Classifications

• • 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
  • C12Q1/6844—Nucleic acid amplification reactions
  • C12Q1/6858—Allele-specific amplification
• • 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
  • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
• • 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 a method for analyzing length polymorphisms in areas of simple or cryptically simple DNA sequences.
• the invention is therefore based on the object of providing a method for analyzing length polymorphisms in DNA regions which is highly sensitive, delivers reliable results in a short amount of time, is also suitable for serial examinations and routine tests, and if appropriate also can be done automatically.
• This object is achieved according to the invention by providing a method for analyzing length polymorphisms in DNA regions, in which the following steps are carried out:
• the primer-controlled chain reaction is known as such from EP-A2 0 200 362 (1) and from (2).
• This is a method for the amplification of specific DNA fragments, in which a PCR (polymerase chain reaction) is carried out.
• the specific amplification is achieved by using oligonucleotide primers which flank the target molecule antiparallel.
• DNA fragments are formed by a polymerase, which fragments themselves again serve as templates for renewed use Cycle are available.
• DNA synthesis is initiated by heat denaturation of the starting molecule, addition of the corresponding primers and chain extension with a polymerase. The next cycle is started by renewed heat denaturation.
• thermostable synthetic components allows the process to be controlled by simple and easily automatable heating and cooling cycles.
• the “antiparallel flanking” of the target molecule by oligonucleotide primers means the attachment of one of the two primers of a pair of primers to one of the complementary strands of the target molecule, so that the 3 ends of the pair of primers point towards one another.
• Simple and cryptically simple DNA sequences are repetitive components of all eukaryotic genomes, some of which also occur in prokaryotic genomes (6-9). Simple DNA sequences include short DNA motifs that have at least one nucleotide and at most about 6 to 10 nucleotides included and repeated a dozen to about a hundred times in tandem. These simple DNA sequences have been found by hybridization with synthetic DNA sequences and by direct sequencing in all previously analyzed eukaryotic genomes and also in the human genome (8, 10). Presumably all possible permutations of short motifs occur with different frequencies (9). Cryptically simple DNA sequences are characterized by a randomly frequent, but irregular, direct repetition of short DNA motifs (9). Cryptically simple DNA sequences are usually only found indirectly with an appropriate computer program in already sequenced DNA areas. However, they occur at least as often or even more frequently than simple DNA sequences.
• the simple and cryptically simple DNA sequences may have arisen through genomic mechanisms which have a tendency to double again existing short duplications of any DNA sequence motifs or longer regions of already existing simple or cryptic in any DNA sequence motifs partially delete simple DNA sequences (8-10). It can therefore be assumed that these areas are usually length-polymorphic.
• the method according to the invention is based on this length polymorphism.
• Simple or cryptically simple DNA sequences suitable for the method according to the invention can be found in already known DNA sequences with or without the aid of a computer program (9).
• a simple or cryptically simple DNA sequence is suitable if it has a length of approximately 20 to 300 nucleotides and is flanked by random sequences, that is to say by DNA sequences without internal repetitions. Pieces are then selected from the range of flanking DNA sequences without internal sequence repeats, for which suitable complementary, synthetic oligonucleotides are produced.
• An oligonucleotide is suitable for this Its purpose is when its nucleotide composition and its nucleotide sequence are only likely to occur once in the genome to be examined and are therefore specific to the DNA region to be analyzed individually.
• length polymorphisms of simple or cryptically simple DNA sequences are preferably investigated, which are essentially composed of trinucleotide motifs.
• the simple or cryptically simple DNA sequence is essentially composed of the trinucleotide motif
• Primer pairs are preferably used in the method according to the invention, the individual molecules of which are attached to the DNA region to be examined at a distance of 50 to 500 nucleotides from one another, that is to say they span this, so to speak, at the specified distance.
• the DNA region to be examined is surrounded by the attached molecules of the primer pair.
• Two pairs of primers are preferably used in the method according to the invention. In a particularly preferred embodiment, 2 to 50 primer pairs are used.
• the primers used in the method according to the invention preferably have a length of 15 to 25 nucleotides.
• the individual primer pairs when using several primer pairs, are selected such that the corresponding specific polymerase chain reaction products of the individual primer pairs can be separated into individual bands on a suitable gel.
• the detection of the specific polymerase chain reaction products is carried out by radioactive labeling or by non-radioactive labeling, e.g. with fluorescent dyes.
• the oligonucleotide pairs can be labeled radioactively or, as described in (12), with a fluorescent dye.
• cutlery (kits) with which the method according to the invention can be carried out are the subject of the present invention.
• the primers contained therein are optionally radioactive, for example 35 S or 1 C, or fluorescence-labeled.
• the synthesis products obtained in the process according to the invention can be separated using high-resolution gel systems, such as conventional sequencing gels.
• the length of the synthesis products can also be determined.
• Polymorphisms which are formed by insertions or deletions of individual or multiple motifs of the simple or cryptically simple DNA sequence, are indicated by a changed position of the syn these products are recognizable in the gel.
• approximately 20 to 50 independent polymorphic regions can be examined simultaneously with a suitable resolution of the gel system. The identity of an individual can thus be reliably established on the basis of the individual combination of length distributions of the synthesis products obtained.
• DNA sequences suitable for the DNA areas to be examined can be identified as follows.
• a genomic DNA to be examined is subjected to a partial restriction cleavage. Restriction enzymes are used which usually do not cleave in simple or cryptically simple DNA sequences.
• the DNA fragments obtained are in a suitable vector, e.g. cloned in lambda phage derivatives or in M13 phages and then searched in a conventional manner by hybridization for simple or cryptically simple DNA sequences; see. (11).
• the probe molecules used are synthetic DNA molecules which contain various permutations of simple or cryptically simple DNA sequences. In this way, hybridizing plaques can be identified.
• the recombinant DNA contained therein can then be isolated and characterized by sequencing. The DNA sequence obtained in this way can then be searched for DNA sequences suitable for the test method according to the invention.
• the method according to the invention was carried out using Drosophila DNA as a model system.
• Drosophila DNA as a model system.
• simple and cryptic simple DNA sequences occur in all eukaryotic genomes and in some cases also in prokaryotic genomes, it can be assumed that the results obtained in the Drosophila model system are also achieved when examining other genomes, in particular when examining the human genome can be.
• the method according to the invention is thus suitable for determining the identity and relationship of organisms, for example humans.
• paternity tests and forensic tests for identifying offenders can be carried out using the method according to the invention; see. also example 4.
• the method is also suitable for determining the mode of inheritance for genetic diseases for which the locus is known and sequenced. For this, one or more simple or cryptically simple sequences are selected that are in or near the locus to be analyzed. The specific length pattern of these areas is correlated with the mutated locus, as is usual with conventional RFLP markers; see. (14). With this information, a genetic counseling or a prenatal diagnosis can then be carried out in the families concerned, in a manner analogous to that used for RFLP markers.
• the use of the method according to the invention for this purpose is particularly useful because it is based on DNA regions that are polymorphic with a predictable probability, while the RFLP analysis relies on randomly found variations that are often far from the locus themselves, which reduces the reliability of diagnosis.
• the method according to the invention is suitable for determining polymorphisms in simple or cryptically simple DNA sequences of animals and plants. Therefore, in animal breeding e.g. in horses, dogs or cattle, the relationship to high-quality breeding individuals can be reliably proven.
• the advantage of the method according to the invention over the previously known methods lies in its broad applicability, quick feasibility and high sensitivity.
• the in the inventive The amplification step carried out for the length-polymorphic simple or cryptically simple DNA sequences makes an independent detection step, such as a subsequent hybridization reaction, superfluous.
• the method according to the invention is particularly well suited for automation and for routine tests and serial examinations.
• Figure 1 Hybridization of a gene bank with a simple DNA sequence as a probe molecule.
• Figure 2 Sequence of the region tested for polymorphism in Example 2.
• the areas to which complementary oligonucleotides have been synthesized are underlined with a wavy line.
• the area of the simple DNA sequence is underlined with a double label.
• the direct repetition of 8 nucleotides is indicated by two arrows.
• the Haelll gap is marked in italics.
• Figure 5 Sequence of the DNA region used in Example 4. The areas to which complementary oligonucleotides have been synthesized are underlined with a wavy line.
• Figure 6 Human paternity analysis.
• the DNA fragments amplified by PCR and separated on the gel are shown.
• the DNA of the mother is plotted in the first lane, the DNA of the father to be tested and the three children tested are plotted in the following lanes.
• the sixth lane (marked with "C") a control DNA is plotted which is only intended to indicate size classes.
• the main bands and their size classes are marked on the right margin.
• Drosophila DNA is completely cleaved with the restriction endonuclease EcoRI and the resulting fragments are cloned into the lambda vector 641.
• a more detailed description of the methods used can be found in (11).
• a gene bank is thus obtained, from which approximately 20,000 phages are plated out.
• the corresponding isolated plaques are transferred to a nitrocellulose filter and hybridized with a probe molecule which contains the simple DNA sequence motif CAG / CTG.
• the filters are hybridized and washed at 65 ° C.
• the hybridization solution contains 5x SSPE, 5x Denhardt's solution, 0.1% sodium dodecyl sulfate (SDS) and about 1 x 10 6 cp / ml radioactive ( 32 P) labeled DNA as a probe molecule.
• the wash solution contains 2 x SSPE and 0.1% SDS (the composition of Denhardt's solution and SSPE is described in (11)).
• plaques show a positive signal; see. Figure 1.
• Some of these plaques are purified, DNA is isolated and sequenced. Areas can be identified in the DNA sequences obtained which contain the simple DNA sequence CAG / CTG; see. (7).
• Oligonucleotide 1 5 • -TAAGCTTGGGAATCA-3 'Oligonucleotide 2: 5'-ATTGAACTTTGTATC-3 •
• DNA sequences are located directly at the beginning or at the end of the sequence shown in FIG. 2.
• the synthesized oligonucleotides are labeled with 32 P at their 5 'end.
• a PCR reaction is then carried out with the labeled primers.
• a total of 20 cycles are carried out, de-naturing in each case at 95 ° C. for 90 seconds, addition at 90 ° C. for 90 seconds and synthesis at 120 ° C. for 120 seconds.
• the genomic DNAs from 11 wild-type strains of Drosophila are used as the DNAs to be examined melanogasters from different areas of the whole world, these Drosophila wild-type strains originally come from individual fertilized females and have been collected over the past 10 years.
• the amplified fragments are cleaved with the Hae III restriction endonuclease. This should normally result in two fragments that are 202 nucleotides or 177 nucleotides in length. This step is usually not necessary for routine experiments. It only serves to refine the analysis here.
• the resulting fragments are separated on a percentage sequencing gel, the gel is then dried and an X-ray film is exposed to the dried gel.
• the two expected DNA fragments show a pronounced polymorphism in the different .Drosophila wild-type strains.
• the 202 nucleotide fragment containing the simple DNA sequence shows four different size classes; see. Figure 3. These size classes are each shifted by three nucleotides.
• the majority of the strains examined in this simple experiment can already be distinguished from one another. Only the strains 2, 7 and 11 and 3 and 4 cannot be distinguished. Therefore, additional primer pairs would be used for an actual test. For example, 20 to 50 independent DNA regions could be examined in order to find a unique one Allow identification. Since the size classes of the individual Drosophila wild-type strains are uniform in themselves, it can be assumed that the observed polymorphisms do not occur with such a high frequency that family relationships could no longer be determined. The Drosophila wild-type strains all originate from a single starting pair and the DNA of several 100 individuals was combined for the test. If a change in pattern had occurred within these "families", one would have to expect more than a maximum of two bands. However, this is not the case here. It follows that the observed length classes are stable at least for a few dozen generations.
• Example 2 The observed length variations could also be caused by polymerase errors occurring during the experiment.
• the experiment carried out in Example 2 is repeated with two different DNA preparations of Drosophila strain No. 3, each in 10 independent batches. From Figure 4 it can be seen that all approaches lead to the same bands. Similar experiments were also carried out for other loci. However, no change in band length was observed in any case. This shows that the method can be reliably reproduced.
• a pair of primers is used which flanges a sequence region from the autosomal human cardiac muscle actin gene kiert.
• This sequence contains a simple sequence with a GT / CA dinucleotide repeat structure ( Figure 5).
• the following oligonucleotides are used as primers:
• Primer 1 5'-CTCCCCCACACAAAGAAG-3 • Primer 2: 5 • -TTCCATACCTGGGAACGA-3 '
• Primer 2 is labeled with 32 P at its 5 'end and both oligonucleotides are then used for a PCR reaction. A total of 25 cycles are carried out with a denaturation phase of 1 min at 94 ° C., an addition phase of 2 min at 45 ° C. and a synthesis phase of 1 min at 72 ° C. (last synthesis phase for 5 min). The reaction products are then carried out The gel is dried and exposed on a 6% denaturing acrylamide gel, the result is shown in Fig. 6.
• a cloned control DNA which has only one length variant was treated in lane "C". Like the other samples, it shows one main band and several sub-bands. These are due to PCR artifacts that arise during amplification. There are two types of artifacts. The first type is due to the fact that the Taq poly erase has the tendency to add a further nucleotide to the completely synthesized DNA strand. This creates the band that runs one nucleotide above the main band. This effect differs from reaction to reaction, but is not disturbing for the analysis of the band pattern. A second type of artifact is created by "slippage" during the amplification process. This creates the bands that can be seen at the dinucleotide spacing below the main bands.

Landscapes

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

Priority Applications (3)

Applications Claiming Priority (2)

Related Child Applications (1)

Publications (1)

Family

ID=6364896

Family Applications (1)

Country Status (7)

Cited By (6)

Families Citing this family (22)

Citations (1)

Family Cites Families (7)

• 1988
  • 1988-10-11 DE DE3834636A patent/DE3834636A1/de active Granted
• 1989
  • 1989-10-11 JP JP51115289A patent/JP3218318B2/ja not_active Expired - Lifetime
  • 1989-10-11 AT AT89912096T patent/ATE161585T1/de not_active IP Right Cessation
  • 1989-10-11 DE DE58909827T patent/DE58909827D1/de not_active Expired - Lifetime
  • 1989-10-11 WO PCT/EP1989/001203 patent/WO1990004040A1/de not_active Ceased
  • 1989-10-11 EP EP89912096A patent/EP0438512B2/de not_active Expired - Lifetime
  • 1989-10-11 HK HK98103477A patent/HK1004341A1/xx not_active IP Right Cessation
• 2000
  • 2000-06-09 US US09/591,383 patent/USRE37984E1/en not_active Expired - Lifetime

Patent Citations (1)

Non-Patent Citations (2)

Also Published As

Similar Documents

Legal Events