WO1992013104A1 - Progression d'une reaction en chaine de polymerase 5' et 3' a partir de sequences d'adn connues - Google Patents

Progression d'une reaction en chaine de polymerase 5' et 3' a partir de sequences d'adn connues Download PDF

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WO1992013104A1
WO1992013104A1 PCT/US1992/000532 US9200532W WO9213104A1 WO 1992013104 A1 WO1992013104 A1 WO 1992013104A1 US 9200532 W US9200532 W US 9200532W WO 9213104 A1 WO9213104 A1 WO 9213104A1
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dna
strand
stranded dna
double stranded
linker
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PCT/US1992/000532
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English (en)
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Kun C. Wu
Albert B. Deisseroth
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Board Of Regents, The University Of Texas System
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA

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  • this invention is related to DNA libraries and describes methods for producing DNA libraries from different organisms, as well as for cloning and amplifying DNA sequences that flank known genes. More particularly, the instant invention concerns a method of rapid polymerase chain reaction walking.
  • PCR Polymerase chain reaction
  • the conventional method is of limited use when one desires to clone uncharacterized DNA sequences flanking a known region because only one DNA primer can be designated from a known DNA region.
  • a one primer polymerase chain reaction only generates a linear increase in the number of copies; whereas when two primers are employed there is an exponential increase in the number of copies.
  • a polymerase chain reaction method that requires only one specific DNA primer, and exponentially increases the number of copies generated of an uncharacterized DNA sequence would substantially facilitate the cloning and sequencing of unknown DNA molecules.
  • inverse polymerase chain reaction has been used to clone cellular DNA flanking sequences from an inherited murine ecotropic provirus and to isolate DNA sequences flanking the Insertion Sequence I element from the E. coli genome (Silver and Keerikatte, "Novel Use of Polymerase Chain Reaction To Amplify Cellular DNA Adjacent to an Integrated Provirus", J. Virol. 63:1924-1928 (1989) and Och an, et al . r "Genetic Applications of an Inverse Polymerase Chain Reaction", Genetics 120:621-623 (1988)).
  • Inverse polymerase chain reaction involves enzyme restriction digestion and subsequent self circularization of genomic DNA fragments.
  • the circular DNA product is then used as a template for DNA amplification using two DNA primers designed from a known DNA region.
  • the method of inverse polymerase chain reaction requires the digestion of a specific site between the two DNA primers, and the existence of this specific restriction site in the DNA region to be amplified, would abolish the amplification process.
  • the second method is the single-specific primer polymerase chain reaction (Shyamala and Ames, "Genome walking by single-specific-primer polymerase chain reaction: SSP-PCR", Gene 84:1-8 (1989)).
  • This method involves linking a generic vector to a digested DNA fragment and a specific DNA primer complementary to the vector is used to initiate the polymerase chain reaction cycle.
  • single-specific primer polymerase chain reaction has been used to take chromosomal walks in the histidine transport operon in Salmonella typhimurium, it is unclear whether this procedure would be sensitive enough to detect and amplify mammalian genomic sequences.
  • the application of single-specific primer polymerase chain reaction for cloning a desired gene from mammalian genomes is very questionable.
  • This invention describes a method for producing a vector-free DNA library, as well as a method for to clone and amplify DNA sequences adjacent to a specific known DNA primer.
  • the present invention has several advantages over the standard cloning procedures. First, making the vector-free library is significantly less tedious than making a lambda or cosmid library because a vector free library only involves linking a DNA oligonucleotide to the 5' end of the DNA fragment, blocking the 3' OH end by dideoxyribonucleotide and removing the linker oligo.
  • the method provides for rapid isolation of a flanking sequence once the library has been made. It takes about five days to amplify the DNA fragment from the library, blot to the nitrocellulose filter, hybridize the internal probe, isolate the fragment from the gel, and sequence the fragments.
  • genomic DNA sequences are preferentially deleted or rearranged in a cosmid or lambda vector because of the incompatibility of those DNA sequences in the host cell.
  • the present invention can be used to resolve the ambiguities of the DNA sequence resulting from the DNA rearrangement by cloning and sequencing the region directly without resorting to making another vector-based library.
  • the present invention does not contain the limitations of inverse polymerase chain reaction (IPCR) .
  • IPCR inverse polymerase chain reaction
  • IPCR requires a rare cutter site between two primers for amplification to occur efficiently. It is well known in the art that the choice of primers is the limiting step in using PCR to clone DNA sequences because of insufficient data on the effect of DNA sequences on the annealing kinetics. In order for the amplification to be successful, optimization of the PCR is required and can be time consuming.
  • Another advantage of the present invention over IPCR is that only one primer need be tested instead of two.
  • two directions of walking can be performed at the same time, to generate DNA sequences in both directions in the same or separate reactions.
  • the present invention has an advantage over SSP-PCR because the DNA linker in the present invention can be easily incubated in excess of the genomic fragment to prevent ligation among the genomic fragments without increasing the viscosity of the incubation mix. Furthermore, the DNA linker can be removed before the cycles of the amplification to minimize the background.
  • This method can be used in various applications such as cloning the region upstream of a 5' end of cDNA or downstream of the 3' end of the CDNA; intron/exon junctions in the genome using a primer complementary to the exonic sequence; transposon where the end sequence of the transposon is usually available; and translocated breakpoints or deletion junctions when the region of the fused locus has been pinpointed.
  • the present invention can be carried out repeatedly allowing more distant sequences to be determined or the strategy can be modified to make a PCR- based chromosomal jumping library.
  • the use of such a jumping library would enable construction of a physical chromosomal map with known sequences localized on every 200 kb on the average along the human genome.
  • the present invention provides a method for generating a vector-free DNA library and for cloning and amplifying DNA sequences adjacent to a specific DNA primer of known sequence.
  • vector-free DNA libraries may be produced from any source of DNA.
  • the invention relates to the production of vector free DNA libraries representing different organisms.
  • the invention can be used to directly clone and amplify DNA sequences from unknown regions of genomes by using an artificially linked unphosphorylated 5' DNA linker and a specific primer that is complementary to at least part a known DNA region as a primer to amplify a PCR-based walking DNA library.
  • This method can also produce an additional 1000 bases (on the average) of DNA flanking a known cloned sequence every 72 hours.
  • the method of the invention does not use cloning vectors.
  • One embodiment of PCR walking involves using unphosphorylated DNA linkers which will ligate only at the 5' ends, blocking the unligated 3' ends by dideoxyribonucleotide triphosphate, and specifically priming the synthesis of the desired flanking sequence with a primer complementary to the known sequence.
  • the instant invention allows isolation of DNA sequences from regions with no known restriction site.
  • the need to isolate DNA from regions that do not contain a known restriction site is huge.
  • the ability to isolate DNA sequences from regions that contain no known restriction site is immense.
  • the method of this invention and the libraries that are produced by these methods have applications in the fields of medicine, as well as in the area of biological sciences.
  • the methods described in this invention have application to areas ranging from agriculture, food production, diagnostics, therapeutics, as well as personal care products.
  • the present invention provides a method for generating a genomic DNA library from an organism and for cloning and amplifying DNA sequences flanking known DNA sequences.
  • an organism refers to any "thing" that an investigator might want clone and sequence DNA from that "thing.”
  • eukaryote is defined as an organism with cells that have nuclear membranes, membrane-bound organelles, 80S ribosomes, and charactertic biochemistry.
  • prokaryote is defined as a simple unicellular organism, such as bacterium or blue- green algae, that contains no nuclear membrane, no membrane-bound organelles, and possesses no characteristic ribosomal system nor biochemistry.
  • the invention also involves a vector-free DNA library comprising fragments of double stranded DNA having a 5' end ligated to a DNA linker and blocked 3' end.
  • the vector-free DNA library is formed by the following steps:
  • DNA linkers are synthetic oligodeoxyribonucleotides that are used to generate cohesive ends at the termini of DNA fragments.
  • polymerase chain reaction walking library is defined as DNA fragments by which DNA sequences neighboring known DNA regions can be isolated.
  • FIG. 1 schematically illustrates the basic steps described, in this invention, in forming a polymerase chain reaction vector-free walking library, as well as steps involved in amplifying unknown and uncharacterized DNA sequences using a polymerase chain reaction vector-free walking library.
  • PIG. 2 Figure 2 illustrates the zeta-globin promoter region target sites for linker primers and specific primers (Oligo A is the 33 mer oligo and Oligo B is the 17 mer oligo) .
  • FIG. 3 is a photograph of an ethidium bromide- stained 2% agarose gel demonstrating DNA fragments generated from two polymerase chain reactions using either oligo A or oligo B as specific primers and Sau3A linker DNA as the other primer.
  • the amplified DNA products were extracted two times with chloroform, ethanol precipitated and run on 2% agarose gel.
  • Lane M shows DNA size markers (given in nucleotides) .
  • Lanes 1 and 2 show amplified DNA product primed by DNA linker alone and oligo B with DNA linker respectively.
  • Lanes 3 and 4 show polymerase chain reaction DNA generated by oligo A with DNA linker and DNA linker alone respectively.
  • PIG.4 Figure 4 is a photograph of a Southern transfer of a 2% agarose gel transferred to nitrocellulose filter and probed with s P-labelled hybrid oligo. Lanes 2 and 3 are the PCR mix containing oligo A or oligo B, respectively. Lanes 1 and 4 are - ⁇
  • the invention relates to the production of vector free DNA libraries representing different organisms.
  • the invention can be used to directly clone and amplify DNA sequences from unknown regions of genomes by using an artificially linked unphosphorylated 5' DNA linker and a specific primer that is complementary to at least part a known DNA region as primers to amplify a PCR-based walking DNA library.
  • This method can produce an additional 1000 bases (on the average) of DNA flanking a known cloned sequence every 72 hours.
  • the method of the invention does not use cloning vectors.
  • One embodiment of PCR walking involves using unphosphorylated DNA linkers which will ligate only at the 5' ends, blocking the unligated 3' ends by dideoxyribonucleotide triphosphate, and specifically priming the synthesis of the desired flanking sequence with a primer complementary to the known sequence.
  • Figure 1 illustrates the steps involved in forming a single primer polymerase chain reaction walking library and for amplifying DNA sequences using such a polymerase chain reaction walking library using Sau3A as the restriction endonuclease, as well as using unphosphorylated DNA linkers.
  • DNA linkers are synthetic oligodeoxyribonucleotides that are used to generate cohesive ends at the termini of DNA fragments. The steps are divided for purposes of illustration. For example, Steps 6-11 may be performed in one step.
  • Step 1 human genomic DNA is digested by treating the DNA with Sau3A to produce DNA fragments small enough to be amplified by polymerase chain reaction.
  • EcoRI is another restriction enzyme that could be used.
  • any restriction endonuclease enzyme can be used, but an enzyme that frequently cuts the DNA is preferred because the PCR amplification size is generally limited to about 3 to 5 Kb.
  • an enzyme that digests DNA at multiple sites is defined as an enzyme that recognizes four base pairs.
  • restriction endonuclease may be used to digest the DNA.
  • Sau3A, Taq 1, and Msp 1 may be used to generate smaller sized fragments of DNA.
  • Any combination of 2-4 different enzymes that frequently digest the DNA (frequent cutters) could accomplish this objective and are therefore embodied in this invention.
  • Other preferred enzymes include Hhal, Hpa2, Rsal and combinations thereof.
  • an unphosphorylated 20 mer/24 mer DNA linker with GATC cohesive ends (which are complementary to the 5' "CTAG" ends of Sau3A digested DNA fragments) , a cohesive end specific to Sau3A, is annealed to the Sau3A DNA fragment.
  • the preferred DNA linker is a 20 mer/24 mer polymer having 20 mers (or 20 bases) as one strand of the DNA linker and 24 mers (or 24 bases) for the other strand of the linker DNA.
  • "mer” is also defined as a nucleotide "base.” According to this invention, only the 20 mer strand of the 20 mer/24 mer linker DNA ligates to the 5' end.
  • a 20 mer/22 mer DNA linker with GC cohesive ends may be employed as well.
  • cohesive ends specific to Taq 1 and Msp 1 may be employed as well.
  • the DNA linkers should complement the cohesive ends produced by the restriction endonucleases.
  • the length of the DNA linker can vary but the ends of the DNA linkers must be cohesive with the restriction enzyme sites produced by the restriction endonuclease.
  • Non-specific sites are sites where a DNA linker primer or specific primer anneals without being 100% homologous to the sequence it is annealing to. These sites can be found in any region of the DNA molecule including the DNA terminus.
  • the phrases DNA linker, linker DNA and linker are used interchangeably and are double stranded for the purpose of this inventoin.
  • a common feature of primer DNA and specific primer DNA are that both are single stranded for the purpose of this invention. Thus, using DNA linkers smaller than 16 mers are not recommended. DNA linkers 18-30 mers long are preferred.
  • the DNA linker comprises a first strand of 18-30 mer and a second strand of 18-30 mer, with the second strand having an additional portion such that the second strand is longer than the first strand.
  • the additional portion of the second strand is complementary to the sticky ends formed by the restriction endonuclease, thus, allowing the DNA linker to anneal to the DNA fragment.
  • Step 3 the Sau3A DNA fragment is ligated to the 20 mer/24 mer DNA linker with GATC cohesive ends with the enzyme DNA T4 ligase.
  • the unphosphorylated DNA linker will ligate only at 5' phospho-end of the DNA fragment.
  • the preferred ligation method is using unphosphorylated DNA linkers is slightly modified from the protocol described by Seth et al ("A New Method for Linker Ligation", Gene Anal. Tech. 1:99-103 (1984).
  • the DNA linker is added in excess of the Sau3A DNA fragments to minimize annealing and ligation among the genomic DNA fragments.
  • the optimal ratio of DNA linkers to Sau3A DNA fragments is 50 to 1, however, other ratios are also acceptable.
  • the 20 mer sequence should always be unique.
  • Step 4 briefly, the unligated 3' OH ends are blocked by three denaturation cycles (65 * C, 75 * C, and 85 * C) followed by 3 cycles of incubation with Taq polymerase for ten minutes at 73'C, in the presence of 2mM ddG.
  • the excess DNA linkers are removed from the DNA linker-ligated genomic DNA fragment admixture.
  • the 24 mer strand of the DNA linker that is not ligated to one of the strands of the DNA fragment will also be removed along with the excess DNA linkers.
  • the resulting 5' linker-ligated strand and the 3' OH blocked strand of the Sau3A DNA fragments constitutes a vector-free human Sau3A DNA library, vector-free DNA library or a PCR walking library.
  • dideoxyribonucleotide triphosphates are used to block the hydroxy group at the 3' end.
  • the dideoxyribonucleotide triphosphate method is currently the least expensive, and most efficient, and requires reagents that are simple to work with.
  • Step 4 Blocking the hydroxy groups at the 3' end of the DNA fragment is the important aspect of Step 4, whether or not denaturation takes place.
  • the denaturation portion depicted in Step 4 may not be necessary for optimal blocking of the 3' end hydroxy groups.
  • the dideoxyribonucleotide that is to be added to the 3' end can be either G, A, T, or C, depending on the last nucleotide of the cohesive end DNA linker.
  • any polymerase enzyme may be used to add the dideoxyribonucleotide bases, however, the inventors prefer Taq polymerase.
  • Step 4 of Figure 1 the dideoxyribonucleotide G is incorporated into the 3' OH end by Taq polymerase to prevent the synthesis of the 24 mer sequence at the 3' end of the DNA fragment.
  • the blocking step, Step 4 a percentage of the 3' ends might not be blocked using a single dideoxyribonucleotide base because the dideoxy- triphosphate may contain a small impurities of deoxy- triphosphate which may result in synthesis of the 24 mer.
  • the inventors recommend that at least two dideoxyribonucleotide bases be used in blocking the 2 ' end. For example, in the Sau3A digested genomic DNA of Figure 1, optimal blocking would result with all four bases but blocking would result from using only two bases.
  • the excess 24 mer may be removed by denaturing the 24 mer at 85'C, then putting in an excess amount of 20 mer to remove the 24 mer by the formation of 24/20 mer duplex. This step prevents the 24 mer from reannealing back to the DNA fragments.
  • the excess 24/20 mer duplex DNA is removed by column filtration. Column filtration is a standard method routinely employed by those skilled in this art.
  • the 24 mer strand of the DNA linker that is not ligated to one of the strands of the DNA fragment will also be removed along with the excess DNA linkers.
  • dd-N refers to either of the 4 dideoxyribonucleotides dd-G, dd-A, dd-C or dd-T.
  • the product is called a vector-free DNA library or a PCR walking library.
  • Amplification of DNA sequences flanking known DNA sequences in the vector-free DNA library using specific primers is the next part of the invention.
  • Step 5 is crucial because if denaturation is not done first, any unblocked ends will be amplified, resulting in amplification of impurities.
  • the denatured fragments provide independent upper and lower strands with each strand having a 5' end and a 3' end. For the purpose of this invention, these denatured fragments are called strands.
  • a specific DNA primer of interest is annealed to the upper strand.
  • the specific DNA primer is defined as a complementary sequence to a known DNA sequence in the upper strand.
  • a 20 mer DNA primer is employed. The inventors prefer to employ a 20 mer but other sequences in the range of 16 to 28 mers would also be effective.
  • Synthetic reaction by Taq polymerase is performed in step 7 to produce a complementary template strand adjacent to the DNA primer of interest (as depicted in Figure 1, the complementary template strand is being generated from the 3' end to the 5 # end).
  • the newly synthesized template strand and the upper strand form a duplex.
  • Step 8 involves denaturing the duplex product generated in Step 7.
  • This denaturation step yields two strands: one strand is the original strand with the ligated DNA linker to it and the other strand is the newly synthesized complementary template strand with the DNA primer of interest at one end of this strand.
  • Denaturation is a standard technique known to those skilled in this art. For effective denaturation, the inventors prefer a temperature range from 90"C to 97 * C with 94'C being the most optimal temperature. At lower- temperatures, denaturation may be incomplete.
  • a DNA linker is annealed to the template strand produced in Step 9.
  • This DNA linker is the same 20 mer DNA linker that was ligated to the 5' end of the original DNA fragment (see Step 2).
  • Step 10 synthesis, by Taq polymerase, off the newly annealed 20 mer strand of DNA linker is achieved.
  • a polymerase effective at about 90'C and above is required.
  • Taq polymerase is the only polymerase presently effective at this temperature. These high temperatures are required to denature the double-strand amplified DNA product so that DNA primer can anneal to the single stranded template from synthesis of DNA in the next round. At higher temperatures, greater than 97"C, the Taq polymerase activity will be inactivated at a faster rate.
  • Step 11 30-40 cycles, with specific DNA primers, DNA linkers, and Taq polymerase, are repeated to generate upwards of about 10 5 copies of the 5'-flanking DNA sequences. Less cyles can be performed but the inventors prefer at least 30-45 cycles for optimal synthesis of the desired 5-flanking DNA sequences.
  • a second specific DNA primer of interest complementary to a sequence on the lower strand of the original DNA fragment, can be annealed as described in Steps 6-11.
  • each walk is limited by the location of the restriction endonuclease sites 5' upstream and downstream of the known sequence. All the steps may be performed on both strands simultaneously, thus, the upper strand need not be separated from the lower strand for optimal synthesis of desired flanking regions.
  • phosphorylated DNA linkers are used instead of unphosphorylated DNA linkers.
  • Lambda exonuclease is used to enrich the target DNA products primed by the specific DNA primer. This combination of phosphorylated DNA linkers and lambda exonuclease eliminates any nonspecific DNA resulting from the blocking step by dideoxyribonucleoside triphosphate. Non-specific amplification can decrease the yield of the desired DNA fragments.
  • the following scheme is proposed to circumvent the problem by enriching the DNA fragments generated by the linker primer and specific primer.
  • the approach makes use of the phosphorylated 20 mer/24 mer linker DNA, with the 5' end of the 20 mer kinased (another term for phosphorylated) in the step 2 of the scheme.
  • the steps 3-12 are essentially identical as the non-kinased DNA linker 20 mer/24 mer.
  • the lambda exonuclease is added to the reaction mix ("Production of Single-Stranded DNA Templates by Exonuclease Digestion Following Polymerase Chain Reaction", Nucleic Acid Res. 17:5865 (1989)).
  • the specificity of the lambda exonuclease allows it to degrade DNA from 5' to 3 ' only if the 5' end is phosphorylated.
  • DNA linker primer is defined as the 20 mer strand of the DNA linker (as shown in Figure 1, Step 9) . DNA fragments produced by specific priming where only the strand initiated by the linker primer are degraded while the strand initiated by the specific primer remain intact.
  • This intact strand can be used for generating more targeted DNA fragments in the next round of PCR reaction.
  • the procedure of PCR amplification and the lambda exonuclease reaction can be repeated a few times to ensure the amplification of the specific DNA products, i.e. the DNA fragments primed by the specific primer. gSW P ?II
  • Example III is presents the necessary steps for producing a polymerase chain reaction human vector-free chromosomal walking library derived from human genomic DNA (isolated from human peripheral blood) .
  • High molecular weight DNA was isolated from human peripheral blood. Briefly, the cells were lysed in Triton-X 100 buffer and digested with proteinase K in the presence of 0.5% sodium dodecyl sulfate overnight. The proteinase K was removed by two rounds of phenol extraction and RNAs were removed by digesting the phenol- extracted sample with 50 ⁇ g/ml pancreatic RNase A for 1 hour at 37 * C. The resulting sample was re-extracted with 2 rounds of phenol and ethanol precipitated and ready for restriction endonuclease digestion.
  • the high molecular weight DNA was digested with Sau3A in medium buffer overnight for at least 4 hours.
  • the average size of the Sau3A digested DNA fragments were in the range of 0.1 Kb (100 base pairs) to 10 Kb.
  • the digested DNA was treated with 0.5% sodium dodecyl sulfate at 65 * C for 20 hours to inactivate the enzyme, ethanol precipitated, and dissolved in IX Tris/EDTA.
  • the dissolved DNA was ligated to unphosphorylated 20 mer/24 mer DNA linker with GATC cohesive end overnight at 20'C in standard ligation buffer in the presence of T4 DNA ligase.
  • the inventors prefer a 50 to 1 ratio of DNA linkers to Sau3A digested DNA fragments, but 5 to 1 or 100 to 1 would also suffice.
  • the DNA was now ready for amplification by the polymerase chain reaction met Only the 20 mer strands of the DNA linkers were ligated to the genomic DNA fragments.
  • the unligated 3 ' OH ends were blocked by three cycles of denaturation at 65'C, 75'C, and 85"C followed by 3 cycles of incubation with Taq polymerase for ten minutes at 73"C or other DNA polymerase at 37 * C in the presence of 2mM ddG.
  • the DNA linker was removed from the linker- ligated genomic fragment by column filtration. Column filtration is a standard protocol known to those skilled in this art.
  • the 5' linker-ligated and the 3' OH blocked (five L-3B) Sau3A DNA fragments constitute the vector free human Sau3A DNA library.
  • promoter specific primer is defined as a DNA primer complementary to the promoter region
  • DNA sequences in the zeta-globin promoter region were amplified using the methods of the invention. It is appreciated that virtually any region 5' to a known sequence may be cloned by employing the methods described in this invention.
  • oligo A 3 33 mer and a 17 mer oligomer (oligo B 17 mer”) which were complementary to the sequences located, respectively, -302 to -270 and -415 to -399 upstream of the CAP cite in the zeta-globin region as illustrated in Figure 2.
  • oligo B 17 mer a 17 mer oligomer oligomer which were complementary to the sequences located, respectively, -302 to -270 and -415 to -399 upstream of the CAP cite in the zeta-globin region as illustrated in Figure 2.
  • a portion of the 5' DNA linker ligated and the 3' OH blocked Sau3A fragments (the Sau3A vector free library; 0.5 ⁇ g) were used as templates and the 20 mer from the 20 mer/24 mer DNA linker, as well as oligo A 33 mer or oligo B 17 mer, were used as the specific DNA primers.
  • Two polymerase chain reactions were performed as described in steps 6-11
  • polymerase chain reaction amplification was performed by an automated method using a Perkin-Elmer DNA thermal cycler.
  • 0.5 ⁇ g of genomic DNA as prepared above was incubated in 100 ul of IX PCR buffer (10 mM Tris-HCl, pH 8.3/50 mM KCl/1.5 mM MgCl 2 /0.1% gelatin/dNTP at 200 ⁇ each, with each primer at 1 ⁇ M) .
  • Two units of Taq polymerase were added and the reaction mixture was heated to 94 * C for 5 minutes, cooled to 55 * C for 2 minutes and brought to 73 * C for 3 minutes for the first cycle.
  • FIG. 3 is a photograph of an ethidiu bromide stained agarose gel demonstrating DNA fragments from two reactions using oligo A 33 mer and oligo B 17 mer as specific DNA primers and Sau3A linker DNA as the other primer.
  • Lanes 1 and 2 show amplified DNA product primed by DNA linker alone and oligo B 17 mer plus DNA linker, respectively.
  • Lanes 3 and 4 show polymerase chain reaction DNA generated by oligo A 33 mer with DNA linker and DNA linker alone, respectively.
  • Lane M shows marker DNA (size given in nucleotides) .
  • the hybridized DNA band was then excised from the gel and the DNA was eluted by a standard protocol known to those skilled in this art.
  • the DNA fragment was re- amplified under the same conditions as described above.
  • the second cycle DNA products were digested with restriction enzyme, and run on 2% agarose gel to check the yield and quality of the DNA product.
  • the primers were removed from the products by column filtration.
  • the purified amplified DNA products were ethanol precipitated, dissolved in IX Tris/EDTA and were ready for DNA sequencing.
  • a small amount of human genomic DNA was digested with Sau3A, Msp 1 and Taq 1, consecutively in appropriate buffers.
  • the size population of the DNA fragments was mostly less than 2.5kb.
  • This sized DNA can readily be amplified by polymerase chain reaction.
  • Other enzymes such as Hhal, Hpa2 and Rsal may also be employed to digest the genomic DNA.
  • the DNA fragments were ligated to 22 mer/20 mer linker DNA with GC cohesive ends and 24/20 mer linker DNA with GATC cohesive ends overnight at 20 C in a standard ligation buffer.
  • a small quantity of the linker ligated DNA prepared above was incubated in a PCR buffer in the presence of 20 mer primer. (Note: The DNA sequence of the two 20 mer was identical in the linker DNA and this linker DNA may be synthetically or randomly designed.)
  • the mix was denatured at 94° C for 1 min. , annealed at 55°C for 1 minute, and elongated at 73° C for 15 minutes in sequential order. The cycle of denaturing, annealing and elongating was repeated 30 times.
  • the amplified DNA product was extracted one time with chloroform, ethanol precipitated and dissolved in IX Tris/EDTA buffer.
  • the size distribution of amplified DNA fragments was almost the same as the original DNA digest suggesting the amplification may be homogeneous.
  • the gel analysis of the DNA fragments suggested that the original composition of the human DNA digest can be regenerated by this non- primer specific PCR procedure.
  • This technology can also be applied to quantitate the copy number of an amplified gene in small number of tumor cells.
  • Another application is the use of the three restriction endonucleases to digest DNA to yield DNA fragments for the production a polymerase chain reaction walking library, where the average DNA size is amenable to polymerase chain reaction amplification (at least 98% DNA fragments will amplify by the polymerase chain reaction method) .
  • An additional application is the use of this strategy to homogeneously amplify DNA micro- dissected from a specific chromosomal region.

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Abstract

L'invention se rapporte à un procédé de progression d'une réaction en chaîne de polymérase, destinée à produire des bibliothèques non vectorielles d'ADN génomiques, à partir desquelles des séquences latérales d'un ensemble de séquences d'ADN connues peuvent être clonées et amplifiées. Ce procédé de progression d'une réaction en chaîne de polymérase consiste à utiliser des agents de liaison d'ADN non phosphorylés ou phosphorylés, qui se joignent uniquement aux extrémités 5', en bloquant les extrémités 3' non jointes, et en amorçant spécifiquement la synthèse des séquences latérales désirées au moyen d'une amorce complémentaire de la séquence d'ADN connue. Le clonage de séquences d'ADN situées en position 5' par rapport à la région promotrice de zêta-globine, grâce à l'utilisation d'une bibliothèque de progression de réaction en chaîne de polymérase, est présenté à titre d'exemple.
PCT/US1992/000532 1991-01-22 1992-01-22 Progression d'une reaction en chaine de polymerase 5' et 3' a partir de sequences d'adn connues WO1992013104A1 (fr)

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WO1994017203A1 (fr) * 1993-01-29 1994-08-04 The Governors Of The University Of Alberta Procede d'analyse d'empreinte d'adn amplifie permettant de detecter les variations du genome
WO1995027079A2 (fr) * 1994-03-31 1995-10-12 Perkin Elmer Corp Procede de reduction de signaux de fond dans des techniques de replication/detection d'adn
WO2000024929A2 (fr) * 1998-10-26 2000-05-04 Christof Von Kalle Reaction pcr etablie par amplification lineaire
WO2000060121A1 (fr) * 1999-04-06 2000-10-12 Genome Technologies, Llc Marche le long d'un genome par pcr a l'aide d'une amorce synthetique
US7026115B1 (en) 1991-09-24 2006-04-11 Keygene N.V. Selective restriction fragment amplification: fingerprinting
US7935488B2 (en) 1991-09-24 2011-05-03 Keygene N.V. Selective restriction fragment amplification: fingerprinting

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ATE191510T1 (de) * 1991-09-24 2000-04-15 Keygene Nv Selektive restriktionsfragmentenamplifikation: generelles verfahren für dns-fingerprinting

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EP0356021A2 (fr) * 1988-07-28 1990-02-28 Zeneca Limited Procédé pour l'amplification de séquences nucléotidiques

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7026115B1 (en) 1991-09-24 2006-04-11 Keygene N.V. Selective restriction fragment amplification: fingerprinting
US7572583B2 (en) 1991-09-24 2009-08-11 Keygene N.V. Selective restriction fragment amplification: fingerprinting
US7935488B2 (en) 1991-09-24 2011-05-03 Keygene N.V. Selective restriction fragment amplification: fingerprinting
WO1994017203A1 (fr) * 1993-01-29 1994-08-04 The Governors Of The University Of Alberta Procede d'analyse d'empreinte d'adn amplifie permettant de detecter les variations du genome
WO1995027079A2 (fr) * 1994-03-31 1995-10-12 Perkin Elmer Corp Procede de reduction de signaux de fond dans des techniques de replication/detection d'adn
WO1995027079A3 (fr) * 1994-03-31 1995-11-23 The Perkin-Elmer Corporation Procede de reduction de signaux de fond dans des techniques de replication/detection d'adn
WO2000024929A2 (fr) * 1998-10-26 2000-05-04 Christof Von Kalle Reaction pcr etablie par amplification lineaire
WO2000024929A3 (fr) * 1998-10-26 2000-09-21 Kalle Christof Von Reaction pcr etablie par amplification lineaire
US6514706B1 (en) 1998-10-26 2003-02-04 Christoph Von Kalle Linear amplification mediated PCR (LAM PCR)
WO2000060121A1 (fr) * 1999-04-06 2000-10-12 Genome Technologies, Llc Marche le long d'un genome par pcr a l'aide d'une amorce synthetique

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