microtiter plate for at least the digestion step, and the second amplification step.

5. A process as claimed in claim 1, wherein steps i - iv are performed in a polymerase chain reaction microcycler, wherein the detection step comprises denaturing the uncleaved duplexes bound to the supporting surface and performing hybridization with a labelled probe to detect said mutant nucleotide sequence.

6. A process as claimed in claim 1, wherein the first and second amplification steps and the first and second digestion steps are performed in a single reaction mix, wherein the region comprising the mutant nucleic acid sequence comprises codon 12 of a human K-ras gene, and wherein the restriction enzyme is BstN1, the polymerase is Taq polymerase and the buffer is a diluted solution comprising 60 - 80% of BstN1 buffer.

7. A process as claimed in claim 6, wherein the region comprising the mutant nucleic acid sequence comprises codon 12 of a human K-ras gene, and wherein the restriction enzyme is BstN1, the polymerase is Taq polymerase and the buffer is a diluted solution comprising 60 - 80% of BstN1 buffer.

8. A process as claimed in claim 1, wherein the region comprising the mutant nucleic acid sequence, is a codon of K-ras, H-ras or N-ras gene selected from the group consisting of codon 12, codon 13 and codon 61.

9. A process for detecting the presence of a mutant nucleotide sequence in a specific region of a first genomic nucleic acid strand of a genomic nucleic acid duplex, said genomic duplex consisting of the first genomic nucleic acid strand and a first complementary nucleic acid strand, said process comprising:
i) amplifying the genomic and complementary nucleic acid strands in a polymerase chain reaction in which upstream and downstream primers, four different nucleoside triphosphates, a DNA polymerase and at least one buffer are used in a repetitive series of reaction steps involving template denaturation, primer annealing and extension of annealed primers to form synthesized nucleic acid duplexes having upstream and downstream ends and consisting of first synthesized and first synthesized complementary nucleic acid strands, said upstream primers comprising upstream nucleotide sequences which are sufficiently complementary to nucleotide sequences in a first end of said first complementary nucleic acid strand to enable the upstream primers to anneal thereto and to be used to initiate synthesis of the first synthesized strands, the upstream nucleotide sequences being selected such that synthesized nucleic acid duplexes formed in the polymerase chain reaction using the upstream primers have a restriction site cleavable by a first restriction enzyme if and only if the first synthesized strands have said region with the wild-type nucleotide sequence, said upstream primers being labelled with a first substance that binds tightly with a second substance such that the upstream ends of the first synthesized duplexes bind to a supporting surface coated with the second substance, said downstream primers comprising downstream nucleotide sequences which are sufficiently complementary to nucleotide sequences in a first end of said first genomic strand to enable said downstream primers to anneal hereto and thereby to initiate synthesis of said first complementary synthesized strands, said downstream primers being labelled with a fluorescent label such that synthesized duplexes formed using said downstream primers can be detected by assaying for the fluorescent label;
ii) causing contact between the synthesized nucleic acid duplexes formed in step i and the supporting surface whereby to bind the synthesized nucleic acid duplexes to the supporting surface, iii) treating the synthesized nucleic acid duplexes bound in step ii with the first restriction enzyme to cleave nucleic acid duplexes having a wild-type sequence in said region, and washing to remove cleaved duplexes, and iv) assaying bound synthesized duplexes for the presence of the fluorescent label.

10. A process as claimed in claim 9, wherein the synthesized nucleic acid duplexes formed in step i are treated with the first restriction enzyme and synthesized in a second amplification step using a polymerase chain reaction prior to step ii.

11. A process as claimed in claim 10, wherein the upstream and downstream primers in step i are long tail upstream and downstream primers and wherein upstream and downstream short tail primers are used in the second amplification step, the upstream and downstream long and short tail primers comprising nucleotide sequences which are selected such that synthesized nucleic acid strands formed in the first polymerase chain reaction using the upstream and downstream long tail primers can anneal with one of either the upstream or downstream short tail primers and such that the upstream and downstream short tail primers do not anneal with either strand of the genomic duplex.

12. A diagnostic kit for use in an assay for detecting the presence or absence on a first genomic nucleic acid strand of a genomic region containing a mutant nucleotide sequence, wherein said genomic region can contain the mutant nucleotide sequence or a wild-type sequence, wherein said first genomic nucleic acid strand is present in a test sample in the form of a first genomic duplex consisting of the first genomic nucleic acid strand and a first complementary nucleic acid strand, and wherein the assay comprises at least a first and a second amplification step, said kit comprising:

a) a first reagent mixture for use in the first amplification step wherein the first nucleic acid duplexes are synthesized in a polymerase chain reaction with synthesis of a first synthesized duplex, said first mixture comprising upstream and downstream long tail primers, each of said upstream and downstream long tail primers comprising a complementary primer portion and a non-complementary primer portion, the complementary primer portion of the upstream long tail primers being sufficiently complementary to a first end portion of the first complementary nucleic acid strand to enable the upstream long tail primers to anneal thereto and thereby to initiate synthesis of a nucleic acid extension product using the first complementary strand as a template, the complementary primer portion of the downstream long tail primers being sufficiently complementary to a first end portion of the first genomic strand to enable the downstream primers to anneal thereto and thereby to initiate synthesis of a nucleic acid extension product using the first genomic strand as a template, the non-complementary primer portions of said upstream and downstream long tail primers not being sufficiently complementary to either the first genomic strand or the first complementary strand to anneal with either, the non-complementary primer portions of the respective upstream and downstream long tail primers being positioned on the upstream and downstream long tail primers such that a first end portion of the first synthesized strand of synthesized duplexes formed using the upstream and downstream primers has nucleotide sequences that are identical to the nucleotide sequences of the non-complementary primer portion of the upstream long tail primers and such that a first end portion of the first complementary synthesized strand of synthesized duplexes formed using the upstream and downstream primers has nucleotide sequences that are identical to the nucleotide sequences of the non-complementary primer portion of the downstream primers, and b) a second reagent mixture for use in the second amplification step comprising upstream and downstream short tail primers, each of said upstream short tail primers comprising nucleotide sequences which are sufficiently complementary to the nucleotide sequences in the non-complementary primer portion of the upstream long tail primers to anneal thereto but which are not sufficiently complementary to nucleotide sequences in either the first genomic strand or the first complementary genomic strand to anneal thereto, each of said downstream short tail primers comprising nucleotide sequences which are sufficiently complementary to the nucleotide sequences in the non-complementary primer portion of the downstream long tail primers to anneal thereto but which are not sufficiently complementary to nucleotide sequences in either the first genomic strand or the first complementary genomic strand to anneal thereto, whereby the upstream and downstream short tail primers can be used in the second amplification step selectively to amplify synthesized duplexes synthesized in the first amplification step and none of the first genomic duplexes.

13. A kit as claimed in claim 12, wherein the upstream short tail primers are labelled with biotin and the downstream short tail primers are labelled with a fluorescent label.

14. A kit as claimed in claim 13, wherein the kit further comprises a microtiter plate coated with avidin or strepavidin.

15. A kit as claimed in claim 13, wherein each of the first and second reagent mixtures further comprises four different nucleoside triphosphates, a DNA
polymerase and a buffer.

16. A kit as claimed in claim 14, wherein the kit further comprises a first microtiter plate comprising the first reaction mixture.

17. A reagent mixture for use in an assay for detecting the presence of a mutant nucleotide sequence in a specific region of a first genomic strand of a nucleic acid duplex, said duplex consisting of the first genomic nucleic acid strand and a first complementary nucleic acid strand, said assay comprising amplifying material in the genomic and complementary nucleic acid strands in a polymerase chain reaction so as to form synthesized nucleic acid duplexes having upstream and downstream ends and consisting of first synthesized and first synthesized complementary nucleic acid strands, said mixture comprising:
a) a plurality of upstream primers comprising upstream nucleotide sequences which are sufficiently complementary to nucleotide sequences in a first end of said first complementary nucleic acid strand to enable the upstream primers to anneal thereto and to be used to initiate synthesis of the first synthesized strands, the nucleotide sequences of the upstream primers being selected such that synthesized nucleic acid duplexes formed in the polymerase chain reaction using the upstream primers have a restriction site cleavable by a first restriction enzyme if and only if the first synthesized strands have said region with the mutant nucleotide sequence, said upstream primers being labelled with biotin such that the upstream ends of the first synthesized duplexes containing the first synthesized strands formed using said upstream primers bind to a supporting surface containing a substance that binds tightly with the biotin;
b) a plurality of downstream primers comprising downstream nucleotide sequences which are sufficiently complementary to nucleotide sequences in a first end of said first genomic strand to enable said downstream primers to anneal thereto and thereby to initiate synthesis of said first complementary synthesized strands, said downstream primers being labelled with a fluorescent label such that uncleaved synthesized duplexes bound to the supporting surface at their upstream ends and containing first complementary strands formed using said downstream primers can be detected by assaying for the fluorescent label.

18. A process as claimed in claim 17, wherein the reagent mixture further comprises four different nucleotide triphosphates, a nucleic acid polymerase and a buffer.

19. In a process for detecting mutations in nucleic acid duplexes, wherein synthesized duplexes synthesized from genomic duplexes in a first amplification stage are present in a test sample together with the genomic duplexes, wherein the synthesized duplexes are synthesized in the first amplification stage from the genomic duplexes in a first polymerase chain reaction in which upstream and downstream primers are used to provide the synthesized duplexes with nucleotide sequences which differ from nucleotide sequences in the genomic duplexes, wherein the synthesized duplexes are treated with a restriction enzyme selectively to cleave duplexes containing a specific nucleotide sequence and wherein material in the synthesized duplexes present in the test sample with the genomic duplexes is further amplified in a second amplification stage using said upstream and downstream primers, the improvement wherein the upstream and downstream primers in the first amplification stage are upstream and downstream long tail primers and the upstream and downstream primers in the second amplification stage are upstream and downstream short tail primers, the upstream and downstream long tail and short tail primers being selected such that synthesized nucleic acid strands formed in the first polymerase chain reaction using the upstream and downstream long tail primers anneal with one of either the upstream or downstream short tail primers, and such that the short tail primers do not anneal with any nucleic acid strands in the genomic duplexes whereby the upstream and downstream short tail primers can be used in the second amplification stage selectively to amplify material in duplexes synthesized in the first amplification stage but cannot amplify material in the first or second genomic duplexes.

20. A reagent mixture for use in a polymerase chain reaction for amplifying material in a nucleic acid duplex comprising a first nucleic acid strand having first and second ends and a second nucleic acid strand having first and second ends, the first and second ends of the first nucleic acid strand having nucleotide sequences which are complementary to the nucleotide sequences in the second and first ends of the second nucleic acid strand respectively, the mixture comprising upstream oligonucleotide primers which can anneal with the first end of the first nucleic acid strand and downstream oligonucleotide primers which can anneal with the first end of the second nucleic acid strand, said upstream primers being labelled with biotin such that upstream ends of synthesized nucleic duplexes formed in the polymerase chain reaction using said upstream primers bind to a supporting surface containing a substance that binds tightly with the biotin, said downstream primers being labelled with a fluorescent label such that the synthesized nucleic acid duplexes formed in the polymerase chain reaction using said downstream primers and bound to the supporting surface at the upstream ends can be detected by assaying for the fluorescent label.

21. In a process for detecting mutations in nucleic acid duplexes comprising amplification of material in the duplexes in a polymerase chain reaction in a first reaction mix to form synthesized nucleic acid duplexes and subsequent treatment of the synthesized duplexes with a restriction enzyme in a second reaction mix selectively to cleave duplexes containing a specific nucleotide sequence, the first reaction mix comprising a nucleic acid polymerase with a first buffer suitable for enabling the polymerase to facilitate amplification of material in the nucleic acid duplexes in a polymerase chain reaction, the second reaction mix comprising the restriction enzyme with a second buffer suitable for enabling the restriction enzyme selectively to cleave duplexes containing the specific nucleotide sequence, said polymerase chain reaction comprising repetitive cycles of denaturation, annealing and extension, the improvement comprising: conducting the amplification and treatment steps in a single reaction mix by a) including in the single reaction mix a thermostable nucleic acid polymerase, a thermostable restriction enzyme and an intermediate buffer suitable for enabling the polymerase to facilitate amplification of the material in the nucleic acid duplexes while also enabling the restriction enzyme to cleave duplexes containing the specific nucleotide sequence, said intermediate buffer consisting essentially of BstN1 buffer diluted to between 60 - 80% concentration, said thermostable restriction enzyme being selected such that it can selectively cleave duplexes containing the specific nucleotide sequence when used with said intermediate buffer; and b) regulating the times and temperatures at which the cycles of the polymerase chain reaction are performed so as to amplify the nucleic acid duplexes without inactivating the thermostable restriction enzyme.

22. A process as claimed in claim 21, wherein the material amplified in the polymerase chain reaction comprises codon 12 of the human K-ras gene, the restriction enzyme is BstN1, the polymerase is Taq polymerase and the intermediate buffer is a diluted solution comprising 60 - 80% of BstN1 buffer.

23. A process as claimed in claim 21, wherein the temperature at which the denaturation cycle of the polymerase chain reaction is performed is between about 86°C. - 92°C.

24. A reagent mix for use in amplification of nucleic acid duplexes, said reagent mix comprising a thermostable nucleic acid polymerase, a thermostable restriction enzyme which selectively cleaves the nucleic acid duplexes if and only if the duplexes contain a specific nucleotide sequence, upstream and downstream primers for annealing to each strand of the nucleic acid duplexes whereby to initiate formation of nucleic acid extension products, four different nucleotide triphosphates, and a buffer suitable for enabling the restriction enzyme to cleave duplexes containing the specific nucleotide sequence and for enabling the polymerase to catalyze formation of the nucleic acid extension products.

25. A method as claimed in claim 1, wherein the upstream and downstream short tail primers consist of DNA sequences from polyoma virus.

26. A process as claimed in claim 25, wherein the upstream and downstream short tail primers are between about 15 and 25 bases in length, whereby the further synthesized duplexes are longer than the genomic duplexes by between about 30 and 50 bases pairs.

27. A process as claimed in claim 19, wherein the upstream and downstream short tail primers consist of DNA sequences from polyoma virus.

28. A process as claimed in claim 27, wherein the upstream and downstream short tail primers are between about 15 and 25 bases in length, whereby the further synthesized duplexes are longer than the genomic duplexes by between about 30 and 50 bases pairs.