[go: up one dir, main page]

WO1998018966A1 - Amorces pour l'amplification de brca1 - Google Patents

Amorces pour l'amplification de brca1 Download PDF

Info

Publication number
WO1998018966A1
WO1998018966A1 PCT/US1997/019596 US9719596W WO9818966A1 WO 1998018966 A1 WO1998018966 A1 WO 1998018966A1 US 9719596 W US9719596 W US 9719596W WO 9818966 A1 WO9818966 A1 WO 9818966A1
Authority
WO
WIPO (PCT)
Prior art keywords
sequence
exon
seq
primer
mutation
Prior art date
Application number
PCT/US1997/019596
Other languages
English (en)
Inventor
Jennifer Lescallett
Original Assignee
Jennifer Lescallett
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Jennifer Lescallett filed Critical Jennifer Lescallett
Priority to AU50938/98A priority Critical patent/AU5093898A/en
Publication of WO1998018966A1 publication Critical patent/WO1998018966A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the invention relates to the BRCAl gene.
  • it relates to the nucleotide sequences of certain exons of the BRCAl gene and the intronic regions adjacent thereto, and oligonucleotide primers for the amplification of the sequences .
  • BRCAl is the first gene identified with a link to inherited breast and ovarian cancer. Miki et al . , Science 266 : 66-71 (1994).
  • the BRCAl gene is approximately 100,000 base pairs of genomic DNA. Most of the 22 coding exons of BRCAl span regions of between 100-500 basepairs, which in combination result in a protein of 1863 amino acids. Weber, B., Scientific American (January/February 1996) . The exception, exon 11, containing approximately 3600 bp, makes up over half of the coding region of the gene.
  • CDGE constant denaturing gel electrophoresis
  • DGGE denaturing gradient gel electrophoresis
  • Introns located between each pair of exons, contain DNA that is transcribed into RNA, but is not used to synthesize the final protein product.
  • Specific sequence information about an intron enables the construction of primers which can be used for Polymerase Chain Reaction (PCR) technology to produce sufficient copies of a desired region of DNA for further analysis.
  • primers short pieces of DNA that are complementary to the target sequence, are constructed in the intron approximately 100-150 base pairs upstream and downstream from the exon.
  • the resulting PCR product embodies the intron sequence before the exon, the exon, and intron sequence after the exon.
  • BIC Breast Cancer Information Core Database
  • the invention provides sequences and oligonucleotide primers for the amplification of portions of the BRCAl gene using PCR technology.
  • these sequences relate to the regions of Exons 8, 15, 18, 20, 21 and 23 and their surrounding intronic regions (particularly to the region of exon 20) .
  • the primers of the invention will be optimally suitable for amplifying portions of the BRCAl gene for analysis using CDGE, in order to detect mutations in the BRCAl gene.
  • the primers will also be useful in other applications using PCR amplification. These applications include, but are not limited to, DNA sequencing, allele specific oligonucleotide assay, heteroduplex analysis, DNA chip technology and mismatch cleavage methods .
  • forward primer is intended to mean a short complementary single stranded DNA sequence that binds to the DNA strand in the 5' to 3' direction and defines the upper boundaries of amplification.
  • reverse primer is intended to mean a short complementary single stranded DNA sequence that binds to the DNA strand in the 3 ' to 5 ' direction and defines the lower boundaries of amplification.
  • the primers are generally at least about 15 nucleotide bases in length, preferably at least about 20 nucleotide bases in length, and not more than about 65 bases in length (including a GC clamp, if present) .
  • isolated in reference to a DNA sequence, is intended to mean a sequence which is at least free of its natural cellular environment.
  • Example 20 of the BRCAl gene is intended to mean SEQ ID NO:2, and any naturally occuring mutations and variants thereof. It is also intended to mean artificially constructed variants in which no more than two, preferably no more than one, base substitution has been made.
  • Example 21 of the BRCAl gene is intended to mean SEQ ID NO: 38 and any naturally occuring mutations and variants thereof. It is also intended to mean artificially constructed variants in which no more than two, preferably no more than one, base substitution has been made.
  • Exon 8 of the BRCAl gene is intended to mean SEQ ID NOS: 48, 50, 52 and 53 and any naturally occuring mutations and variants thereof as well as artificially constructed variants in which no more than two, preferably no more than one, base substitution has been made .
  • substantially similar as applied to a sequence is intended to include such naturally occuring mutations and variants and artificially constructed variants.
  • expressions “95% (90%) identity” or “95% (90%) identical”, according to the invention are intended to include sequences in which no more than 5% (10%) of the bases are changed, preferably by substitution, as opposed to insertions or deletions.
  • the present invention utilizes CDGE to detect mutations in the BRCAl gene.
  • the BRCAl genomic sequence information containing intron and exon regions around exon 20 was acquired from the Breast Cancer Information Core Database (BIC) . The sequence is listed on the World Wide Web for members of the BIC. Exon 20 of BRCA 1
  • upstream refers to a position on a DNA sequence that is before (5') the exon of interest and “downstream” refers to a position on a DNA sequence that is after (3') the exon of interest.
  • the typed upstream intronic sequence in the BIC of exon 20 (SEQ ID NO : 3 ) was missing nucleotides. These missing nucleotides are identified by the BIC in the typed sequence with the letter *n' . These represent nucleotides which were not identified in the initial sequencing for various reasons including technology hindrance. Resolving these unknown nucleotides was essential for two reasons. First, the accurate melting profile of BRCAl exon 20 was necessary for mutation detection using CDGE. Second, given the best melting profile, the design of primers is facilitated.
  • mutation according to the invention is intended to mean any alteration in the intronic or exonic sequences of BRCAl .
  • alterations to exonic sequences which are known or suspected to be associated with an increased risk of cancer by an individual, particularly of breast cancer.
  • normal sequence unless otherwise specified, is intended to mean a sequence which does not contain such mutations. This may also be referred to as "wild-type sequence” . It is noted that certain variants in the exonic or intronic sequences of BRCAl occur which are not known to confer increased suseptibility to cancer. For example, single base substitutions which do not result in an alteration to the protein produced (normal polymorphisms) are known to occur. While the invention is particularly intended to detect mutations which are known or suspected to be associated with increased cancer risk, it can also be used to detect these variants .
  • sequence is intended to include single- and double- stranded forms as well as sequences which are complementary thereto .
  • FIG. 7 Schematic Diagram of Denaturing Gradient Gel Electrophoresis. The method is performed as detailed by Anne- Lisa Borresen ("Constant Denaturing Gel Electrophoresis (CDGE) in Mutation Screening" in Technologi es for Detection of DNA Damage and Mutations (Ed. G.D. Pfeifer) Chapter 11.22., 1995).
  • CDGE Constant Denaturing Gel Electrophoresis
  • Figure 9 Schematic Diagram of Constant Denaturing Gel Electrophoresis. Method performed as detailed by Anne-Lisa Borresen, cited above.
  • intronic bases are generally shown throughout the application in lower case, whereas exons are indicated in upper case. It is also noted that where dots, dashes or asterisks (*) are inserted into sequences, it is for the purposes of alignment, or to indicate the absence of a particular base which is present in a corresponding sequence .
  • IUB codes have been used in certain locations to designate bases, as will be familiar to those skilled in the art. (Briefly, R designates A or G: Y, C or T; K, G or T; M, A or C; S, G or C; W, A or T; and N, any base.)
  • Genomic DNA was isolated from white blood cells of subjects with a family history of breast cancer using the Blood and Cell Culture DNA Maxi Kit (Qiagen, Germany) .
  • the fact that most breast cancer patients are negative with respect to mutations on BRCAl made it possible to use patient samples for the determination of the correct normal sequence. Samples for the sequencing were selected from 10 patients who were determined to be of normal genetic makeup with respect to Exon 20.
  • Exon 20 of the BRCAl gene was subjected to cycle sequencing with dye-labeled dideoxynucleotides, AmpliTaq ® DNA Polymerase, FS (Roche Molecular Systems, Branchburg, NJ, USA) and primers for amplification.
  • each growing chain is simultaneously terminated and labeled with a dye that corresponds to a particular base (Comparative PCR Sequencing: A Guide to Sequencing-Based Mutation Detection, The Perkin-Elmer Corporation, Applied Biosystems Division, 1995) .
  • primers used for the PCR amplification are suitable for the cycle sequencing reactions. Accordingly, the following primers for BRCAl exon 20 were used: forward primer: 5'- ATA TGA CGT GTC TGC TCC AC -3' [] reverse primer: 5'- GGG AAT CCA AAT TAC ACA GC -3' []
  • PCR Machine PTC 100 Programmmable Thermal Controller, MJ Research, Inc.
  • Fluorescent dye was attached to the PCR product for automated sequencing using the Dye Terminator Cycle Sequence Ready Reaction Kit (Perkin-Elmer ® cat# 402122) . DNA sequencing was performed in both forward and reverse directions on an Applied Biosystems, Inc. (ABI) automated sequencer (Model 377) . The software used for analysis of the resulting data was "Sequence Navigator" purchased through ABI .
  • BIC Sequence CAC TCCATTG (SEQ ID NO: 5)
  • the BIC sequence was determined to have a number of missing or incorrect bases, specifically in the upstream intron.
  • the sequence determined for the 83 bases immediately upstream of Exon 20 are shown in SEQ ID NO: 10.
  • BIC sequence beginning -20 bp upstream: g agtgtktctc attctgcag (SEQ ID NO: 19) Sequence of the invention: g agtgtttttc attctgcag (SEQ ID NO: 20) "k" and "c" have each been changed to "t", as indicated.
  • BIC sequence (downstream 10 bp from Exon 21) : gcctcgggag (SEQ ID NO: 26) Sequence of the invention: gcct*gggag
  • the BIC sequence tttttaaata (SEQ ID NO:30) is replaced by tttt*aaata in the invention.
  • Theoretical analysis of the exon with flanking intron is performed prior to running samples through the CDGE system. Initial optimization is critical for maintaining the highest degree of sensitivity possible.
  • the sequence is entered into computer software created to analyze the melting profile of an exon.
  • the software was designed by Bio-Rad Laboratories, Hercules, California, titled, MacMel t Software - DNA Mel t Profile Macintosh Software for the D GENE System Version 1 . 0.
  • the term melting profile refers to the change in structure of the PCR product when it transitions from a double-stranded molecule to a single-stranded molecule.
  • the melting profile is characteristic of the DNA sequence residing in a particular PCR product and is demonstrated visually on the computer software by way of a graph.
  • the profile theoretically predicts regions where base changes, differing from the original sequence, can be detected.
  • one nucleotide change will create a different curve on the graph.
  • the altered curve is compared to a "normal" or wild-type curve on the same graph. If it is possible to resolve the altered curve from the normal curve, then it is possible to resolve the two different sequences on a polyacrylamide gel with a constant denaturant (CDGE) .
  • CDGE constant denaturant
  • the PCR product processed from genomic DNA, migrates to a certain position, in a defined time period, on the gradient gel. This position is based solely on the sequence of the PCR product. If one base change has occured, the band will migrate to a different position when compared to a normal control.
  • Figure 2 illustrates the melt profile for Exon 20 obtained using the primers of the invention.
  • the y-axis is in units of °C and the x-axis defines the basepairs .
  • the highest domain on the graph is a result of the GC clamp primer.
  • the exon spans a region between 124 basepairs and 207 basepairs. This is an optimal melt profile for two reasons. First, there are not any significant deviations from the 72 °C mark. Deviations would include inclines or declines along the length of the profile. These inclines or declines represent higher and lower melting domains in the sequence.
  • a control sample that has a single base change in that area must be run through the system to ensure sensitivity of strand separation.
  • the entire profile melts in a staircase fashion. There is sequential melting from the upstream (GC-clamp region) portion of the exon to the downstream except for small deviations that are less than 0.25°C (these can be seen when the melt profile is magnified) . These very small deviations in temperature are negligible.
  • the staircase fashion melt profile is important because higher domains at the downstream end of the sequence might not be detected if the upstream portion denatures first in the electrophoresis system.
  • the melt sequence for Figure 3 incorporates primers taken from the BIC sequence referred to as MY-20F (SEQ ID NO: 39) and MY-20R (SEQ ID NO:40) .
  • the sequence includes the MY-20F (SEQ ID NO: 39) primer, the intron sequence following the primer, Exon 20, intron sequence before the MY-2 OR primer and the MY- 2 OR primer (SEQ ID NO: 40) .
  • the exon spans a region between 121 basepairs and 204 basepairs.
  • This sequence is very similar to the sequence used in Figure 2, it gives a very different melt profile. (The two profiles are superimposed in Figure 4 for the purpose of comparison.) There are inclines and declines occuring throughout the profile.
  • each variation would need a different control. Instead of having one or two controls, (i.e. Figure 2), an extensive control panel made up of several mutants would have to be produced and examined for each gel run. This would turn what should be a straightforward mutation analysis procedure into a time consuming task with diminished sensitivity. In contrast, the present invention results in a straightforward efficient procedure which produces superior results.
  • the primers were designed using the primer analysis software, Oligo 4.03 developed by National Biosciences, Inc., Madison, MN (copyright 1992 Wojciech Rychlik) .
  • the bases making up potential primers were analyzed in the Oligo computer program.
  • the analysis procedures indicate problem areas inherent within the short sequence of bases that would inhibit the PCR reaction.
  • Primers determine the success or failure of a PCR amplification (Erlich, Henry A. , PCR Technology, Principles and Application for DNA Amplification, Stockton Press, 1989) . Problems can arise because of primer characteristics such as (1) stretches of a single nucleotide sequence (e.g. -TTTT-), (2) secondary structure (e.g. hairpin loops) , (3) primers which are complementary to each other or to themselves, and other characteristics which would affect the efficiency of the PCR amplification process.
  • it is important that a primer amplify a DNA sequence which includes the splice sites of the exon in order to be certain that errors in the final protein product do not occur due to premature termination of transcription or other problems in initiating and terminating transcription.
  • a forward primer was developed 83 basepairs before the exon and a reverse primer was developed 63 base pairs after the exon :
  • Forward primer 5'- TAAATATGACGTGTCTGCTC -3' (SEQ ID NO: 31) Forward primer with a CG clamp-: 5'- CGCCCGCCGCGCCCCGCGCCCGTCCCGCCGCCCCCGCCCGTAAATA TGACGTGTCTGCTC -3' (SEQ ID NO: 32) Reverse primer: 5'- TGAAGCGGCCCATCTCTGCA -3' (SEQ ID NO: 33)
  • the forward primer (SEQ ID NO: 31) is 20 bases in length, and has optionally attached at the 5' end a further GC clamp of 40 bases, as shown in SEQ ID NO: 32.
  • a "GC clamp” is a nucleotide sequence containing optionally one thymidine base and otherwise guanine and cytosine bases, which is attached to a primer at the 5' end or 3' end for the purpose of affecting the melt profile of the primer during electrophoresis.
  • 20-25 nucleotides of "unique" sequence is attached.
  • the GC clamp contributes a non-melting region to the PCR product .
  • a PCR product without a GC clamp has its own melting characteristics. For example, a GC rich area in the center of a sequence of interest will create a higher melting domain in that region or an arrangement of bases creating several melting variations. Melting variations in the sequence make it difficult to detect mutations in some areas.
  • the attachment of a GC clamp on one of the ends absolves melting variants and creates a profile that is more sensitive for mutation detection.
  • the GC-clamp enables staining the gel with ethidium bromide or SYBR ® green I nucleic acid stain (Molecular Probes, Inc.) and viewing the migration of the PCR product under a UV light.
  • the PCR primers are useful for amplification and subsequent analysis of the PCR product on a constant denaturing gradient gel (CDGE) .
  • CDGE constant denaturing gradient gel
  • the PCR primers may also be labeled with a radiolabel, a fluorescent label, a bioluminescent label, a chemiluminescent label, or an enzyme label, using means familiar to those of skill in the art.
  • Genomic DNA 100 nanograms extracted from white blood cells of a subject was amplified in a final volume of 50 microliters containing 100 nanograms genomic DNA, IX PCR buffer (Roche Molecular Systems, Branchburg, NJ, USA) , 200 micromolar dNTP mix, 1 mM MgCl 2 , 0.2 micromolar forward primer (designated BRCA1-20-F, SEQ ID NO:32), 0.3 micromolar reverse primer (designated BRCA1-20-R, SEQ ID NO:33), 2.5 units AmpliTaq DNA polymerase (Roche Molecular Systems, Branchburg, NJ, USA) , and deionized water up to 50 ⁇ l .
  • IX PCR buffer Roche Molecular Systems, Branchburg, NJ, USA
  • the primers were synthesized at Bioserve Biotechnologies in Laurel, MD using standard 0.2 ⁇ M synthesis scale. Thirty-five cycles were performed, each consisting of denaturing (95°C; 1 minute), annealing (55'C; 1 minute), and extension (72 °C; 1.5 minute), except during the first cycle in which the denaturing time was 3 minutes. In addition, following the thirty five cycles, there was a 10 minute extension at 72 °C, a three minute denaturation at 94 °C and a 1 hour heteroduplexing step at 65°C.
  • PCR products were purified using an ethanol precipitation protocol .
  • the following reagents were added, in order, into a 1.5ml Eppendorf tube: 30 ⁇ l 3M sodium acetate (Sigma: St. Louis, MO), 2 ⁇ l 20mg/ml glycogen (Boehringer Mannheim: Indianapolis, IN), 45 ⁇ l of PCR product, and 223 ⁇ l of deionized water. After mixing these components, 600 ⁇ l of 100% EtOH was added. The reaction was left at room temperature for 15 minutes and then placed in a centrifuge at 13,000 rpm for 15 minutes. After the spin, the supernatant was drained, leaving a small pellet at the bottom of the microfuge tube. One milliliter of cold 70% ethanol was added to the tube.
  • the reaction was placed in the centrifuge and spun at 13,000 rpm for 5 minutes The liquid was drained, leaving a small pellet on the bottom of the centrifuge. The sample was dried in a speed-vac for 15 minutes. The dried pellet was diluted in lO ⁇ l of deionized water. 10% of the purified PCR product was run on a 2% TAE agarose gel for confirmation that the correct PCR product was produced.
  • DGGE Perpendicular Denaturing Gradient Gel Electrophoresis
  • the denaturant composed of varying concentrations of 7M urea and formamide, ranged from 20% to 60% (left to right) .
  • the gel was stained with SYBR ® green I (Molecular Probes, Inc.: Eugene, OR) and viewed under a UV transilluminator .
  • SYBR ® green I Molecular Probes, Inc.: Eugene, OR
  • CDGE constant denaturing gel electrophoresis
  • mutant and wild-type samples migrate vertically through an electrophoretic field encountering a denaturing environment that is created horizontally across the gel .
  • double stranded DNA undergoes partial melting. Higher denaturants melt the PCR products even further, causing greater changes in conformation and migration rates.
  • a heteroduplex is a double stranded nucleic acid in which each strand is amplified from a different template and not exactly complementary.
  • the term complementary describes the nature of the four bases constituting the DNA double helix (adenine, thymine, cytosine and guanine) and their chemical interaction with one another so that adenine always binds with thymine and guanine always binds with cytosine.
  • a heteroduplex has at least one base pair that does not uphold the normal nucleotide interaction stated above. Homoduplexes have complementary PCR products generated from the same template.
  • the example in Figure 8 demonstrates the use of CDGE for mutation detection using amplified PCR product from BRCAl exon 21.
  • the target DNA used for this experiment was human DNA from a patient sample without a history of inherited breast cancer and human DNA known to have an insertion of a cytosine at nucleotide 5438.
  • the PCR products from these examples are representative of both alleles of exon 21 BRCAl. Therefore, if one allele is normal and one allele has a mutation, then 50% of the PCR product will be amplified from the normal allele and 50% of the PCR product will be amplified from the mutant allele.
  • heteroduplexes and homoduplexes made during the melting and reannealing steps allows for a graphical display of the PCR products.
  • Wild-type homoduplexes and mutant homoduplexes are represented on the gel by a single band. These bands will migrate to a specific point on the gel in a designated period of time. The migration pattern is sequence dependent, therefore the position for each homoduplex band will be different.
  • Heteroduplexes have different pattern bands due to the combination of mutant and wild-type polynucleotide strands (refer to Figure 6) .
  • BRCAl exon 21 shows the heteroduplex bands above the homoduplexes. This occurs because the heteroduplex melts sooner than the homoduplex and migrates at a slower rate.
  • FIG. 9 A schematic diagram illustrating the protocol for CDGE is shown in Figure 9.
  • SEQ ID NO: 16 Sequence of the invention beginning ⁇ 60 bp upsteam of exon 15: gtatgatttg tcctttcaca attggtggcg
  • SEQ ID NO: 17 BIC sequence beginning ⁇ 20 bp downsteam of exon 15: attggarcam acactytgat
  • SEQ ID NO: 18 Sequence of the invention beginning -20 bp downsteam of exon 15: attggaacaa acactttgat
  • SEQ ID NO: 19 BIC sequence beginning ⁇ 20 bp upstream of exon 18: g agtgtktctc attctgcag
  • SEQ ID NO: 20 Sequence of the invention beginning ⁇ 20 bp upstream of exon 18: g agtgtttttc attctgcag
  • SEQ ID NO: 22 Sequence of the invention beginning -91 bp downstream of exon 18: ttgctgatgc tgagtctgag ttaccaaaaggt ctttaattgt aatactaaact SEQ ID NO: 23 Variant portion of SEQ ID NO: 21: vvw gctgatgctt gagtctgagt cncnaaagnc
  • SEQ ID NO: 24 Sequence of the invention corresponding to SEQ ID NO: 23 tgag ttaccaaaaggt
  • SEQ ID NO: 26 BIC sequence downstream 10 bp from exon 21: gcctcgggag
  • SEQ ID NO: 28 Sequence of the invention beginning -11 bp upstream of exon 23 : tggggatccagGGTGTCC
  • SEQ ID NO: 29 53 bp sequence of the invention directly downstream of exon 23: gtaaggtgcctgcatgtacctgtgctatatggggtccttttgcatgggtttgg SEQ ID NO:30
  • SEQ ID NO: 33 Reverse primer for exon 20 of the invention:
  • SEQ ID NO: 54 Forward primer of the invention for exon 8:
  • SEQ ID NO: 56 Forward primer of the invention for exon 15
  • SEQ ID NO: 60 Forward primer of the invention for exon 20

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Immunology (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Biophysics (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Pathology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Hospice & Palliative Care (AREA)
  • Physics & Mathematics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Oncology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

On a modifié plusieurs parties des régions à introns entourant les exons 8, 15, 18, 20, 21 et 23 de BRCA1 ainsi que leurs régions introniques adjacentes. Lesdites modifications permettent de construire des amorces oligonucléotidiques qui sont capables d'amplifier les exons avec une fidélité supérieure à ce qu'il était possible d'obtenir jusqu'à maintenant, et qui sont particulièrement utiles dans l'amplification de séquences pour l'électrophorèse sur gel à dénaturation constante dans le gène BRCA1.
PCT/US1997/019596 1996-10-31 1997-10-31 Amorces pour l'amplification de brca1 WO1998018966A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU50938/98A AU5093898A (en) 1996-10-31 1997-10-31 Primers for amplification of brca1

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US2920896P 1996-10-31 1996-10-31
US60/029,208 1996-10-31

Publications (1)

Publication Number Publication Date
WO1998018966A1 true WO1998018966A1 (fr) 1998-05-07

Family

ID=21847811

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1997/019596 WO1998018966A1 (fr) 1996-10-31 1997-10-31 Amorces pour l'amplification de brca1

Country Status (2)

Country Link
AU (1) AU5093898A (fr)
WO (1) WO1998018966A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001057272A3 (fr) * 2000-02-04 2003-01-03 Aeomica Inc Sondes d'acide nucleique a un seul exon derivees du genome humain utiles pour analyser l'expression genique dans le placenta humain

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5622829A (en) * 1993-12-08 1997-04-22 The Regents Of The University Of California Genetic markers for breast, ovarian, and prostatic cancer
US5654155A (en) * 1996-02-12 1997-08-05 Oncormed, Inc. Consensus sequence of the human BRCA1 gene
US5693473A (en) * 1994-08-12 1997-12-02 Myriad Genetics, Inc. Linked breast and ovarian cancer susceptibility gene

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5622829A (en) * 1993-12-08 1997-04-22 The Regents Of The University Of California Genetic markers for breast, ovarian, and prostatic cancer
US5693473A (en) * 1994-08-12 1997-12-02 Myriad Genetics, Inc. Linked breast and ovarian cancer susceptibility gene
US5654155A (en) * 1996-02-12 1997-08-05 Oncormed, Inc. Consensus sequence of the human BRCA1 gene

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF THE AMERICAN MEDICAL ASSOCIATION, 15 February 1995, Vol. 273, No. 7, SHATTUCK-EIDENS et al., "A Collaborative Survey of 80 Mutations in the BRCA1 Breast and Ovarian Cancer Susceptibility Gene", pages 535-541. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001057272A3 (fr) * 2000-02-04 2003-01-03 Aeomica Inc Sondes d'acide nucleique a un seul exon derivees du genome humain utiles pour analyser l'expression genique dans le placenta humain
WO2001057270A3 (fr) * 2000-02-04 2003-02-13 Aeomica Inc Sondes d'acide nucleique a un seul exon derivees du genome humain utiles pour analyser l'expression genique dans des cellules hbl 100
GB2385053B (en) * 2000-02-04 2004-12-22 Aeomica Inc Human genome-derived single exon nucleic acid probes useful for analysis of gene expression in human breast and HBL 100 cells

Also Published As

Publication number Publication date
AU5093898A (en) 1998-05-22

Similar Documents

Publication Publication Date Title
Zeviani et al. Rapid detection of the A----G (8344) mutation of mtDNA in Italian families with myoclonus epilepsy and ragged-red fibers (MERRF)
Jensen et al. High sensitivity of the single-strand conformation polymorphism method for detecting sequence variations in the low-density lipoprotein receptor gene validated by DNA sequencing
US5741645A (en) Gene sequence for spinocerebellar ataxia type 1 and method for diagnosis
JPH0630636B2 (ja) 遺伝的特性決定方法
KR20010024597A (ko) 만성 폐쇄성 기도 질환에 대한 진단방법 및 치료방법
PL162338B1 (pl) do wykrywania sekwencji nukleotydów PL PL PL PL PL
Moraes et al. Techniques and pitfalls in the detection of pathogenic mitochondrial DNA mutations
JPH07136000A (ja) 欠失を検出するための複数染色体ｄｎａ増幅法
US6007231A (en) Method of computer aided automated diagnostic DNA test design, and apparatus therefor
CN111269978B (zh) 一种人ApoE基因分型检测试剂盒
Tesoriero et al. Molecular characterization and cancer risk associated with BRCA1 and BRCA2 splice site variants identified in multiple‐case breast cancer families
US6063567A (en) Method, reagents and kit for diagnosis and targeted screening for retinoblastoma
DE60128434T2 (de) Polymorphismen im menschlichen KDR Gene
CN115976182B (zh) 一种检测脊髓性肌萎缩症致病基因smn1的引物探针组以及试剂盒
US20140148344A1 (en) Association markers for beta thalassemia trait
US5712098A (en) Hereditary hemochromatosis diagnostic markers and diagnostic methods
US20160032405A1 (en) Method for identifying or detecting genomic rearrangements in a biological sample
US6462190B1 (en) Polynucleotides and kits for detection of an age-related mutation
CN114317727B (zh) 用于smn基因拷贝数分析的荧光定量检测试剂及应用
WO1998018966A1 (fr) Amorces pour l'amplification de brca1
KR100552684B1 (ko) 인간 ｍｏｄｙ 1, 4, 5, 6 및 7 유전자 증폭용 다중ｐｃｒ 프라이머 세트
Martins et al. Optimization of a genotyping screening based on hydrolysis probes to detect the main mutations related to Leber hereditary optic neuropathy (LHON)
EP1029079B1 (fr) Diagnostics et therapies des maladies obstructives chroniques des voies respiratoires
WO2005123961A2 (fr) Approches pour identifier des mutations associees au cancer colorectal hereditaire sans polypose
US20070117095A1 (en) Diagnostic method for neonatal or infantile epilepsy syndromes

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE GH HU ID IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZW AM AZ BY KG KZ MD RU TJ TM

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH KE LS MW SD SZ UG ZW AT BE CH DE DK ES FI FR GB GR IE IT LU MC

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA