WO1993011261A1

WO1993011261A1 - A novel pcr method with a single primer for nucleic acid analysis

Info

Publication number: WO1993011261A1
Application number: PCT/EP1992/002722
Authority: WO
Inventors: George Rudenko; John Nijkamp; Jacques Hille
Original assignee: Keygene N.V.
Priority date: 1991-11-25
Filing date: 1992-11-25
Publication date: 1993-06-10
Also published as: AU2945792A

Abstract

The invention relates to a process for the amplification of a nucleotide fragment contained in multiple copies of a nucleic acid sample, obtained after digestion of the nucleic acid sample with at least one restriction endonuclease, the nucleotide sequence of the fragment to be amplified being unknown, except for a short nucleotide sequence sufficient to hybridize specifically with an oligonucleotide sequence used as primer in an amplification reaction.

Description

A NOVEL PCR METHOD WITH A SINGLE PRIMER FOR NUCLEIC ACID ANALYSIS

1. FIELD OF THE INVENTION

This invention relates to applications of PCR methods for DNA analysis in a number of different fields including, but not limited to, plant and animal breeding, diagnostic medicine, disease diagnosis in animals and plants, identification of genetically inherited diseases in humans and icrobial typing.

More specifically, this invention relates to PCR methods for detecting anonymous DNA sequences in genomes ranging from microorganisms to higher plants, animals and humans.

2. BACKGROUND OF THE INVENTION

The molecular characterization of anonymous DNA sequences adjacent to sequences with known primary structure are of great importance in applications on DNA or RNA molecular biology. There is an urgent need to develop quick, sensitive, and efficient general procedures to produce such anonymous DNA sequences in vitro. The techniques currently used to this end involved the creation of different types of genomic libraries which are very laborious, followed by cloning, screening and/or dot/blot hybridization. The Polymerase Chain Reaction (PCR) technique is a well known and broadly used method for synthesizing specific DNA fragments in vitro and the technology can be used to amplify single copy genomic DNA fragments. The PCR method which relies on the use of specific oligonucleotides which will attach to unique sequences on a DNA molecule and a thermostable DNA polymerase is schematically presented in figure l. The oligonucleotides are designed in such a way that they can anneal to the opposite strands of the DNA and serve as primers in a DNA synthesis reaction in such a way that each will direct the synthesis of new DNA strands. Hence, in one round of synthesis a copy of the DNA molecule between the primers will be made, so that the DNA compound between the primers is duplicated. Each round of DNA synthesis results in the doubling of the amount of DNA, hence leading to the amplification of the DNA sequence comprised between the two primers. Consequently, the PCR technique allows one to synthesize a precise DNA segment using a small amount of "substrate DNA". The sole prerequisite for PCR amplification is that the DNA sequence of the region to synthesized is known in advance. Consequently, the PCR method can not be used directly to amplify DNA segments lying outside boundaries of DNA regions with known sequence because the second primer which is needed is lacking. A few different modifications of basic PCR protocol have been reported up to date to overcome these difficulties. The approaches involve the construction of artificial boundaries on a region of interest, which can then be used as binding site for the second primer. These techniques include inverse PCR, utilizing the inversion of the genomic fragment with known sequence information together with flanking region by circularization and re-opening at the different site, and oligo-cassette mediated PCR based on ligation of oligo-cassette adaptors to the ends of the amplified fragment of question. Yet another approach utilizes a random primer which do not require an absolutely sequence-specific interaction with template DNA as the second primer for amplification, a technique called targeted gene walking PCR.

The method of the present invention is a noveltechnigue based on polymerase chain reaction for efficient amplification of genomic DNA sequences flanking small DNA stretches with known sequence information. Sequence information, 25-30 bp, is the only prerequisite for amplifying single copy DNA of unknown sequence that occurs either upstream or downstream from the known sequence.

3. SUMMARY OF THE INVENTION

In the present invention we have devised a new PCR method to amplify anonymous DNA fragments of which only a limited segment of 15 to 25 bases is sequenced and which can not be amplified using standard PCR method. The standard PCR method requires that one knows either the entire nucleotide sequence of the DNA fragment that is to be amplified or at least the sequences of two small segments flanking the DNA fragment to be amplified. The reason is that in the PCR reaction two oligonucleotides are used which correspond to the sequences located at the ends of the DNA fragment. If only one of these sequences is known, only one oligonucleotide primer can be designed and consequently the DNA fragment can not be amplified. We have devised a new method which relies on the use of a single PCR primer to amplify DNA segments adjacent to one side of this primer. The advantage of our new method is that we can obtain specific amplification of anonymous DNA segments flanking short known sequences of 18 to 24 bases. 4. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 schematically outlines the principle ofthe Polymerase Chain Reaction (PCR) . The linesrepresent DNA strands and the arrows represent the PCR primers. The boxes represent the regions of knownsequence, based upon which the primers are designed.

Figure 2 depicts the different steps in the general method of the invention in which a single PCR primer is used to amplify anonymous DNA adjacent to a known DNA sequence represented by the box. The tagged line represents the newly synthesized DNA strand containing bio-11-dUTP, which is immobilized on the straptavidin substrate.

Figure 3 depicts the general method of the invention in which a single PCR primer is used to amplify anonymous DNA adjacent to a known DNA sequence represented by the box, and in which a linker is added to the DNA molecule in step 4. In this case the PCR reaction is carried out using two primers.

Figure 4: A structure of the T-DNA segment which is engineered in the transgenic plants. The left and right borders are indicated by the boxed areas.

B: structure of the integrated T-DNA segment

C: illustrates the sPCR amplification steps and the primers used in the reaction.

Figure 5: The results of sPCR amplification of the genomic fragments flanking the borders of an original insertions in tomato plants. Transgenic tomato plants have been obtained via T-DNA mediated transformation and contain one genomic insertion (AAT6514-30,-33) or three (AAT6514-44) . Restriction endonucleases used were: T-TaqI, HII-Hae II, HIII-Haelll, A-Avail.

A: shows the sPCR products detected on a 2% agarose gel.

B: shows the southern blot results obtained after hybridization with a T-DNA probe.

Figure 6: Southern blot analysis of L. esculentum and L. penelli using the TaqI sPCR fragment isolated from plant AAT6514-33.

Figure 7: Direct PCR analysis done on genomic DNA from the plant AAT6514-44, digested by TaqI. The middle band indicated on fig. 1 has been sequenced. The primary structure of the region is presented on fig. 3.A. Two primers (BP1 and BP2) have been synthesized and border fragments have been amplified (fig. 3.B.) in the presence of different compositions of primers: line 1-P2/BP2 (expected size 120 bp) , line 2-P1/BP1 (expected size 136 bp) , line 3-P1/BP2 (expected size 186 bp) .

Figure 8: Analysis of inverse PCR products obtained with DNA from plants AAT6514-33 and AAT6514-44 digested with TaqI.

Figure 9: Southern blot hybridization data showing the correlation between inverse PCR results and situation in vivo.

Figure 10: Shows the PCR product obtained with DNA from plant AAT6514-02 digested with EcoRI and amplified as described in the text.

Figure 11: Shows the two PCR products obtained after amplification of EcoRI digested AAT 6514-02 DNA. 5. DETAILED DESCRIPTION OF THE INVENTION

5.1. DEFINITIONS

In the description and examples that follow, a number of terms are used herein. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided:

Restriction Endonuclease. A restriction endonuclease or restriction enzyme is an enzyme that recognizes a specific base sequence (target site) in a double-stranded DNA molecule, and will cleave both strands of the DNA molecule at every target site.

Restriction Fragments. The DNA molecules produced by digestion with a restriction endonuclease are referred to as restriction fragments. Any given genome will be digested by a particular restriction endonuclease into a discrete set of restriction fragments.

Synthetic oligonucleotides. The single-stranded DNA molecules having preferably from almost 10 to almost 50 bases long, which can be synthesized chemically are referred to as synthetic oligonucleotides. In general, these synthetic DNA molecules are designed to have a unique nucleotide sequence, although it is possible to synthesize families of molecules having related sequences and which have different nucleotide compositions at specific positions within the nucleotide sequence. Ligation. The enzymatic reaction catalyzed by the enzyme ligase in which two double-stranded DNA molecules are covalently joined together is referred to as ligation. In general, both DNA strands are covalently joined together, but it is also possible to prevent the ligation of one of the two strands, through chemical or enzymatic modification of one of the ends. In that case the covalent joining will occur in only one of the two DNA strands.

Linkers. Short double-stranded DNA molecules, with limited number of base pairs, e.g. 10 to 20 base pairs long, which are designed in such a way that they can be ligated to the ends of DNA fragments. Linkers are composed of two synthetic oligonucleotides which have nucleotide sequences which are in part complementary to each other. When mixing the two synthetic oligonucleotides, they will form a double-stranded structure in solution under appropriate conditions. One of the ends of the linker molecule is designed so that it can be ligated to the end of a DNA fragment, the other end is designed so that it cannot be ligated.

Polymerase Chain Reaction (PCR) . The enzymatic reaction as depicted schematically in figure 1 in which DNA fragments are synthesized from a substrate DNA in vitro is referred to as PCR. The reaction typically involves the use of two synthetic oligonucleotides, which are complementary to nucleotide sequences in DNA molecules which are separated by a short distance of a few hundred to a few thousand base pairs, and the use of a thermostable DNA polymerase. The chain reaction consists of a series of 10 to 30 cycles. In each cycle the substrate DNA is first denaturated at high temperature. After cooling down the synthetic oligonucleotides which are present in vast excess will form double-stranded structures with the substrate DNA molecules in solution at specific sites on the substrate DNA molecule that have complementary nucleotide sequences. The oligonucleotide-substrate DNA complexes will then serve as initiation sites for the DNA synthesis reaction catalyzed by the DNA polymerase, resulting in the synthesis of a new DNA strand complementary to the substrate DNA strand.

DNA amplification. The term DNA amplification will be used to denote the synthesis of double-stranded DNA molecules in vitro using Polymerase Chain Reaction (PCR) . The products of the PCR reaction will be referred to as amplified DNA fragments. Primers. In general, the term primer refers to a DNA strand which can prime the synthesis of DNA. DNA polymerase cannot synthesize DNA de novo without primers: they can only extend an existing DNA strand in a reaction in which the complementary strand is used as a template to direct the order of nucleotides to be assembled. We will refer to the synthetic oligonucleotide molecules which are used in the PCR reaction as primers.

5.2. DESCRIPTION OF THE PREFERRED EMBODIMENTS

This invention is based on the use of thepolymerase chain reaction (PCR) to detect DNA fragments of which only a small part of its nucleotide sequence is known, sufficient to design only one primer and for which typical PCR reactions can not be used because the sequence at the other end of the fragment is not known, so that a second primer cannot be used. The invention consists of a novel PCR method in which a single PCR primer is used to amplify and isolate anonymous DNA sequences adjacent to the primer. The invention also consists of methods for analyzing DNA's of organisms based on short sequence, 15 to 20 base, information, and of applications of the method of the invention for diagnostic purposes in different fields of plant and animal breeding, human genetic desease diagnostics and microbial diagnostics.

The general method of the present invention presented in figure 2 involves in a first step the use of restriction endonucleases to cleave genomic DNA into discrete fragments. In a next step (step 2, figure 2) a single synthetic oligonucleotide primer is added to the restriction fragments and the solution is first heated to allow denaturation of the DNA and then cooled to allow the synthetic oligonucleotide to anneal with its complementary sequence in the genomic DNA. Next a DNA polymerase is added to the reaction mixture. The complex of the synthetic oligonucleotide bound to the restriction fragment will serve as a primer template complex for DNA synthesis, which is carried out in the presence of the tree normal deoxynucleotide triphosphates dATP, dGTP and dCTP and a biotinilated derivative of dUTP (bio-11-dUTP) , which results in the synthesis of a novel DNA strand containing biotinilated uridine instead of thymidine. After the synthesis of the primed DNA strand the newly synthesized biotinilated DNA are specifically purified from the DNA mixture by immobilizing the biotiniliated DNA strands on a substrate containing streptavidin (step 3, figure 2) . It is known that streptavidin makes a higly specific complex with biotin which is extremely stable. In this way one obtains a very strong enriche ent for the newly synthesized DNA fragments which are the only DNA fragments that will bind to the matrix. In the next step (step 4, figure 2) ligase is added and the newly synthesized DNA fragments are allowed to ligate to one another. These molecules contain at one end a single stranded protrusion corresponding to the DNA region upstream of the primer, and are fluss ended at the other end. Consequently only the flush ends will be reactive to the ligase so that the ligation reaction will result in the production of di eric molecules in which the newly synthesized DNA fragments are joined tail to tail (see figure 2). In the last step (step 5, figure 2) the ligated molecules are used as DNA templates for a PCR reaction in which the single synthetic oligonucleotide , used to produce the first strand, is used. In this PCR reaction only the ligated DNA fragments will be amplified exponentially, whereas the unligated DNA's are amplified linearly, and hence will remain undetected at the end of the PCR reaction. The PCR reaction will thus specifically amplify the dimeric ligated molecules which can therafter be identified by gelelectrophoresis.

It should be obvious that the above procedure could also be used to amplify cDNA fragments either by using the cDNA mRNA hybrid molecules as a substrate for the synthesis of the biotinilated strand or by using ds cDNA cleaved with a restriction enzyme.

In another embodiment of the method of the invention depicted in figure 3 the immobilized fragments are ligated in the presence of two partially complementary synthetic oligonucleotides which, when they form double stranded DNA complex, will have at one end a flush end and at the other end a single stranded protrusion. These double stranded synthetic oligonucleotides are refered to as linkers. Due to the differences in their ends these linker molecules will ligate in a specific way to the immobilized DNA fragments: normely ligation will only take place with the flush end of the linker ligating to the flush end of the immoblized DNA fragment. Since all the immobillized DNA fragments have linkers ligated to one end, one can then use either a conventional two primer PCR reaction or a single primer PCR reaction, depending on the nucleotide sequence of the linker which is added to the newly synthesized DNA to amplify the immobilized DNA fragments.

The PCR products obtained in accordance with the invention can be identified using standard fractionation methods for separating DNA molecules according to size followed by staining of the DNA molecules with appropriate agents. Alternatively, the primer used in the PCR amplification can be tagged with a suitable chromophore thus allowing the identification of the reaction products after size fractionation. In a preferred embodiment of the invention the PCR products are fractionated by gel electrophoresis using standard gel matrices such as, but not limited to, agarose or polyacrylamide.

The method of the present invention can be used to generate sets one or more DNA fragments from restriction digests of genomic DNA comprizing the short (15 to 25 base pairs) known DNA sequence, upon which the primer oligonucleotide was designed. Since these DNA fragments are highly specific for the particular genome under study, the method of the invention can be used to detect and analyze variations in the nucleotide sequences flanking the known sequence.

One particular application of the methods of the invention involves the use of primers corresponding to DNA sequences which are repeated more then once in the genome. Such primers will yield characteristic patterns of fragments after separation by gelelectrophoresis which constitute unique and reproducible fingerprints of the genomic DNA. Such fingerprints can have different applications such as, but not limited to, forensic typing, the diagnostic identification of organisms,and the identification of species, races, varieties or individuals. This identification will be possible when different members of a specific group exhibit differences in these DNA patterns.

One particular application of the present invention involves the detection of DNA polymorphisms associated with a particular DNA sequence. Changes in the nucleotide composition of genomic DNA often result in polymorphisms of restriction fragments : insertions or deletions affect the size of the restriction fragments containing them (figure 10) , nucleotide changes can result in the elimination of restriction endonuclease target sites or the creation of new restriction endonuclease target sites. In accordance with the method of the present invention, polymorphisms can be identified by comparing the DNA fragments obtained from different genomes. When modifiactions of the type described above occur in the DNA sequences adjacent to the primer, these will influence the length of the DNA fragments produced with the method of the invention. These polymorphisms can then be used to trace the inheritance of the genomic locus associated with a particular nucleotide sequence. Applications include forensic typing, monitoring of genetically inherited diseases in humans and monitoring the inheritance of agronomic traits in plant and animal breeding. The underlying principle is that certain DNA polymorphisms which are closely linked with specific genetic traits can be used to monitor the presence or absence of specific genetic traits. According to the method of the present invention, the analysis of the amplified DNA products can be used to define the genetic linkage of polymorphisms with specific genetic traits.

Another application of the methods of the invention involves the detection and analysis of transgenic organisms obtained with commonly used genetic engineering methods. In this application DNA sequences corresponding to the DNA segment that is introduced into the genetic material of the organism are used to design the primer oligonucleotides which are used in the methods of the present invention. When these sequences are appropriately located within the engineered gene and when appropriate restriction enzymes are used the amplification products will contain chi eric sequences comprizing sequences of the engineered gene and sequences of the genomic DNA adjecent to the engineered gene. In this way one is able to analyse how the engineered gene has been integrated into the genome of the transgenic organism, identify the number of gene copies and identify the location of the gene in the genome by analyzing the amplified genomic sequences. Yet, another application of the present invention involves the detection and analysis of rearrangements in genomic DNA resulting from the movement of so-called movable elements. Movable elements, or transposons, are genetic elements (DNA segments) which exhibit the property that they can move from one site in the genome into another site. While moving to another site these elements can cause genetic changes due to the disruption of the normal DNA sequences in which they integrate. Since such genetic changes can be associated with observable phenotypes, the movable elements can be used to tag and locate genetic functions in the genome of an organism. This method is generally referred to as transposon tagging. When the nucleotide sequence of such movable elements is known the methods of the present invention can be used to detect and analyse mutant organisms in which the movable element has moved to a new location, in the same way as described above for the analysis of transgenic organisms.

Finally the methods of the invention can be used to isolate unknown sequences which are linked to a particular known sequence. In the particular applications listed hereunder, examples are given for which the methods of the invention can be used :

(1) direct gene isolation without necessity of creation of genomic libraries,

(2) cDNA isolation without necessity of creation cDNA libraries,

(3) genomic walkings based on sequence information on newly amplified flanking regions,

(4) heterologous gene isolation based on sequence homology with a known sequence,

(5) development of in vivo or in vitro footprinting techniques to identify DNA protein interactions at particular genomic regions, (6) isolation of unknown sequences from the ends of DNA fragments cloned in lambda, cosmid, PI or YAC libraries. These applications allow the isolation of missingparts of genes, upsteam regulatory sequences or homologous genes in other organisms.

6. EXAMPLES

Having now defined the invention, the same will be understood by means of specific examples which are, however, not intended to be limiting, unless otherwise specified.

6.1. Example l: Identification of foreign genes in transgenic tomato plants using the linker supported PCR method.

In this example we will demonstrate the application of the methods of the invention to analyze the insertion of foreign genes in transgenic tomato plants, obtained by transformation with Agrobacterium tumefaciens. Such transgenic plants contain a foreign DNA segment which becomes integrated at various places in the genome. The foreign DNA segments transfered by Agrobacterium are contained within a specific DNA segment usually refered to as the T-DNA segment. Upon integration the T-DNA segment becomes fused to plant DNA sequences at the border sequences of the T-DNA segment. The structure of the T-DNA segment used to produce the transgenic plants is presented in figure 4. We have taken adventage of the fact that the nucleotide sequence of the T-DNA borders are known to design two primers PI and P2 based on the nucleotide sequence of the right T-DNA border. The sequences of the Pi and P2 primers are listed under respectively 7.1 and 7.2.. In order to analyze the inserted DNA in the transgenic plants we have used the PI and P2 primers to amplify the plant DNA segments adjacent to the right border.

In a first step the genomic DNA was prepared of 4 transgenic tomato plants: AAT6514 - 02, AAT6514 - 30, AAT6514 - 33, AAT6514 - 44, using standard DNA purification procedures, whereafter 2 microgram of each DNA was digested with the following restriction enzymes TaqI, Haell and Haelll using digest conditions as suggested by the supplier. Each of the 12 samples was then treated as follows : The restricted DNA was extracted with phenol/chlorophorm, precipitated with 2.5 vol. of EtOH in the presence of 0.1 M NaOAc (pH 4.8-5.2), washed twice with 70-80% EtOH, once with EtOH, dried and resuspended in water (to final concentration, approx. 0.2 mg/ml) . Following general recommendations should be taken in consideration:

i) There should be no sites for selected restriction endonucleases between the primer and the unknown sequence. ii) It is better to use restriction enzymes recognizing 4 or 5 bases. iii) The presence of RNA does not play an important role during the procedure but it is prefered to perform RNAse treatment using standard procedures.

Once the restricted DNA is prepared the next steps in the procedure are as follows : Step 1) Preparation of the biotinilated fragment.

To 10 mkl of restricted DNA solution was added 40 mkl of biotinilation mix: 5 mkl primer 1 (10 mkm) with the sequence depicted in 7.1, 5 mkl TaqI buffer (10 x) , 0.1 mkl TaqI DNA polymerase (5u/mkl) , 5 mkl "dXTP-mix" containing 2 mM each of dATP, dGTP, dCTP, 1.5 mM dTTP, 0.05 mM bio-11-dUTP, and 24.9 mkl water.

Mix by vortexing, add 3 drops of mineral oil.

Start the DNA synthesis reaction in thermocycler: 6-7 min at 94 oC, 2 in at 55 oC, and 10 min at 72 oC, then cooling to 0-4 oC.

Stop the reaction by addition of 2 mkl of 0.5 M EDTA, pH 8.0.

Step 2) Immobilization of the fragment.

Add to each tube approx. 12-14 mkl of streptavidin agarose (Dynabeads can also be used) .

Mix and incubate 30 min at 37 oC.

Wash agarose particles 3 times with TBS and 3 times with water (add, shake and spinn down for 20-30 seconds in centrifuge, remove water phase) . TBS = 25 mM Tris HC1, pH 7.4-7.6, 150 mM NaCl, 0.1 mM EDTA.

Step 3) Ligation of the fragment with linker primer.

The nucleotide sequence of the ligated primer is presented in 7.3.

Add to agarose pellet 10 mkl of ligation mix : 2.5 mkl ligase buffer, 8 units of ligase, approx. 1000-1100 pM of linker primer (between 600-1100) , water to 10 mkl.

Mix and overlay with 2 drops of mineral oil. The ligation is performed at 12-14 oC for 20-24 hours.

Step 4) Amplification (1st round) .

Wash the agarose particles with TBS and twice with water

Add 40 mkl of PCR mix: 5 mkl primer PI (10 mkM) with the sequence depicted in 7.1., 5 mkl of linker primer (10 mkM) with the sequence depicted in 7.4., 5 mkl TaqI polymerase buffer (10 x) , 0.5 mkl dNTPs (20 mM of each), 0.1 mkl TaqI DNA polymerase (5u/mkl) , 23.4 mkl water.

Amplify in a thermocycler using th following conditions : denaturation at 94 oC for 5 min, then 35 cycles of 30 seconds at 94 oC, 60 seconds at 55 oC, 90 seconds at 72 oC.

Step 5) Final amplification (2nd round - optional) .

Precipitate 20 mkl of 1st round PCR products with an equal volume of isopropanol in the presence of IM ammonium acetate, wash 2 times with 70 oC EtOH, once by EtOH, dry, resuspend in 50 mkl of water.

Use 1-10 mkl of products for 2nd round of amplification following the procedure as described in step 4 and using primer P2 with sequence depicted in 7.2..

Step 6) Run the products on 2% agarose gel.

The results presented in figure 5 show the sPCR products of the transgenic plants AAT6514 - 30, AAT6514 - 44 and AAT6514 - 33, obtained after amplification of the genomic DNAs digested with the enzymes TaqI, Haell and Haelll. Plant AAT6514 - 33 gives one band for each enzyme: an 0,3 Kb fragment with TaqI, a 1,6 Kb fragment with Haell and an 0,6 Kb fragment with Haelll. Plant AAT6514 - 30 and AAT6514 - 44 gave several PCR products in each reaction, indicating that there are several copies of the T-DNA inserted. In conclusion, the results show that the supported PCR method yields specific products upon amplification with the T-DNA specific primers. The experiments described below will demonstrate that the PCr products are indeed derived from plant DNA sequences fused to the T-DNA. To this end the TaqI sPCR fragment has been used further for analysis :

(1) Southern blot analysis of the DNA from the transgenic plant AAT6514 - 33 digested by the enzymes TaqI, Haelll and Haell, was carried out in order to confirm that the sPCR fragments are derived from the segment in the plant DNA in which the T-DNA is fused to the plant DNA (fig. 5) . The sizes of the bands detected after hybridization with a T-DNA probe were : TaqI: 850 bp, Haell: 2.1 kb, Haelll: 1 kb. These sizes are in good agreement with the sizes of sPCR fragments confirming that the PCR fragments contain plant DNA flanking the right border. The southern blot was also hybridized with TaqI sPCR fragment as a probe and yielded the same bands.

(2) The TaqI sPCR fragment was the used to locate the position of the integrated DNA segment on the genetic map of tomato using DNA polymorfisms between L. esculentu and L. penelli (fig. 6) . The position on the tomato genome was deduced from the analysis of a segregating F2 population of 36 plants, derived from a L. esculentum x L. penelli cross, using the restriction enzyme EcoRV in a standard RFLP analysis. The "Mapmaker" software package has been used to calculate the positon which was found to be on chromosome I at position 7.5.

(3) The TaqI fragment has been sequenced to confirm that it contains the boundary between the T-DNA and the plant DNA as well as the linker primer, after recloning into vector pTZ19R. The nucleotide sequence shown in fig. 7 confirms the above.

(4) To verify further the above results we have performed inverse PCR. The results presented in figure 8 show inverse PCR products obtained with plant 33 and plant 44 after TaqI digestion. SacII has been used for relinarization of template DNA before the inverse PCR. The inverse PCR fragments are larger than sPCR bands, the difference being about 300 bp. The inverse PCR reaction yielded only 2 out of 3 border fragments of plant AAT6514-44 (figure 8). Figure 9 is demonstrating the presence of 3 T-DNA borders in the plant-44 (the probe was border fragment according to figure on the page 2 of this letter) . The sizes of sPCR fragments (all 3) are in good agreement with Southern data. It was impossible to get the highest TaqI by using iPCR. The small 44/Taq sPCR and iPCR as well as middle bands have been sequenced. The smaller fragment has been further mapped on chromosome I, pvs 136.5 cM.

6.2. Example 2

In essence the experiment was carried out using exactly the same procedure as described under example 1, except that in step 3 the linker was omitted from the ligation mixture. Also in this example the PI primer has been used in the biotinilaton step and the P2 primer in the amplification steps and plant AAT6514-02 DNA was digested with EcoRI was used. The results presented in fig. 10 show that a unique PCR fragment is obtained with a size of about 150 bp. This fragment is about twice as large as the fragment which was obtained from the same plant DNA using the linker supported PCR technique, and in which the sequence analysis demonstrated that the EcoRI site is located 88 bp from the T-DNA border. This result indicates that the product generated in this PCR reaction could be a tail to tail di er of the original fragment. Sequence analysis has confirmed that this is indeed the case. In certain case we find that the single primer reaction yields two bands (see figure 11) : These represent presumably the dimer in two forms: a fully double stranded form and a folded back form which has the size of a monomer.

7. LISTS OF NUCLEOTIDE SEQUENCES

7.1. Nucleotide sequence of primer PI

S'-CGGCTTGTCCCGCGTCATCGGC-S¹ (22 mer)

7.2. Nucleotide sequence of primer P2

BamHI P2 (T-DNA primer) rest of R border 5^«-CCCCTAGGGATTGTCGTTTCCCGCCTTCAG TTTAAACTA-

7.3. Nucleotide sequence of linker used in the ligation

5'-OH-ACCCGTGGATCAGTACCGCGACTTG-OH-3 ' (25 mer)

3'-OH-TGGCGCTGAAC-P-5' (11 mer)

7.4. Nucleotide sequence of primer P3

-CAAGTCGCGGTACTGATCCACGGGT-3 •

< linker primer

Claims

1. Process for the amplification of a nucleotide fragment contained in multiple copies of a nucleic acid sample, obtained after digestion of the nucleic acid sample with at least one restriction endonuclease, the nucleotide sequence of the fragment to be amplified being unknown, except for a short nucleotide sequence sufficient to hybridize specifically with an oligonucleotide sequence used as primer in an amplification reaction, which process comprises : a) adding to the enzyme-digested sample copies of a single stranded oligonucleotide which is complementary to the known nucleotide sequence contained in the fragment to be amplified, or which is at least capable to specifically and stably anneal with this fragment in order to form a oligonucleotide-template complex for an amplification reaction, the oligonucleotide being used as a primer in such an amplification reaction, b) allowing the copies of the fragment which includes said above known nucleotide sequence, to hybridize with the primer, if required after a denaturation step, thus forming a primer-template complex for DNA synthesis, c) elongating the DNA from the template, in the presence of a DNA polymerase and of four different deoxynucleotide - or deoxyribonucleotide- triphosphates dXTP, X representing A, C, G, T ou U, one of these dXTP being labelled, and preferably using a labelled dUTP derivative, preferably a biotinylated derivative of dUTP (bio-11-dUTP) , in order to synthetise copies of a DNA strand containing labelled, preferably biotinylated uridine, d) immobilizing the copies of the labelled double stranded DNA obtained after the elongation

(elongated fragment) , on supports carrying substrates specific for he label contained in the synthetised DNA strand, of the elongated fragment (especially on supports carrying substrates containing streptavidin when biotinylated dUTP) has been used, e) adding a ligase to the immobilized double stranded DNA elongated fragments, in conditions allowing the flush ends of different copies of elongated fragments immobilized on different supports to ligate to one another , thus forming double stranded molecules comprising two (dimeric) elongated fragments, this double stranded molecules, comprising at each extremity, on the opposite strands, the oligonucleotide used as primer, f) using the above double stranded dimeric molecules, as template for the amplification reaction with a primer consisting of the above single stranded oligonucleotide, g) amplifying the dimeric molecules to obtain an amplification sufficient to detect the amplified product, h) recovering the product of the amplification.

2. Process according to claim 1, characterized in that the fragment to be amplified is a double stranded DNA and in that a step of denaturation of this DNA is performed in order to allow the hybridization of the primer with this DNA.

3. Process according to claim 1 or 2, characterized in that the fragment to be amplified is a restriction fragment obtained after the reaction of one or several determined endonucleases, and in that the known nucleotide sequence of the fragment to be amplified does not contain a sequence corresponding to a restriction site of the endonuclease (s) used.

4. Process according to claim 1, characterized in that the fragment to be amplified is a RNA fragment, and in that a step of production of cDNA complementary to this RNA is performed before the addition of the primer.

5. Process according to anyone of claims 1 to 4, characterized in that the fragment to be amplified contains 10 to 25, preferably 18 to 24 known consecutive nucleotides.

6. Process according to anyone of claims l to 5 characterized, in that the ligation steps a) to e) is performed in the presence of a linker consisting of two partially complementary oligonucleotides which form a duplex having at one end, a flush end, and at the other end, a single stranded protrusion.

7. Process according to one of claims 1 to 6, characterized in that the label is an hapten or an antigen and the substrate is an immunoglobulin.