A process for analyzing length polymorphism in dna regions wherein the following steps are carried out:
(a) annealing at least one primer pair to the dna to be analyzed, wherein one of the molecules of the primer pair is substantially complementary to one of the complementary strands of the 5' or 3' flank of a simple or cryptically simple dna sequence, and wherein the annealing occurs in such an orientation that the synthesis products obtained by a primer-controlled polymerisation reaction with one of said primers can serve as template for annealing the other primer after denaturation;
(b) primer-controlled polymerase chain reaction; and
(c) separating and analyzing the polymerase chain reaction products.
|
0. 42. A kit for analyzing polymorphism in at least one locus in an dna sample, comprising:
a) at least one vessel containing a mixture of primers constituting between 1 and 50 of said primer pairs; b) a vessel containing a polymerizing enzyme suitable for performing a primer-directed polymerase chain reaction; c) a vessel containing the deoxynucleotide triphosphates adenosine, guanine, cytosine and thymidine; d) a vessel containing a buffer solution for performing a polymerase chain reaction; e) a vessel containing a template dna comprising i) a simple or cryptically simple nucleotide sequence having a repeat motif length of 3 to 10 nucleotides and ii) nucleotide sequences flanking said simple or cryptically simple nucleotide sequence that are effective for annealing at least one pair of said primers, for assaying positive performance of the method.
0. 35. A kit for performing analysis of polymorphism in simple or cryptically simple sequences, comprising:
a) at least one vessel containing a mixture of primers constituting 1 to 50 primer parts; wherein each of said primer pairs is composed of a first primer complementary to a nucleotide sequence flanking said simple or cryptically simple dna sequence on the 5' side of said simple or cryptically simple dna sequence and a second primer complementary to a nucleotide sequence flanking the simple or cryptically simple dna sequence on the 3' side of said simple or cryptically simple dna sequence; wherein said first and second primers each anneal to a single site in said dna template and wherein the annealing sites are separated by 50 to 500 nucleotides of template dna; b) a vessel containing a template dna that has a nucleotide sequence including a simple or cryptically simple sequence for assaying positive performance of the method.
0. 15. A method for analyzing length polymorphisms in at least one locus in an dna sample obtained from at least one subject, wherein said dna sample comprises a dna template having at least one locus comprising a simple or cryptically simple dna sequence, said method comprising:
a) annealing said dna template with at least one pair of primers, wherein said primer pair is composed of a first primer complementary to a nucleotide sequence flanking said simple or cryptically simple dna sequence on the 5' side of said simple or cryptically simple dna sequence and a second primer complementary to a nucleotide sequence flanking the simple or cryptically simple dna sequence on the 3' side of said simple or cryptically simple dna sequence; wherein said first and second primers each anneal to a single site in said dna template and wherein the annealing sites are separated by 50 to 500 nucleotides of template dna; b) performing at least one primer-directed polymerase chain reaction upon said template dna having said primers annealed thereto, so as to form at least one polymerase chain reaction product; c) separating the products of each polymerase chain reaction according to their lengths; and d) analyzing the lengths of the separated products to determine the length polymorphisms of said simple or cryptically simple sequences; wherein said simple or cryptically simple sequence has a repeat length of 3 to 10 nucleotides.
1. A method for determining length polymorphisms in a simple or cryptically simple sequence in one or more dna regions of one or more subjects, which comprises:
a) providing at least one dna sample, comprising a template dna having a nucleotide sequence that includes a simple or cryptically simple sequence comprising trinucleotide repeats, from at least one subject; b) annealing at least one primer pair to the template dna of each of said dna samples, wherein said primer pair is composed of a first primer complementary to a nucleotide sequence flanking the simple or cryptically simple dna sequence on the 5' side of said simple or cryptically simple dna sequence and a second primer complementary to a nucleotide sequence flanking the simple or cryptically simple dna sequence on the 3' side of said simple or cryptically simple dna sequence; wherein said first and second primers each anneal to a single site in said template dna and the sequence of the template dna between the sites where said primers anneal is 50 to 500 nucleotides in length; c) performing at least one primer-directed polymerase chain reaction upon said template dna having said primers annealed thereto, so as to form at least one polymerase chain reaction product; d) separating the products of each polymerase chain reaction according to their lengths; and e) analyzing the lengths of the separated products to determine the length polymorphisms of the simple or cryptically simple sequences.
0. 41. A method for analyzing polymorphism in at least one locus in an dna sample comprising a dna template, said method comprising:
a) annealing said dna template with at least one pair of primers, wherein said primer pair is composed of a first primer complementary to a nucleotide sequence flanking said simple or cryptically simple dna sequence on the 5' side of said simple or cryptically simple dna sequence and a second primer complementary to a nucleotide sequence flanking the simple or cryptically simple dna sequence on the 3' side of said simple or cryptically simple dna sequence; wherein said first and second primers each anneal to a single site in said dna template and wherein the annealing sites are separated by 50 to 500 nucleotides of template dna; b) performing at least one primer-directed polymerase chain reaction upon said template dna having said primers annealed thereto, so as to form at least one polymerase chain reaction product; c) separating the products of each polymerase chain reaction product according to their lengths; and d) analyzing the lengths of the separated products to determine the length polymorphisms of said simple or cryptically simple sequences, wherein said dna template includes at least one sequence consisting essentially of a simple or cryptically simple dna sequence having a repeat motif length of 3 to 10 nucleotides and nucleotide sequences flanking said simple or cryptically simple dna sequence effective for annealing said at least one pair of primers.
0. 40. A method for determining length polymorphisms in a simple or cryptically simple sequence in one or more dna regions of one or more subjects, which comprises:
a) providing at least one dna sample, comprising a template dna consisting essentially of a nucleotide sequence that includes i) a simple or cryptically simple sequence having a trinucleotide repeat motif and ii) nucleotide sequences flanking the simple or cryptically simple sequence, from at least one subject; b) annealing at least one primer pair to the template dna of each of said dna samples, wherein said primer pair is composed of a first primer complementary to the nucleotide sequence flanking the simple or cryptically simple dna sequence on the 5' side of said simple or cryptically simple dna sequence and a second primer complementary to the nucleotide sequence flanking the simple or cryptically simple dna sequence on the 3' side of said simple or cryptically simple dna sequence; wherein said first and second primers each anneal to a single site in said template dna and the sequence of the template dna between the sites where said primers anneal is 50 to 500 nucleotides in length; c) performing at least one primer-directed polymerase chain reaction upon said template dna having said primers annealed thereto, so as to form at least one polymerase chain reaction product; d) separating the products of each polymerase chain reaction according to their lengths; e) analyzing the lengths of the separated products to determine the length polymorphisms of the simple or cryptically simple sequences.
0. 2. The method according to
3. The method according to
6. The method according to
7. The method according to claim 2 1, wherein the annealing position of the primers of each pair is selected such that each of the primer-directed polymerase chain reaction products are separable one from the other as individual bands on a suitable electrophoretic gel.
8. The method according to
9. The method according to
10. The method according to
11. The method according to
12. A kit for performing the method of
a) at least one vessel containing an equimolar mixture of primers constituting between 1 and 50 of said primer pairs; b) a vessel containing a polymerizing enzyme suitable for performing a primer-directed polymerase chain reaction; c) a vessel containing the deoxynucleotide triphosphates adenosine, guanine, cytosine and thymidine; d) a vessel containing a buffer solution suitable for performing a polymerase chain reaction, or a concentrate of said buffer solution; e) a vessel containing a template dna that has a nucleotide sequence including a simple or cryptically simple sequence for assaying positive performance of the method, wherein each simple or cryptically simple dna sequence comprises at least one trinucleotide motif.
13. The kit of
14. The method of
0. 16. The method of
0. 17. The method of
0. 18. The method of
0. 19. The method of
0. 20. The method of
0. 21. The method of
0. 22. The method of
0. 23. The method of
0. 24. The method according to
0. 25. The method according to
0. 26. The method according to
0. 27. The method according to
0. 28. The method according to
0. 29. The method according to
0. 30. The method of
0. 31. The method of
0. 32. The method of
0. 33. The method of
0. 34. The method of
0. 36. The kit of
0. 37. The kit of
0. 38. The kit of
0. 39. The kit of
|
|||||||||||||||||||||||||||||||||||||
This application is
The examples illustrate the invention.
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). By this way a gene library is obtained of which about 20,000 phages are plated out. The corresponding independent plaques are transferred to a nitrocellulose filter and hybridized with a probe molecule containing the simple DNA sequence motif CAG/CTG.
The filters are hybridized and washed at 65°C C. The hybridizing solution contains 5 × SSPE, 5 × Denhardt's solution, 0.1% sodium dodecyl sulfate (SDS) and approximately 1×106 cpm/ml of radioactively labelled (32P) DNA as probe molecule. The wash solution contains 2 × SSPE and 0.1% SDS (the reaction product of Denhardt's solution and SSPE is described in (11)).
About 300 to 400 of the plaques formed show a positive signal; cf. FIG. 1. Some of these plaques are purified, DNA is isolated and sequenced. In the obtained DNA sequences regions can be identified containing the simple DNA sequence CAG/CTG; cf. (7).
For this experiment the DNA sequence illustrated in FIG. 2 and published in (13) was chosen. Two oligonucleotides with the following sequences were synthesized:
Oligonucleotide 1 (SEQ. ID No. 1): 5'-TAAGCTTGGGAATCA-3'
Oligonucleotide 2 (SEQ. ID No. 2): 5'-ATTGAACTTTGTATC-3'
These DNA sequences are located immediately at the beginning or at the end of the sequence shown in FIG. 2. For use as primers the synthesized oligonucleotides are labelled with 32P at their 5' end. Then a PCR reaction with the labelled primers is carried out. On the whole 20 cycles are carried out by denaturating at 95°C C. for 90 seconds, hybridizing at 45°C C. for 90 seconds and then synthesizing at 72°C C. for 120 seconds. As DNAs to be examined the genomic DNAs of 11 wild type strains of Drosophila melanogaster from various regions all over the world are employed. These Drosophila wild type strains originally are descendants of individual fertilized females and have been collected during the last 10 years. After the PCR reaction the amplified fragments are cleaved with the restriction endonuclease HaeIII. This should normally yield two fragments having a length of 202 and 177 nucleotides, respectively. This step is normally not necessary for routine experiments. Here it only serves to refine the analysis. The resulting fragments are separated on a 5% sequencing gel, the gel is subsequently dried and an X-ray film is exposed to the dried gel. Both DNA fragments expected show a marked polymorphism in the various Drosophila wild type strains. The 202 nucleotide fragment which contains the simple DNA sequence shows four different size categories; see FIG. 3. These size categories are shifted by three nucleotides each. Starting from frameshifts within the repeat of the trinucleotides this is to be expected. In three cases two different bands appear at the same time; cf.
The majority of the strains examined in this simple experiment are readily distinguishable from one another. Only strains 2, 7 and 11 as well as 3 and 4 cannot be distinguished from each other. To distinguish these strains one would therefore employ further primer pairs. For example, 20 to 50 independent DNA regions could be tested, in order to allow a definite identification. As the size categories of the fragments of the individual Drosophila wild type strains are homogenous per se, one has to start from the assumption that the polymorphisms observed are not so frequent that it would no longer be possible to ascertain a kinship. The Drosophila wild type strains all descend from one single original pair and the DNA of several 100 individuals was combined for the test. If a change of the pattern had taken place within these "families", one should expect more than maximal two bands. This is, however, not the case here. From this follows that the length categories observed are stable for at least some dozens of generations.
The variations in length observed could also be caused by polymerase errors during the experiment. In order to exclude this possibility and to simultaneously prove the general reproducibility, the experiment carried out in Example 2 is repeated with two different DNA preparations of the Drosophila strains No. 3 in 10 independent reaction mixtures. It can be taken from
A primer pair is used which flanks a sequential region from the autosomal human heart muscle actin gene. This sequence contains a simple sequence with a GT/CA dinucleotide repeat structure (FIG. 5). As primers the following oligonucleotides are used:
Primer 1 (SEQ ID No. 3): 5'-CTCCCCCACACAAAGAAG-3'
Primer 2 (SEQ ID No. 4): 5'-TTCCATACCTGGGAACGA-3'
Primer 2 is labelled at its 5' end with 32P and both oligonucleotides are then used for a PCR reaction. On the whole 25 cycles with a denaturation phase of 1 min. at 94°C C., an hybridizing phase of 2 min. at 45°C C. and a synthesis phase of 1 min at 72°C C. (last synthesis phase for 5 min) are carried out. The reaction products are then separated on a 6% denaturating acrylamide gel, the gel is dried and exposed. The result can be seen in FIG. 6. Each of the tested individuals shows two main bands (for explanation of the further bands, see below), i.e. it is heterozygous for different length variants of this locus. Mother and father have the length variant "109 nt" in common, they do, however, differ in the other variant, with the mother having a "127 nt" and the father a "121 nt" variant. The children must have inherited one of each of these variants from father and mother. For two of the children this is actually the case, whereas the third child (labelled with "?") shows a new "113 nt" variant, which can neither be derived from the mother nor from the tested father. Therefore, one has to assume that this child had another father.
In lane "C" a cloned control-DNA having only one length variant has also been treated. Like the other samples it shows a main band and several secondary bands. The secondary bands are caused by PCR artifacts formed during the amplification. In this context, there are two types of artifacts. The first type results from the fact that the Taq-polymerase has the tendency to attach an additional nucleotide to the completely synthesized DNA strand. Thereby the band is formed which runs a nucleotide above the main band. This effect varies from reaction to reaction, but does not disturb the analysis of the band pattern. A second type of artifact is formed by "slippage" during the amplification process. This leads to the bands which can be seen at the dinucleotide distance below the main bands. These artifact bands could have a disturbing effect on the analysis, if they overlap actual length variants.
Simple sequences with trinucleotide repeat motifs do not show these artifact bands (cf. Example 2), as with these sequences "slippage" occurs less frequently during amplification.
EP-A2 0 200 362
1.a EP-A1 0 237 362
2. R. K. Saiki et al., Science 239 (1988), 487-491
3. A. J. Jeffreys et al., Nature 314 (1985), 67-73
4. A. J. Jeffreys et al., Nature 316 (1985), 76-79
4.a Gill et al., Nature 318 (1985), 577-579
4.b Nakamura et al., Science 235 (1987), 1616-1622
5. EP 87 11 6408.3
5.a Higuchi et al., Nature 332 (1988), 543-546
6. D. Tautz, PhD-Thesis (Doktorarbeit) University of T übingen (1983)
7. D. Tautz and M. Renz, J. Mol. Biol. 172 (1984), 229-235
8. D. Tautz and M. Renz, Nucleic Acids Research 12 (1984), 4127-4138.
9. D. Tautz et al., Nature 332 (1986), 652-656
10. G. Levinson and G. A. Gutman, Mol. Biol. and Evolution 4 (1987), 203-221
11. T. Maniatis, E. F. Fritsch and J. Sambrook, "Molecular Cloning, a Laboratory Manual", Cold Spring Harbor Laboratory, New York, 1982
12. L. M. Smith et al., Nature 321 (1986), 674-679
13. K. A. Wharton et al., Cell 40 (1985), 55-62
14. Y. W. Kan and A. M. Dozy, Proc. Natl. Acad. Sci. USA 75 (1978), 5631-5635
15. Marx, Science 240 (1988), 1408-1410
16. Rollo et al., Chem. Abstr. 108(1) (1988), 154, Abstract No. 1552s
Tautz, Diethard, Jäckle, Herbert
| Patent | Priority | Assignee | Title |
| 7645580, | Jun 28 1997 | Qiagen GmbH | Forensic identification |
| 8399191, | Jun 28 1997 | Qiagen GmbH | Forensic identification |
| 8940484, | Jun 28 1997 | Qiagen GmbH | Forensic identification |
| Patent | Priority | Assignee | Title |
| 5075217, | Apr 21 1989 | MARSHFIELD MEDICAL RESEARCH FOUNDATION A DIVISION OF MARSHFIELD CLINIC, | Length polymorphisms in (dC-dA)n ·(dG-dT)n sequences |
| 5364759, | Jan 31 1991 | Baylor College of Medicine | DNA typing with short tandem repeat polymorphisms and identification of polymorphic short tandem repeats |
| EP186271, | |||
| EP200362, | |||
| EP237362, | |||
| EP266787, | |||
| EP342717, | |||
| WO8907658, |
| Executed on | Assignor | Assignee | Conveyance | Frame | Reel | Doc |
| Jun 09 2000 | Max-Planck-Gesellschaft zur Forderung der Wissenschaften e.V. | (assignment on the face of the patent) | / |
| Date | Maintenance Fee Events |
| Nov 15 2005 | M1552: Payment of Maintenance Fee, 8th Year, Large Entity. |
| Dec 30 2009 | M1553: Payment of Maintenance Fee, 12th Year, Large Entity. |
| Dec 30 2009 | M1556: 11.5 yr surcharge- late pmt w/in 6 mo, Large Entity. |
| Date | Maintenance Schedule |
| Feb 11 2006 | 4 years fee payment window open |
| Aug 11 2006 | 6 months grace period start (w surcharge) |
| Feb 11 2007 | patent expiry (for year 4) |
| Feb 11 2009 | 2 years to revive unintentionally abandoned end. (for year 4) |
| Feb 11 2010 | 8 years fee payment window open |
| Aug 11 2010 | 6 months grace period start (w surcharge) |
| Feb 11 2011 | patent expiry (for year 8) |
| Feb 11 2013 | 2 years to revive unintentionally abandoned end. (for year 8) |
| Feb 11 2014 | 12 years fee payment window open |
| Aug 11 2014 | 6 months grace period start (w surcharge) |
| Feb 11 2015 | patent expiry (for year 12) |
| Feb 11 2017 | 2 years to revive unintentionally abandoned end. (for year 12) |