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WO1993011266A1 - Procede de detection de mutations et de lignees monoclonales par analyse des polymorphismes de la configuration d'arn - Google Patents

Procede de detection de mutations et de lignees monoclonales par analyse des polymorphismes de la configuration d'arn Download PDF

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Publication number
WO1993011266A1
WO1993011266A1 PCT/US1992/010518 US9210518W WO9311266A1 WO 1993011266 A1 WO1993011266 A1 WO 1993011266A1 US 9210518 W US9210518 W US 9210518W WO 9311266 A1 WO9311266 A1 WO 9311266A1
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dna
rna
gel
cells
dna sequence
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PCT/US1992/010518
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English (en)
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Olaf M. Koch
Peter V. Danenberg
Matthias Volkenandt
Kathleen Danenberg
Joseph Bertino
Testsuro Horikoshi
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Sloan-Kettering Institute For Cancer Research
University Of Southern California
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Publication of WO1993011266A1 publication Critical patent/WO1993011266A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism

Definitions

  • Point mutations of a single base pair are often associated with the pathogenesis of both inherited and acquired diseases.
  • Recent well-known examples are that of the ras oncogene, which is activated by point mutations (1) and the p53 tumor suppressor gene, which in many common types of cancers has been found to contain single base mutations (2,3).
  • strategies for rapid and reliable detection of point mutations in specific genes would obviously be of great value.
  • a worthwhile goal also is to find techniques that are adaptable for gene analysis in cleanicla laboratories to allow routine and inexpensive screening of individuals for the presence of specific mutations in their genes. Numerous methods are currently available for detection of point mutations, each of them having advantages as well as drawbacks (4).
  • DNA sequencing is the most direct method and permits unequivocal identification of the specific base substituted at the point mutation.
  • sequencing is very labor intensive and is not practical either for screening a large number of samples or for searching long stretches of DNA for mutations.
  • Sequencing as a tool for characterizing mutations is most effective when used in conjunction with a non-specific screening method that detects and fixes the approximate location of the mutations. Most such methods rely on oligonucleotide mismatch analysis.
  • a probe of known sequence is hybridized to a region of the gene of interest. Base pair mismatches within the region covered by the probe can be detected by various chemical and physical methods.
  • RNase treatment of the duplex does not cleave all mismatches (5).
  • Denaturing gradient gel electrophoresis (6) or chemical cleavage (7) may detect a greater percentage of mismatches but these methods are more complicated and difficult to use.
  • the base substitution causes the DNA segment to assume a unique folded conformation, which alters its mobility on a non-denaturing gel compared to the corresponding unmutated DNA segment.
  • the strategy is to amplify the desired segment of a gene by the PCR (10), and then to compare the migration pattern of the denatured DNA with that of a reference segment of known sequence.
  • the "single strand conformational polymorphism" (SSCP) assay is simple, rapid, and sensitive, and has now been used for detection of point mutations in several studies (11, 12, 13, 14, 15). However, because this is still a relatively new method, it is uncertain what percentage of mutations will be. detected by DNA SSCP analysis (12). Cawthon et al.
  • DHFR dehydrofolate reductase
  • clonal lymphoproliferative disorders are diagnosed by morphology, immunogistochemistry and fluorescence activated cell sorting.
  • monoclonal populations can be detected by Southern blot analysis of T-Cell receptor (TCR) or immunoglobulin gene rearrangements.
  • TCR T-Cell receptor
  • Southern blot analysis is associtaed with severe limitations and is not feasible in many clinical situations. Relatively large amounts (several micrograms) of well preserved DNA are required and it is often difficult to obtain, e.g., from paraffin-embedded material).
  • this assay is labor and time intensive.
  • a method to detect within a predetermined DNA sequence from a subject, the presence at a predefined position, a mutation comprises obtaining from the subject a sample containing the predefined DNA sequence, amplifying this DNA sequence, transcribing the amplified DNA to RNA, separating this RNA on a non-denaturing gel so as to separate the RNA into one or more bands having a defined pattern, comparing the pattern of bands obtained with the pattern of bands obtained by separating on a non-denaturing gel RNA transcribed from a sample known to contain the predetermined DNA sequence without the mutation at the predefined position, and detecting any differences in theses patterns, the presence of a difference in the patterns indicating the presence of a mutation at the predefined position of the predetermined DNA.
  • This invention also provides a method for detecting in a monoclonal lymphoid population of cells from a subject, the presence at a predetermined position of a clonespecific DNA sequence, which comprises obtaining from the subject a DNA sample containing the predetermined position, amplifying this DNA, transcribing the amplified DNA to RNA, separating this RNA on a non-denaturing gel so as to separate the RNA into one or more bands having a defined pattern, comparing the pattern of bands obtained by separation on non- denatureing gel to RNA transcribed from a DNA sample known to contain the predetermined position without the clonespecific sequence and detecting any differences in the patterns, the presence of a difference in the patterns indicating the presence of the monoclonal lymphoid population of cells.
  • Figure 1 Comparison between DNA SSCP and RNA SSCP analysis of p53 gene point mutations.
  • Wild-type p53 plasmid, p53-SN3 (lanes 2, 4, 6) and a synthetic 95 mer mutant p53 template (codon 175, CGC ⁇ CAC, Arg ⁇ His) (lanes 1, 3, 5) were PCR-amplified using the primer pair p53-75 and p53-76 (see Materials and Methods - Experiment No. 1).
  • the predicted DNA fragment would be 95 bp spanning positions +1036 to +1130 plus the T7 promoter sequence for a total of 118 bp.
  • RNA SSCP (lanes 1-4) the 32 P-end-labeled primers were used for PCR and for RNA SSCP (lanes 5 and 6), PCR amplified fragments were transcribed into a 101 nucleotide (95 nucleotide coding sequence plus 6 nucleotide transcription start sequence) fragment of uniformly 32 p-labeled RNA.
  • Non-denatured (lanes 1 and 2) and denatured PCR-amplified DNA products were applied to a native 8% polyacrylamide gel containing 10% glycerol and electrophoresed at room temperature (20-22°C) at 25 W constant power according to the conditions of Orita et al. (8).
  • RNA (lanes 5 and 6) was applied to the same gel without denaturation.
  • FIG. 1 Comparison of DNA SSCP and RNA SSCP analysis of a DHFR gene mutation.
  • the cDNA libraries from HCT-8 cells (lanes 1, 4, 7) and from HCT-8R4 cells (lanes 2, 5, 8) containing a DHFR gene with a T ⁇ C mutation at position 91 (codon 31, TTC ⁇ TCC, Phe ⁇ Ser), and plasmid pHD84 (lanes 3, 6, 9) containing the same mutation as the HCT-8R4 cells were PCR-amplified using primer pair DHFR-70 and DHFR-72.
  • the anticipated fragment would have 163 bp of coding sequence spanning positions +38 to +200, plus 20 bases from the T7 promoter sequence for a total of 183 bp.
  • each fragment was the same as in Figure 1.
  • Denatured (lanes 1-3) and non-denatured (lanes 4-6) PCR-amplified DNA products were applied to a native 6% polyacrylamide gel containing 10% glycerol, and electrophoresed at room temperature (21-31°C) at 20 W constant power.
  • Transcribed RNAs of 166 nucleotides (coding sequence plus 3 bases from the transcription start site) (lanes 7-9) were applied to the same gel without denaturation.
  • Direct sequence analysis of the two lower bands of the RNA of HCT-8 cells (lane 7) showed that the lower band had a wild-type sequence while the upper band has a silent A ⁇ G transition at codon 32(AGA ⁇ AGG, Arg ⁇ Arg).
  • Figure 3 Demonstration of conformational equilibration of RNA.
  • Uniformly 32 P-labeled RNAs were applied to a native 8% polyacrylamide gel without glycerol and electrophoresed at 5-7°C at 25 W constant power (A).
  • the bands numbered 2, 3, and 4 from wild-type RNA and band 1 (small arrow in A) from mutant RNA were excised and each RNA was extracted from the gel.
  • Genomic DNA containing the wild-type DHFR gene from mouse L1210 cells (lanes 1 and 4) and genomic DNA from two individual cell lines containing identically mutated DHFR genes (23) (codon 15, GGG ⁇ TGG, Gly ⁇ Trp) (lanes 2 and 3) were PCR-amplified using the primer pair DHFR-85 and DHFR-86 and then transcribed to RNA. These primers are expected to generate a fragment of 177 nucleotides spanning a region of the genomic DNA from positions -77 to +100 plus a 6 base transcription start sequence for a total of 183 nucleotides.
  • cDNA library from mouse mammary adenocarcinoma FM3A wild-type (lanes 1 and 3) and from FM3A (TK-) cells (lane 2) containing a mutated thymidine kinase gene (codon 158, G ⁇ C, Arg ⁇ Pro) were PCR-amplified using primer pair TK-5 and TK-43 and then transcribed to RNA.
  • the anticipated fragment would be 257 nucleotides, corresponding to positions +319 to +575 of the thymidine kinase cDNA plus a 6 base transcription start sequence for a total of 263 nucleotides.
  • RNA SSCP analysis of several mutations in the human DHFR gene was applied to a native 8% polyacrylamide gel without glycerol and electrophoreses at 5-7oC at 25 W constant power. Figure 5. RNA SSCP analysis of several mutations in the human DHFR gene.
  • the plasmids were PCR-amplified using primer pair DHFR-70 and DHFR-71.
  • the PCR-amplified DNAs were transcribed into uniformly-labeled 32 P-RNA.
  • the anticipated fragment of 134 nucleotides would span positions +38 to +171 of the DHFR cDNA plus a 3 base transcription start sequence for a total of 137 nucleotides.
  • the RNAs were applied to a native 8% polyacrylamide gel without glycerol and then electophorased at 6-7°C at 25 W constant power.
  • Lane wt (wild-type) wild-type DHFR plasmid pHD84.
  • Lane 1 Phe 22.
  • Lane 2 Met 22.
  • Lane 3 Trp 22.
  • Lane 4 double mutant Phe 22/Ser 31.
  • Lane 5 Ser 31.
  • Lane 6 Ser 34.
  • FIG. 6 Non-radioactive visualization of RNA SSCP analysis gels. Wild-type p53 plasmid p53-SN3 (lane 1) and synthetic mutant p53 templates p53-80 (lane 2) were PCR-amplified and transcribed in Figure 1. Wild-type DHFR plasmid pHD84 (lane 1)
  • RNAs were applied to a native 8% polyacrylamide gel without glycerol and electrophoresed at 6-7oC at 25 W constant power. The gel was then stained with ethidium bromide and the photograph was taken of the gel on top of a UV- transilluminator.
  • FIG. 7 TCR-gamma junctional sequences were amplified by PCR, the reaction product was transcribed into complementary RNA. The different cRNA conformational polymorphisms were analyzed by electrophoresis on a 8% non-denaturing polyacrylamide gel electrophoresis. Lane 1: Marker substance. Lane 2, 3: Analysis of polyclonal lymphoid populations of 2 healthy individuals. Lane 4, 5, 6: three patients with acute leukemia. Lane 7: patient with mycosis fungoides, DNA isolated from paraffin embedded, formalin fixed tissue. The rearrangement of the TCR genes was proven by cloning and direct sequence analyses prior done to this experiment. Figure 8.
  • the junctional sequences of IgH gene rearrangements were amplified by PCR and transcribed into complementary RNA.
  • the reaction products were analyzed by electrophoresis on an 8% polyacrylamide gel.
  • Lane 1, 2, 3 patients with acute lymphocytic leukemia.
  • Lane 4 Marker substance.
  • This invention provides a method to detect within a predetermined DNA sequence from a subject, the presence at a predefined position, a mutation, which comprises obtaining from the subject a sample containing the predefined DNA sequence, amplifying this DNA sequence to obtain multiple copies of the sequence, transcribing the amplified DNA to RNA, separating this RNA on a non-denaturing gel so as to separate the RNA into one or more bands having a defined or "characteristic" pattern, comparing this pattern with the pattern of; RNA obtained by separating on a non-denaturing gel RNA transcribed from a sample known to contain the predefined DNA sequence without the mutation at the predetermined position, and detecting any differences in these two patterns, the presence of a difference in the patterns indicating the presence of a mutation at the predetermined position of the predefined DNA.
  • mutation means any change in the sequence of DNA.
  • this DNA is genomic DNA, single-stranded DNA, double-stranded DNA, plasmid DNA, bacterial DNA, parasitic DNA or viral DNA.
  • a point mutation is an alteration that changes a single base pair in the DNA sequence. Historically, such single or point mutations have been difficult to detect.
  • a suitable DNA sample is any sample containing the DNA sought to be assayed.
  • amplified means multiply the copy number of the sequence either by enzymatic amplification (polymerase chain reaction or "PCR") or by traditional cloning techniques.
  • PCR polymerase chain reaction
  • the predetermined sequence is isolated, utilizing restriction enzymes that cut the DNA at predetermined, specific locations to create restriction enzyme fragments. These fragments are isolated by separation techniques well known to those of skill in the art, such as separating the restriction enzyme fragments by running them on an agarose gel.
  • the fragments are purified from the gel, by techniques well known to those of skill in the art, and the fragment containing the predefined position is inserted into a recombinant cloning vector such as a virus or plasmid. Suitable host cells are then transformed with these cloning vectors. The host cells are then grown under suitable conditions such that the DNA fragment is multiplied. The vector DNA is then isolated from the host cell and the inserted DNA is then removed from the vector by the use of restriction endonucleases, run out on an agarose gel and subsequently purified from the gel.
  • a recombinant cloning vector such as a virus or plasmid.
  • Suitable host cells are then transformed with these cloning vectors.
  • the host cells are then grown under suitable conditions such that the DNA fragment is multiplied.
  • the vector DNA is then isolated from the host cell and the inserted DNA is then removed from the vector by the use of restriction endonucleases, run out on an agarose gel and subsequently pur
  • the DNA is amplified utilizing polymerase chain reaction ("PCR").
  • PCR polymerase chain reaction
  • a suitable primer is selected which neighbors the predefined position.
  • This primer may include a T7 polymerase promoter sequence on one end of the primer.
  • the DNA is then amplified and transcribed to RNA using methods well known to those of skill in the art.
  • the primer contains not only the T7 polymerase sequence but also a "clamping sequence" which increases the yield of the RNA product and is transcribed along with it.
  • RNA polymerase Methods of transcribing a DNA sample to RNA using RNA polymerase enzymes are well known to those of skill in the art.
  • RNA obtained either through traditional cloning techniques or PCR, is then run on a non-denaturing gel, such as a polyacrylamide gel (PAGE), to obtain an RNA pattern characteristic of the DNA sample.
  • a detectable marker such as a radioisotope or a fluorescent label.
  • the resulting RNA gel may be dyed with ethidium bromide so that the RNA pattern may be visualized. This pattern is then compared to the pattern of RNA obtained from "wild-type" or "normal” DNA, i.e., the predetermined DNA sequence without the mutation at the predefined position.
  • the RNA pattern resulting from the DNA sample will be different from the wild-type.
  • the subject is an animal or a mammal. In the preferred embodiment of this invention, the subject is a human patient.
  • This method is particularly useful for the detection of point mutations in small samples of DNA, for example, DNA obtained from paraffin-embedded material.
  • Inherited or acquired diseases may be detected, at the genetic level, utilizing the subject invention.
  • activation of the ras oncogene, which is activated by point mutations may be detected utilizing the subject invention.
  • Also provided by this invention is a method for detecting a monoclonal lymphoid population of cells, wherein the monoclonal population is the result of the presence of variable idiotypic DNA sequences between lymphoid populations.
  • This also is a method for detecting in a monoclonal lymphoid population of cells from a subject, the presence of a predetermined position of a clonespecific DNA sequence.
  • This method comprises obtaining from the subject a DNA sample from a lymphoid cell, containing the predetermined position of a clonespecific DNA sequence, amplifying this DNA sequence, transcribing the amplified DNA to RNA, separating the resulting RNA on a non-denaturing gel so as to separate the RNA into one or more bands having a defined pattern, comparing the pattern of bands, obtained by separation on non-denaturing gel to RNA transcribed from a DNA sample known to contain the predetermined position without the clonsespecific (i.e., DNA obtained from a polyclonal lymphoid population), and detecting any differences in the RNA patterns, the presence of a difference indicating that the sample of DNA contains monoclonal lymphoid population of cells.
  • the band pattern on the gel is clonotypic for each individual lymphocytic clone. With this method it is possible to distinguish different clones from the same precursor.
  • the monoclonal lymphoid population of cells are T-cells and in another embodiment of this invention the lymphoid population of cells are B cells.
  • the predetermined position is the DNA sequence corresponding to the T-cell receptor gamma gene or it may be, but is not limited to, the DNA sequence corresponding to the T-cell receptor gamma junctional gene.
  • the variation giving rise to the clonespecific population of cells of the predetermined sequence is in the DNA corresponding to the T-cell receptor gamma variable region.
  • "clonespecific" means the presence of variable sequences generated during the process of rearrangement of genes, creating unique sequences for individual lymphoid clones.
  • the predetermined position is the DNA sequence corresponding to the variable DNA sequences in the junctional region of immunoglobulin gene.
  • the DNA sequence correspond to CDR I, CDR II, CDR III or IgH. "Amplified" has been described hereinabove.
  • this method further comprises labeling the RNA obtained after amplification and subsequent to transcription.
  • the RNA may be labeled with a radioisotope or fluorescent label by methods known to those of skill in the art.
  • Suitable lymphoid cell samples for the practices in this invention include, but are not limited to, lymphocytic paraffin-embedded material, lymph node specimens, biopsies and peripheral blood lymphocytes.
  • the gel may be stained with ethidium bromide so that characteristic bands or patterns may be identified.
  • primers are created which are complementary to conserved sequences of these genes, flanking the predetermined position in the DNA sequence.
  • the primers have joined to the 5' end, DNA sequences corresponding to a T7 polymerase promoter. This enables the transcription of the PCR amplified junctional sequences into RNA.
  • the amplified DNA when the DNA is amplified by traditional cloning techniques, the amplified DNA must be transcribed to RNA by the use of RNA polymerases, using methods known to those of skill in the art. Tables I and II, below, schematically describe this invention.
  • variable DNA sequences in the junctional regions of immunoglobulin genes may be detected by this method of conformational polymorphisms of complementary RNA of variable gene segments.
  • disorders such as lymphomas of B cell and T-cell lineages as well as acute lymphocytic leukemias of both lineages may be detected in various clinical specimens (biopsies, paraffin embedded, formalin fixed tissue, aspirates, and exfoliating cells).
  • the practice of this invention is particularly useful for the detection of monoclonal populations of B cell and T-cell lineages. It enables one of skill in the art, to compare different biopsies or samples of the same patient, obtained from different localisations or taken at different timepoint of the disease. By this method it is particularly easy to detect subclone or new clone formation in acute lymphocytic leukemias at various timepoints of disease. In cases of relapse after therapy of a lymphoma or an acute lymphocytic leukemia it is possible to compare the relapse pattern with the initial pattern. If a new clone is causing the relapse, there might be still a response to a therapeutical regiment which has been used primarily to eradicate the primary clone. If the same clone is still present, a resistance to the initial therapy is suggestive.
  • this invention is a method to detect a mutation or variation in a sample of DNA, wherein the DNA sample is characterized by the presence of conserved and variable DNA sequences.
  • This method is as the method described above for the detection of mutations.
  • the mutation is present in the variable sequence.
  • the DNA sequence is amplified and transcribed by PCR, and the primer is complementary to the conserved sequence flanking the variable DNA sequence.
  • This method is useful to distinguish different viral, bacterial or parasital strains from each other, especially when such strains cannot be distinguished easily be conventional methods.
  • the information derived from this method is useful for epidemiological studies, for diagnostics, in the treatment of infectious diseases, as well as for the development of vaccines.
  • RNA can assume elaborate secondary and tertiary structures (18); 2) RNAs can simultaneously exist in a number of different conformations which do not equilibrate among each other unless denaturing or high temperature conditions are applied (19, 20); and 3) that the conformations even of large RNA molecules appear to be sensitive to single-base substitutions. (21).
  • RNA instead of DNA RNA instead of DNA.
  • electrophoretic patterns of RNA molecules corresponding to normal and single-base mutated segments of the genes of DHFR, p53, and thymidine kinase were compared. It was found that the RNA generated from amplified DNA usually had numerous conformational forms that were observable by native gel electrophoresis and that single base mutations changed the distribution and the number of these forms. RNA SSCP analysis was able to detect single base substitutions in all of the segments tested including samples in which the DNA SSCP method failed to detect any change.
  • Human colon carcinoma cell line HCT-8 was obtained from the American Type Culture Collection.
  • the HCT-8R4 cell line was developed from HCT-8 cells by stepwise adaptation to methotrexate (MTX) (22).
  • MTX-resistant mouse L1210 cells containing a G ⁇ T(Gly ⁇ Trp) transition at nucleotide 46 of the DHFR gene were developed in vivo (23).
  • the mouse adenocarcinoma ceil line FM3A (TK ⁇ ) was developed from wild-type FM3A cells by stepwise treatment with 5- fluorodeoxyuridine (24).
  • p53 plasmids pC53-SN3 (wild-type) and pC53-SCX3 (Val ⁇ Ala mutant at codon 143) (25) were obtained from Dr. B. Vogeistein.
  • Plasmid pHD84 containing the wild- type human DHFR sequence (26) was: obtained from Dr. G. Attardi.
  • the DHFR mutants Phe 22 (Leu 22 ⁇ Phe, CTG ⁇ TTT) Met 22 (Leu 22 ⁇ Met, CTG--ATG), Trp 22 (Leu 22 ⁇ Trp, CTG ⁇ TGG), Phe 22/Ser 31 (Leu 22-Phe, CTG-TTT; Phe 31 ⁇ Ser, TTC ⁇ TCC), Ser 31 (Phe 31 ⁇ Ser, TTC ⁇ TCC), and Ser 34 (Phe 34 ⁇ Ser, TTC ⁇ Tcc) were synthesized by site-directed mutaginesis of wild-type DHFR cDNA contained in expression vector pKT7HDR (27) and furnished to us by Dr. Adam Dicker, Memorial Sloan-Kettering Cancer Center.
  • Template p53-80 containing the p53 gene sequence from positions +1036 to +1130 with a G ⁇ A substitution at nucleotide 1084 (codon 175) was synthesized on an Applied Biosystems model 391 PCR-MATE DNA synthesizer by the phosphoramidite method.
  • the sequence of p53-80 was:
  • DNA and RNA isolation DNA was isolated according to the procedure of Blin and Stafford (29). RNA was isolated by the AGPC method of Chomczynski and Sacchi (30).
  • PCR PCR.
  • the primers listed below were synthesized for use in the PCR.
  • Each 5' primer had a T7 polymerase promoter sequence on its 5' end, indicated by the prefix T7.
  • the segments in quotation marks are not part of the target gene sequence, but represent a "clamping sequence" which increases the yield of the RNA product and is transcribed along with it.
  • T7 - TAA TAC GAC TCA CTA TA p53-75: T7-"GGGAGA" CC ATG GCC ATC TAC AAG CAG TCA
  • p53-76 AGG GGC CAG ACC ATC GCT AT
  • DHFR-70 T7-"GGG" AGA ACA TGGG CAT CGG CAA GAA CG
  • DHFR-72 GG TCG ATT CTT CTC AGG AAT GG
  • DHFR-85 T7-"GGGAGA" TCA GGG CTG CGA TGT CGC GCC AAA
  • DHFR-86 AGC CCG GCC AAT ACC TGA GCG GA
  • TK-5 T7-"GGGAGA" TC GAT GAG GGG CAG TTT TTT CC
  • TK-43 ATA CTT GTC GGC TCC GCC AAT CA
  • the PCR reactions contained 12.5 pmol of each of the primers, 2.5 ul of 10X TAQ buffer (500 mM KCI, 100 mM Tris- HCl, pH 8.3, and 0.01% gelatin), 200 uM deoxyribonucleotide triphosphates, 1.87 mM MgCl 2 , 10-100 ng of template DNA and 0.63 units of TAQ polymerase (Cetus) in a total volume of 25 ul.
  • the reaction mixture was overlaid with mineral oil. Before the TAQ polymerase was added, the mixture was heated to 95oC for 5 minutes.
  • the PCR conditions were 30 cycles of 1 minute at 93.5°C, 1 minute at 55°C, and 1 minute at 72°C in an Ericomp Twinblock Temperature Cycler.
  • both PCR primers were 5' end- labeled according to the forward reaction conditions in the BRL T4 polynucleotide kinase kit.
  • the primers (75 pmol) were added to 30 pmol of [ ⁇ -32 P]ATP (5000 C i /mmol) (Amersham), 5.6 ul of BRL 5X forward kinase reaction buffer (300 mM Tris-HCI, pH 7.8, 75 mM 2-mercaptoethanol, 50 mM MgCl 2 and 1.65 uM ATP) and 10 unites of T4 polynucleotide kinase (BRL) in a total volumn of 27.4 ul.
  • the reaction was incubated for 30 minutes at 37°C and then heated for 5 minutes at 65oC.
  • the primers were precipitated with ethanol before use in the PCR reaction.
  • RNA SSCP was performed, the PCR primers were not labeled, but the PCR reaction was transcribed with T7 polymerase.
  • RNA were electrophoresed on a 6 or 8% polyacrylamide gel with or without 10% glycerol in a Hoeffer SE-600 dual-cooled PAGE unit.
  • the gel dimensions were 140 x 135 x 1.5 mm.
  • the running buffer was 89 mM Tris-borate, pH 8.3, and 2 mM EDTA.
  • the gels were run at 25 watts constant power per plate at either 20-22°C or 6-7°C by circulating tap or ice water, respectively, through the cooling tubes of the gel apparatus.
  • the gel running buffer was pre-equilibrated at the correct temperature for at least 1 hour.
  • DNA SSCP analysis was performed according to Orita et al. (16).
  • RNA SSCP analysis 4 ul of the T7 transcription reaction were mixed with 1 ul of 50% glycerol containing 0.25% bromophenol blue dye and electrophoresed as described above. The gel was then dried and exposed to Kodak XAR-5 film or stained wet with ethidium bromide and photographed with Polaroid 665 film.
  • RNA sequencing RNA fragments were extracted from the gels by exising the portions of the gel containing the desired bands and shaking the gel pieces overnight in 0.5 M ammonium acetate. The RNA was precipitated with ethanol and then sequenced with AMV reverse transcriptase according to Stoflet et al. (17).
  • RNA and DNA SSCP analysis To compare RNA and DNA SSCP patterns, a 118 bp wild-type and mutant fragment of the p53 gene using the PCR was amplified. The fragments corresponded to positions +1036 to +1130 of the p53 coding region and the mutant DNA had a G to A substitution at codon 175. A portion of the double-stranded DNA was transcribed to RNA using T7 polymerase. Electrophoresis was performed using the optimal conditions suggested by Orita et al. (8). There was no discernible difference in migration between the separated wild-type and the mutant DNA strands (lanes 3 and 4 of Figure 1). On the other hand, the RNA patterns (lanes 5 and 6) were quite different. Although the major RNA bands migrated identically, the wild-type RNA displayed at least four more bands than did the RNA generated from the mutant DNA.
  • Wild-type and mutated DHFR gene segments were further compared by RNA and DNA SSCP analysis. The following was used: 1) a 183 bp DHFR segment spanning positions +38 to +200 of cDNA prepared from HCT-8R4 cells, which contains a known mutation at position 91 of the coding region (resulting in a substitution of serine for phenylalanine at codon 31) (22); 2) the same fragment amplified from cDNA of HCT-8 cells, which presumably had the wild-type sequence; and 3) the same segment from a vector containing the DHFR gene carrying the same mutation as the HCT-8R4 cells (27) as a control.
  • the PCR amplified double-stranded DNA was remarkably homogenous and showed no visible secondary bands (lanes 4-6 of Figure 2).
  • the denatured DNA did not display any noticeable difference in migration between the wild-type (lane 1) and the mutants (lanes 2 and 3). Only two bands were seen instead of the expected three bands, probably because one of the single-stranded DNA molecules migrated identically with the double-stranded DNA (the lower band).
  • the RNA SSCP showed a distinct difference between migration of the HCT-8 RNA (lane 7) and the two mutant RNAs (lanes 8 and 9). It was originally thought that the HCT-8 cells would contain only the wild-type sequence, and thus that the two bands visible in lane 7 were different conformations of wild-type RNA.
  • RNA SSCP Interconversion of RNA conformations.
  • RNA SSCP shows the RNA SSCP patterns of the wild-type and mutant p53 segments with the wild-type RNA run in two lanes to show that the pattern of bands was reproducible.
  • Figure 3B shows the re-electrophoresis of some of the bands designated by numbers in Figure 3A.
  • band 2 which actually consists of two closely spaced bands
  • band 2 was re-electrophoresed without any heating
  • small amounts of bands 5 and 6 were visible (lane a). Heating the material (lane b) caused more formation of products 5 and 6 as did treatment with formamide, a mild denaturing agent (lane c).
  • band 2 did not give rise to any appreciable amounts of band 3 or 4.
  • Band 3 when re-electrophoresed (lanes d-f) also gave rise mostly to band 5 and a small amount of both 4 and 6 but none of band 2. Heating also increased the conversion of band 3 to band 5.
  • Band 4 apparently was stable conformation, because only a small amount of conversion to band 5 was observed under conditions where the other conformations underwent substantial re- equilibration.
  • RNA SSCP The upper band of the mutant RNA (shown by the small arrow in Figure 3A) was also isolated and was found to interconvert to bands 5 and 6, more so under mild heating (Figure 3C, lane k) or denaturing conditions (lane 1). These results demonstrate that the bands seen in the RNA SSCP gels consist of RNA conformational isomers. More examples of RNA SSCP analysis.
  • a 177 bp fragment of the DHFR gene was amplified by the PCR suing as templates genomic DNA from wild-type L1210 cells and two MTX-resistant cell lines known to contain the same mutation in codon 15 (GGG to TGG, resulting in a Gly to Trp substitution in the protein) (230.
  • Figure 4A shows that the pattern of the major bands of the wild-type RNA (lanes 1 and 4) differs from that of the two resistant cell lines (lanes 2 and 3). There is also a pattern of slower-migrating minor bands (not very visible in the photograph) which vary between the cell lines.
  • FIG. 4B shows a clear difference in migration between the fragment from the wild- type cells (lanes 1 and 3) the mutant fragment containing a G to C substitution at position 505 (Arg ⁇ Pro at codon 158) (lane 2).
  • Figure 4C analyzes a mutation in the p53 gene consisting of a C to T substitution at codon 143 (Val ⁇ Ala). In this case, the major band of the two fragments migrated identically but the wild-type (lane 2) showed several additional minor conformations. This difference is more subtle than seen in the other examples, but nevertheless diagnostic of a different sequence.
  • DHFR mutants were compared in Figure 5. These mutants had been generated by site-directed mutagenesis of DHFR cDNA cloned into an expression vector. The sites of mutation were close enough so that the same set of PCR primers could be used to generate fragments containing the mutation. Each of the segments containing a single-base substitution gave a unique pattern of RNA conformations.
  • RNA bands One of the favorable consequences of converting PCR-amplified DNA into RNA is that a further 500-fold amplification of the PCR amplified oligonucleotide occurs in one step (17).
  • An advantage of this amplification for SSCP analysis is that the large amount of oligonucleotide thus generated permits one to load enough RNA onto the gel so that the bands can be conveniently visualized by ethidium bromide staining.
  • the ethidium bromide stained gels are shown in Figure 6 and correspond to lanes 5 and 6 of Figure 1 and lanes wt (wild- type) and 5 of Figure 5. Except for very faint bands, the same pattern that is seen by autoradiography is also visible by ethidium bromide staining.
  • RNA bands can also be visualized by UV shadowing, which provides a very rapid means of monitoring the results of the experiment.
  • the wet gel is placed on top of a commercial silica gel plate containing fluorescent indicator, and the RNA bands become visible when placed under a hand-held UV light.
  • the DNA SSCP method for detecting point mutations as it is currently used depends on a single base substitution in the DNA strand causing the formation of a novel conformation sufficiently different from that of the wild-type to cause an observable change in electrophoretic migration (8, 9). Thus, a small change in the conformation of the DNA may not be detected by one-dimensional gel electrophoresis and the analysis would therefore give a false negative answer as to the presence of a mutation.
  • RNA may have multiple stable conformational states available to it (19, 20) and therefore, the probability of seeing a change in one or more of these states due to a point mutation would be greater than for the single conformation that is normally seen in DNA SSCP analysis. It was observed, for example, that human 7SL RNA is separated into 4 different conformational states by gel electrophoresis and that mutation of the RNA by deletion of small segments from certain regions eliminated 3 of the 4 conformations (19).
  • RNA SSCP analysis was able to detect single-base substitutions in several cases where no change in migration of the analogous mutated DNA was observed.
  • RNA intrinsically has much more conformational polymorphism than DNA or whether the denaturing conditions used to separate the double-stranded DNA into single stands promotes equilibration of the molecule to a single most stable conformation.
  • the RNA generated by T7 polymerase transcription was not subjected to denaturation before gel electrophoresis so it is possible that single-stranded DNA generated by asymmetric PCR would also display many conformational states.
  • the size limit of the fragments in which DNA SSCP analysis can detect single-base substitutions is about 250-330 bp (8, 9).
  • the mutation in the 250 nucleotide thymidine kinase fragment was readily observed, but a 350 nucleotide fragment of wild-type and mutant DHFR did not show any difference. However, it can be possible to analyze larger fragments by changing the PAGE conditions.
  • RNA-SSCP has several important practical advantages.
  • the RNA molecules are always single- stranded and non-complementary to each other. Thus, it is not necessary to dilute the RNA before gel electrophorese to prevent reannealing as frequently occurs with denatured double-stranded DNA molecules. Much more material can therefore be loaded onto the gels, permitting the use of non-radioactive visualization methods such as ethidium bromide staining. Some sensitivity may be lost by non- radioactive visualization, but in most cases it should be sufficient to detect differences between electrophoretic patterns.
  • the transcription reaction to convert the PCR-amplified DNA to RNA gives uniformly radiolabeled RNA of high specific activity and thus permits the PCR reaction to be performed without radioactivity.
  • RNA SSCP method was not as critically dependent on gel electrophoretic conditions gave the best results (7-8% PAGE, 4°C, no glycerol, run at a constant 25 watts per gel) the differences between wild-type and mutant RNA could still be observed under practically all conditions that were tried.
  • RNA SSCP is as simple and rapid as DNA SSCP and may detect point mutations more reliably.
  • This assay can be performed on any clinical specimen, where a lymphocytic population is present and the DNA can be extracted and amplified by polymerase chain reaction. This is possible for biological materials, such as peripheral blood lymphocytes, DNA extracted from paraffin embedded histological specimens, lymph node or other biopsies.
  • junctional DNA sequences of the T-cell receptor (gamma) genes and of the immunoglobulin heavy chain genes (CDR III) which are unique for an individual lymphocytic clone are amplified by polymerase chain reaction, using primers annealing to conserved sequences of these genes.
  • the 5' primer of the PCR reaction contains a T7 RNA polymerase promoter sequence (5' TAA TAC GAC TCA CTA TAG GGA G). This enables the transcription of the PCR amplified junctional sequences of T-cell receptor gamma genes and immunoglobulin heavy chain genes (CDR III regions) into RNA by a T7 RNA polymerase.
  • the synthesized complementary RNA of the hypervariable regions of the genes forms multiple conformations, that can be separated by a non-denaturing polyacrylamide gel electrophoresis.
  • RNAse inhibitor (40,000 U/ml) 0.25 ul
  • a radioactive labelled ribonucleotide is used, if there is need to document the final reaction product by autoradiography.
  • the mixture is incubated for one hour at 37oC and the enzymatic process is stopped by adding 0.75 ul of 0.5 M EDTA.
  • Ten to 20 ul of the reaction product are loaded on an 8% non-denaturing polyacrylamide gel with a cross-linkage of acrylamide/bisacrylamide of 19;1 and run at 15 Watt ("W") at 4oC.
  • W 15 Watt
  • the gel is stained in an ethidium bromide solution and the bands are visualized on an UV screen.
  • T-cell receptor gamma TCR-gamma genes rearrange in T-and preB-ALL.
  • the junctional structures of V(N)J rearrangements are clonespecific and allow for the detection of a monoclonal population by different breakpoints in the rearrangement of different V and J genes and by the random nucleotide sequence of the inserted N-region.
  • Junctional sequences of TCR-gamma were amplified in ALL by PCR using primers annealing to highly conserved regions of the variable and joining genes.
  • the 5' primer annealing to the v genes contained a T7 RNA polymerase promoter sequence:
  • the PCR product was transcribed into cRNA and electrophoreses on a 8-16% non-denaturing polyacrylamide gradient gel. Because of the clonespecific structure and the unique sequence of the highly variable N-region, multiple conformations of cRNA can be detected as distinct bands on the gel. This presents an idiotypic molecular "fingerprint" of the particular predominant clone.
  • a brad smear on the gel is formed representing numerous conformations of different cRNA molecules. This method can detect as few as 1% monoclonal lymphoid cells. This novel and non-radioactive technique allows the detection of a clonal lymphoid population.
  • Figure 7 shows the results of this experiment.
  • junctional sequences of IgH gene rearrangements were amplified by PCR and transcribed into complementary RNA.
  • the reaction products were analyzed by electrophoresis on an 8% polyacrylamide gel. See Figure 8 for results.

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Abstract

Un procédé permet de détecter une mutation à une position prédéfinie dans une séquence prédéterminée d'ADN d'un sujet. Selon ce procédé, on amplifie et on transcrit la séquence prédéterminée d'ADN dans un échantillon prélevé du sujet et on compare le schéma des bandes d'ARN dans un gel non dénaturant avec le schéma de bandes d'ARN de la même séquence prédéterminée d'ADN sans la mutation. Toutes différences détectées entre les schémas comparés indiquent la présence d'une mutation à la position prédéfinie de la séquence prédéterminée d'ADN. L'invention concerne également un procédé de détection d'une séquence d'ADN spécifique de clones à une position prédéterminée dans une population de cellules lymphoïdes monoclonales prélevées d'un sujet. Selon ce procédé, on amplifie et on transcrit l'ADN contenant la position prédéterminée dans un échantillon prélevé du sujet et on compare le schéma des bandes d'ARN dans un gel non dénaturant avec le schéma de bandes d'ARN transcrit à partir d'un échantillon d'ADN dont on sait qu'il contient la position prédéterminée sans la séquence spécifique de clones. Lorsque des différences sont détectées entre les schémas de bandes comparés, la présence d'une population de cellules lymphoïdes monoclonales est indiquée.
PCT/US1992/010518 1991-12-05 1992-12-07 Procede de detection de mutations et de lignees monoclonales par analyse des polymorphismes de la configuration d'arn WO1993011266A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995017523A3 (fr) * 1993-12-15 1995-09-28 Univ Johns Hopkins Diagnostic moleculaire de la polypose adenomateuse hereditaire
WO2008121899A2 (fr) 2007-03-29 2008-10-09 Vanda Pharmaceuticals Inc. Procédé permettant de prédire une prédisposition à la prolongation du qt
EP3018144A1 (fr) 2006-08-18 2016-05-11 XOMA Technology Ltd. Anticorps prlr spécifique et ses utilisations

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BIOCHEMISTRY, Vol. 20, No. 14, issued 1981, T.O. SITZ et al., "Effect of Point Mutations on 5.8S Ribosomal Ribonucleic Acid Secondary Structure and the 5.8S-28S Ribosomal Ribonucleic Acid Junction", pages 4029-4033. *
CELL, Vol. 61, issued 01 June 1990, M. DEAN et al., "Multiple Mutations in Highly Conserved Residues are Found in Mildly Affected Cystic Fibrosis Patients", pages 863-870. *
GENOMICS, Vol. 8, issued 1990, ORITA et al., "DNA Sequence Polymorphisms in Alu Repeats", pages 271-278. *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES USA, Vol. 86, issued April 1989, ORITA et al., "Detection of Polymorphisms of Human DNA by Gel Electrophoresis as Single-Strand Conformation Polymorphisms", pages 2766-2770. *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995017523A3 (fr) * 1993-12-15 1995-09-28 Univ Johns Hopkins Diagnostic moleculaire de la polypose adenomateuse hereditaire
US5709998A (en) * 1993-12-15 1998-01-20 The Johns Hopkins University Molecular diagnosis of familial adenomatous polyposis
US5760207A (en) * 1993-12-15 1998-06-02 The Johns Hopkins University Primers for amplifying APC gene sequences
EP3018144A1 (fr) 2006-08-18 2016-05-11 XOMA Technology Ltd. Anticorps prlr spécifique et ses utilisations
WO2008121899A2 (fr) 2007-03-29 2008-10-09 Vanda Pharmaceuticals Inc. Procédé permettant de prédire une prédisposition à la prolongation du qt

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